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Volume 20,Issue 1,2013 Table of Contents
2013, 20(1):1-12.
DOI: 10.3872/j.issn.1007-385X.2013.1.001
Abstract:
The development of cancer is a complex multistage process, which is primarily due to the unbalance of cellular homeostasis. In addition to the well-characterized protein coding dysfunction and DNA mutation, dysregulation of non-coding RNAs including microRNA (miRNA), long non-coding RNA (lncRNA) and circulating miRNA was found in the initiation and progression of cancer. Moreover, aberrant RNA transcription, processing and regulation, such as RNA alternative splicing, RNA editing and competing endogenous RNA regulation, have emerged as important regulatory molecules or mechanisms in cancer cells. These non-coding RNAs may play vital roles in tumorigenesis by regulating oncogene and tumor suppressor gene at either post-transcriptional level or epigenetic level, and act as potential targets for tumor diagnosis, prognosis and cancer therapy. Through competing endogenous RNA, the lncRNA cross-talk with mRNA in a miRNA-dependent manner, and thus form a regulatory network. Via RNA alternative splicing, oncogene and tumor-suppressor gene could generate tumor-specific alternative-splicing transcripts, and therefore affect their biological functions. However, several questions remain to be elucidated: What are the underlying mechanisms of abnormal expression pattern and dysregulation of RNA in cancer? How do the aberrant expression and function of RNAs affect tumorigenesis and progression? Which abnormal RNA can be used as a biomarker for tumor diagnosis, prognosis, as well as the therapeutic target for cancers? Taken together, RNA dysregulation in cancer has become a new research frontier in cancer research.
The development of cancer is a complex multistage process, which is primarily due to the unbalance of cellular homeostasis. In addition to the well-characterized protein coding dysfunction and DNA mutation, dysregulation of non-coding RNAs including microRNA (miRNA), long non-coding RNA (lncRNA) and circulating miRNA was found in the initiation and progression of cancer. Moreover, aberrant RNA transcription, processing and regulation, such as RNA alternative splicing, RNA editing and competing endogenous RNA regulation, have emerged as important regulatory molecules or mechanisms in cancer cells. These non-coding RNAs may play vital roles in tumorigenesis by regulating oncogene and tumor suppressor gene at either post-transcriptional level or epigenetic level, and act as potential targets for tumor diagnosis, prognosis and cancer therapy. Through competing endogenous RNA, the lncRNA cross-talk with mRNA in a miRNA-dependent manner, and thus form a regulatory network. Via RNA alternative splicing, oncogene and tumor-suppressor gene could generate tumor-specific alternative-splicing transcripts, and therefore affect their biological functions. However, several questions remain to be elucidated: What are the underlying mechanisms of abnormal expression pattern and dysregulation of RNA in cancer? How do the aberrant expression and function of RNAs affect tumorigenesis and progression? Which abnormal RNA can be used as a biomarker for tumor diagnosis, prognosis, as well as the therapeutic target for cancers? Taken together, RNA dysregulation in cancer has become a new research frontier in cancer research.
2013, 20(1):13-19.
DOI: 10.3872/j.issn.1007-385X.2013.1.002
Abstract:
Objective:To explore the biological properties of leukemia cell-derived exosomes (LEXs) and the anti-leukemia immunological effect of LEX-sensitized dendritic cells (DCs). Methods: LEX from BCR-ABL positive leukemia K562 cells was separated and purified by ultracentrifugation. The expressions of heat shock protein 70 (HSP70) and BCR-ABL were investigated by immuno-electron microscopy and Western blotting. The kinetics of LEXK562 targeted binding to DCs was examined by laser scanning confocal microscopy and flow cytometry. The anti-leukemia immunological effect of LEX and its sensitized DCs was explored by LDH release assay and in vivo mouse model studies. Results: K562 cell-derived exosomes (LEXK562) had cystic structures between 50 and 100 nm diameter as the other cell-derived exosomes, and expressed K562 cell specific proteins, HSP70 and BCR-ABL. LEXK562 could target and bind DCs in vitro, and reached a plateau after 3 to 4 h of co-culturing. LEXK562 uptaken in DCs was quite stable for over 72 h. Cytotoxic T lymphocytes (CTLs) induced by LEXK562-sensitized DCs (DC/LEXK562) could effectively kill K562 target cells, and their cytotoxicity was significantly higher than that of CTLs induced by LEXK562 (\[68.6±5.7\]% vs \[22.5±2.9\]%, P<0.01) at an effector to target ratio of 50∶1. Furthermore, the in vivo study demonstrated that the incidence of tumor in mice incubation with leukemia L1210 cells after being immunized with L1210-derived exosomes (LEXL1210) was significantly higher than that of LEXL1210-sensitized DCs (\[54.17±8.33\]% vs \[16.67±4.18\]%, P<0.05). Conclusion: LEX expresses antigens associated with leukemia cells and can target bind DCs in vitro. LEX-sensitized DCs can induce a stronger anti-leukemia immunological effect.
Objective:To explore the biological properties of leukemia cell-derived exosomes (LEXs) and the anti-leukemia immunological effect of LEX-sensitized dendritic cells (DCs). Methods: LEX from BCR-ABL positive leukemia K562 cells was separated and purified by ultracentrifugation. The expressions of heat shock protein 70 (HSP70) and BCR-ABL were investigated by immuno-electron microscopy and Western blotting. The kinetics of LEXK562 targeted binding to DCs was examined by laser scanning confocal microscopy and flow cytometry. The anti-leukemia immunological effect of LEX and its sensitized DCs was explored by LDH release assay and in vivo mouse model studies. Results: K562 cell-derived exosomes (LEXK562) had cystic structures between 50 and 100 nm diameter as the other cell-derived exosomes, and expressed K562 cell specific proteins, HSP70 and BCR-ABL. LEXK562 could target and bind DCs in vitro, and reached a plateau after 3 to 4 h of co-culturing. LEXK562 uptaken in DCs was quite stable for over 72 h. Cytotoxic T lymphocytes (CTLs) induced by LEXK562-sensitized DCs (DC/LEXK562) could effectively kill K562 target cells, and their cytotoxicity was significantly higher than that of CTLs induced by LEXK562 (\[68.6±5.7\]% vs \[22.5±2.9\]%, P<0.01) at an effector to target ratio of 50∶1. Furthermore, the in vivo study demonstrated that the incidence of tumor in mice incubation with leukemia L1210 cells after being immunized with L1210-derived exosomes (LEXL1210) was significantly higher than that of LEXL1210-sensitized DCs (\[54.17±8.33\]% vs \[16.67±4.18\]%, P<0.05). Conclusion: LEX expresses antigens associated with leukemia cells and can target bind DCs in vitro. LEX-sensitized DCs can induce a stronger anti-leukemia immunological effect.
2013, 20(1):20-25.
DOI: 10.3872/j.issn.1007-385X.2013.1.003
Abstract:
Objective:To analyze the proliferation and differential expressions of activated and inhibitory surface markers, drug-resistance gene ABCG2 and stem cell transcription factors in umbilical cord blood-derived cytokine induced killer (CIK) cells or peripheral blood-derived CIK (PB-CIK) cells in cancer patients, and to explore the superiority of umbilical cord blood-derived CIK (CB-CIK) cells in adoptive cellular immunotherapy. Methods: Cord blood mononuclear cells and peripheral blood mononuclear cells in cancer patients were isolated using Ficoll-plaque density gradients and incubated with cytokines. The proliferation, immunophenotypes, activated surface markers (CD28, CD27) and inhibitory surface marker (PD-1) were analyzed by flow cytometry, and the drug-resistance gene ABCG2 or stem cell transcription factors ( c-Myc, Nanog, Oct-4 and Sox2 ) were determined by RT-PCR. Results: CB-CIK cells and PB-CIK cells started to proliferate on the fourth day in vitro, and the proliferation index of CB-CIK cells was significantly higher than that of PB-CIK cells on day 10 (\[251.52±16.76\]% vs \[158.00±43.19\]%, P<005\]. Thirteen days after incubation, the proportion of CD3+CD56+ cells in CB-CIK cells was higher than that in PB-CIK cells (\[21.20±4.82\]% vs \[10.06±346\]%, P<0.05). When detecting the status of CIK cells, the proportions of activated CD4+CD28+, CD4+ CD27+ and CD8+CD27+ cells were significantly higher in CB-CIK cells than in PB-CIK cells (\[32.40±16.81\]% vs \[18.65±923\]%; \[27.48±13.53\]% vs \[0.98±0.55\]%; \[41.76±13.98\]% vs \[2.58±2.10\]%，P<0.05 or P<0.01), while the proportion of inhibitory CD8+PD-1+ cells was significantly lower in CB-CIK cells (\[3.25±2.13\]% vs \[8.05±9.23\]%, P<0.01). The stem cell transcription factors ( c-Myc, Nanog, Oct and Sox2 ) were expressed both in CB-CIK cells and PB-CIK cells. Moreover, no significant differences were found between the two kinds of CIK cells. Furthermore, only CB-CIK cells expressed a high level of drug-resistance gene ABCG2 . Conclusion: Compared with the PB-CIK cells, the CB-CIK cells showed increasing proliferation rates, higher expressions of activated surface markers, and lower expression of inhibitory surface marker. Besides, drug-resistant gene ABCG2 is highly expressed in CB-CIK cells, showing several advantages in applying to adoptive cellular tumor immunotherapy.
Objective:To analyze the proliferation and differential expressions of activated and inhibitory surface markers, drug-resistance gene ABCG2 and stem cell transcription factors in umbilical cord blood-derived cytokine induced killer (CIK) cells or peripheral blood-derived CIK (PB-CIK) cells in cancer patients, and to explore the superiority of umbilical cord blood-derived CIK (CB-CIK) cells in adoptive cellular immunotherapy. Methods: Cord blood mononuclear cells and peripheral blood mononuclear cells in cancer patients were isolated using Ficoll-plaque density gradients and incubated with cytokines. The proliferation, immunophenotypes, activated surface markers (CD28, CD27) and inhibitory surface marker (PD-1) were analyzed by flow cytometry, and the drug-resistance gene ABCG2 or stem cell transcription factors ( c-Myc, Nanog, Oct-4 and Sox2 ) were determined by RT-PCR. Results: CB-CIK cells and PB-CIK cells started to proliferate on the fourth day in vitro, and the proliferation index of CB-CIK cells was significantly higher than that of PB-CIK cells on day 10 (\[251.52±16.76\]% vs \[158.00±43.19\]%, P<005\]. Thirteen days after incubation, the proportion of CD3+CD56+ cells in CB-CIK cells was higher than that in PB-CIK cells (\[21.20±4.82\]% vs \[10.06±346\]%, P<0.05). When detecting the status of CIK cells, the proportions of activated CD4+CD28+, CD4+ CD27+ and CD8+CD27+ cells were significantly higher in CB-CIK cells than in PB-CIK cells (\[32.40±16.81\]% vs \[18.65±923\]%; \[27.48±13.53\]% vs \[0.98±0.55\]%; \[41.76±13.98\]% vs \[2.58±2.10\]%，P<0.05 or P<0.01), while the proportion of inhibitory CD8+PD-1+ cells was significantly lower in CB-CIK cells (\[3.25±2.13\]% vs \[8.05±9.23\]%, P<0.01). The stem cell transcription factors ( c-Myc, Nanog, Oct and Sox2 ) were expressed both in CB-CIK cells and PB-CIK cells. Moreover, no significant differences were found between the two kinds of CIK cells. Furthermore, only CB-CIK cells expressed a high level of drug-resistance gene ABCG2 . Conclusion: Compared with the PB-CIK cells, the CB-CIK cells showed increasing proliferation rates, higher expressions of activated surface markers, and lower expression of inhibitory surface marker. Besides, drug-resistant gene ABCG2 is highly expressed in CB-CIK cells, showing several advantages in applying to adoptive cellular tumor immunotherapy.
2013, 20(1):26-29.
DOI: 10.3872/j.issn.1007-385X.2013.1.004
Abstract:
Objective:To explore the effect of DNA methylation inhibitor 5-Aza-2’-deoxycytidine (5-Aza-CdR) on enhancing the sensitivity of human non-small cell lung cancer (NSCLC) cells to gefitinib and its mechanisms. Methods: EGFR mutant NSCLC cell line H1650 and EGFR wild type NSCLC cell line H1299 were used in this study. The changes in sensitivity of H1650 and H1299 cells to gefitinib after 5-Aza-CdR treatment were detected by CCK-8 assay. The expression levels of miR-200c and epidermal growth factor receptor (EGFR) mRNA were evaluated by real-time PCR. Results: EGFR mutant and wild type NSCLC cell lines H1650 and H1299 showed a certain degree of drug resistance to gefitinib. The sensitivity of H1650 and H1299 cells to gefitinib ncreased after 5-Aza-CdR treatment, with IC50 value of gefitinib to cells decreasing significantly (\[1.04±0.35\] vs \[159.37±17.48\] μmol/L, \[6.28±1.02\] vs \[223.76±23.63\] μmol/L, P<0.01). Real-time PCR assay results showed that the expression level of miR-200c was increased in EGFR mutant H1650 cells or wild type H1299 cells after 5-Aza-CdR treatment (\[0.009±0.003\] vs \[0.002±0.001\], \[0.004±0.001\] vs 0, P<0.01), respectively. Besides, the expression level of EGFR mRNA was significantly increased compared with 5-Aza-CdR untreated group (\[0.286±0.037\] vs \[0.015±0.012\], \[0.057±0014\] vs \[001±0.01\], P<0.01). Conclusion: The expressions of miR-200c and EGFR mRNA in human NSCLC H1650 and H1299 cells are up-regulated by DNA methylation inhibitor 5-Aza-CdR, which increases the sensitivity of H1650 and H1299 cells to gefitinib.
Objective:To explore the effect of DNA methylation inhibitor 5-Aza-2’-deoxycytidine (5-Aza-CdR) on enhancing the sensitivity of human non-small cell lung cancer (NSCLC) cells to gefitinib and its mechanisms. Methods: EGFR mutant NSCLC cell line H1650 and EGFR wild type NSCLC cell line H1299 were used in this study. The changes in sensitivity of H1650 and H1299 cells to gefitinib after 5-Aza-CdR treatment were detected by CCK-8 assay. The expression levels of miR-200c and epidermal growth factor receptor (EGFR) mRNA were evaluated by real-time PCR. Results: EGFR mutant and wild type NSCLC cell lines H1650 and H1299 showed a certain degree of drug resistance to gefitinib. The sensitivity of H1650 and H1299 cells to gefitinib ncreased after 5-Aza-CdR treatment, with IC50 value of gefitinib to cells decreasing significantly (\[1.04±0.35\] vs \[159.37±17.48\] μmol/L, \[6.28±1.02\] vs \[223.76±23.63\] μmol/L, P<0.01). Real-time PCR assay results showed that the expression level of miR-200c was increased in EGFR mutant H1650 cells or wild type H1299 cells after 5-Aza-CdR treatment (\[0.009±0.003\] vs \[0.002±0.001\], \[0.004±0.001\] vs 0, P<0.01), respectively. Besides, the expression level of EGFR mRNA was significantly increased compared with 5-Aza-CdR untreated group (\[0.286±0.037\] vs \[0.015±0.012\], \[0.057±0014\] vs \[001±0.01\], P<0.01). Conclusion: The expressions of miR-200c and EGFR mRNA in human NSCLC H1650 and H1299 cells are up-regulated by DNA methylation inhibitor 5-Aza-CdR, which increases the sensitivity of H1650 and H1299 cells to gefitinib.
2013, 20(1):30-36.
DOI: 10.3872/j.issn.1007-385X.2013.1.005
Abstract:
Objective:To investigate the effect of in vitro silencing CCL18 gene expression on invasion and metastasis of ovarian epithelial carcinoma SKOV3 cells. Methods: Three pairs of siRNAs targeting CCL18 (CCL18-siRNA61, CCL18-siRNA127, CCL18-siRNA224) were chemically synthesized, and were transfected into CCL18-positive SKOV3 cells in vitro. RT-PCR was used to detect the expression of CCL18 mRNA in SKOV3 cells. The siRNA sequence with the best interference efficiency was selected to construct interference plasmid targeting CCL18 , named pSilencer4.1-CCL18-siRNA61. After transfection of pSilencer4.1-CCL18-siRNA61 plasmid, the proliferation of SKOV3 cells was detected by MTT assay, the cell cycle was detected by flow cytometry, and the cell invasion, migration and adhesion capacity in vitro was determined by Transwell assay, Migration assay and Fibronectin adhesion method, respectively. Results: CCL18-siRNA61 showed the best interference efficiency in the three CCL18-siRNAs. The interference plasmid pSilencer4.1-CCL18-siRNA61 was then successfully constructed. The expression of CCL18 mRNA was significantly decreased after pSilencer4.1-CCL18-siRNA61 transfection. The proliferation of SKOV3 cells was not affected by pSilencer4.1-CCL18-siRNA61 transfection. However, the proportion of SKOV3 cells in phase (S+G2+M) in the pSilencer4.1-CCL18-siRNA61 transfection group was significantly lower than that in the pSilencer4.1-Ctrl-siRNA transfection group (\[1971±44\]% vs \[26.45±7.91\]%, P<0.05). The invasion, migration and adhesion capacity of SKOV3 cells was effectively inhibited in the pSilencer4.1-CCL18-siRNA61 transfection group compared with that in the pSilencer4.1-Ctrl-siRNA transfection group (\[9.91±3.41\]% vs \[23.75±6.81\]%, \[16.80±8.71\]% vs \[31.74±11.23\]%, \[6.73±4.33\]% vs \[17.53±6.54\]%, P<0.05), respectively. Conclusion: siRNA silencing CCL18 expression can inhibit the invasion, adhesion and migration capacity of ovarian epithelial cancer cell line SKOV3.
Objective:To investigate the effect of in vitro silencing CCL18 gene expression on invasion and metastasis of ovarian epithelial carcinoma SKOV3 cells. Methods: Three pairs of siRNAs targeting CCL18 (CCL18-siRNA61, CCL18-siRNA127, CCL18-siRNA224) were chemically synthesized, and were transfected into CCL18-positive SKOV3 cells in vitro. RT-PCR was used to detect the expression of CCL18 mRNA in SKOV3 cells. The siRNA sequence with the best interference efficiency was selected to construct interference plasmid targeting CCL18 , named pSilencer4.1-CCL18-siRNA61. After transfection of pSilencer4.1-CCL18-siRNA61 plasmid, the proliferation of SKOV3 cells was detected by MTT assay, the cell cycle was detected by flow cytometry, and the cell invasion, migration and adhesion capacity in vitro was determined by Transwell assay, Migration assay and Fibronectin adhesion method, respectively. Results: CCL18-siRNA61 showed the best interference efficiency in the three CCL18-siRNAs. The interference plasmid pSilencer4.1-CCL18-siRNA61 was then successfully constructed. The expression of CCL18 mRNA was significantly decreased after pSilencer4.1-CCL18-siRNA61 transfection. The proliferation of SKOV3 cells was not affected by pSilencer4.1-CCL18-siRNA61 transfection. However, the proportion of SKOV3 cells in phase (S+G2+M) in the pSilencer4.1-CCL18-siRNA61 transfection group was significantly lower than that in the pSilencer4.1-Ctrl-siRNA transfection group (\[1971±44\]% vs \[26.45±7.91\]%, P<0.05). The invasion, migration and adhesion capacity of SKOV3 cells was effectively inhibited in the pSilencer4.1-CCL18-siRNA61 transfection group compared with that in the pSilencer4.1-Ctrl-siRNA transfection group (\[9.91±3.41\]% vs \[23.75±6.81\]%, \[16.80±8.71\]% vs \[31.74±11.23\]%, \[6.73±4.33\]% vs \[17.53±6.54\]%, P<0.05), respectively. Conclusion: siRNA silencing CCL18 expression can inhibit the invasion, adhesion and migration capacity of ovarian epithelial cancer cell line SKOV3.
2013, 20(1):37-42.
DOI: 10.3872/j.issn.1007-385X.2013.1.006
Abstract:
Objective:To investigate the effect of mitofusin-2 ( Mfn-2 ) gene expression on sensitivity of human breast cancer T47D cells to parthenolide. Methods: The expressions of Mfn-2 mRNA in various breast cancer cell lines (T47D, MDA-MB-231, MCF-7, MDA-MB-435 and HCC38) were detected by real-time PCR. Plasmids pEGFP and pEGFP-Mfn-2 were transfected into human breast cancer T47D cells by LipofectamineTM 2000 in vitro. The expression levels of Mfn-2 mRNA and protein in T47D cells were detected by real-time PCR and Western blotting. MTT assay was used to detect the proliferation of T47D cells. The cell apoptotic rate and mitochondrial membrane potential of T47D cells were measured by flow cytometry. Results: Compared with that in normal breast cells, Mfn-2 mRNA was highly expressed in breast cancer HCC38 cell line, and lowly expressed in the other cell lines, such as T47D etc. After pEGFP-Mfn-2 transfection for 48 h, the expression levels of Mfn-2 mRNA and rotein were significantly up-regulated in T47D cells. Compared with the pEGFP transfection group, the pEGFP-Mfn-2 transfection group showed a significant decrease in surivival rate of T47D cells under the treatment of parthenolide (50 mmol/L) (\[47.93±2.21\]% vs \[56.93±2.05\]%, P<0.05). Flow cytometry results showed that the apoptotic rate of T47D cells under the treatment of 0.05 mol/L parthenolide was significantly increased in pEGFP-Mfn-2 transfection group compared with that in pEGFP ransfection group (\[71.2±2.1\] % vs \[38.8±2.6\] %, P<005). However, the mitochondrial membrane potential was significanly decreased in the pEGFP-Mfn-2 transfection group (\[1.6±0.1\] % vs \[5.0±0.5\] %, P<0.05). Conclusion: pEGFP-Mfn-2 transfection can enhance the sensitivity of T47D cells to parthenolide.
Objective:To investigate the effect of mitofusin-2 ( Mfn-2 ) gene expression on sensitivity of human breast cancer T47D cells to parthenolide. Methods: The expressions of Mfn-2 mRNA in various breast cancer cell lines (T47D, MDA-MB-231, MCF-7, MDA-MB-435 and HCC38) were detected by real-time PCR. Plasmids pEGFP and pEGFP-Mfn-2 were transfected into human breast cancer T47D cells by LipofectamineTM 2000 in vitro. The expression levels of Mfn-2 mRNA and protein in T47D cells were detected by real-time PCR and Western blotting. MTT assay was used to detect the proliferation of T47D cells. The cell apoptotic rate and mitochondrial membrane potential of T47D cells were measured by flow cytometry. Results: Compared with that in normal breast cells, Mfn-2 mRNA was highly expressed in breast cancer HCC38 cell line, and lowly expressed in the other cell lines, such as T47D etc. After pEGFP-Mfn-2 transfection for 48 h, the expression levels of Mfn-2 mRNA and rotein were significantly up-regulated in T47D cells. Compared with the pEGFP transfection group, the pEGFP-Mfn-2 transfection group showed a significant decrease in surivival rate of T47D cells under the treatment of parthenolide (50 mmol/L) (\[47.93±2.21\]% vs \[56.93±2.05\]%, P<0.05). Flow cytometry results showed that the apoptotic rate of T47D cells under the treatment of 0.05 mol/L parthenolide was significantly increased in pEGFP-Mfn-2 transfection group compared with that in pEGFP ransfection group (\[71.2±2.1\] % vs \[38.8±2.6\] %, P<005). However, the mitochondrial membrane potential was significanly decreased in the pEGFP-Mfn-2 transfection group (\[1.6±0.1\] % vs \[5.0±0.5\] %, P<0.05). Conclusion: pEGFP-Mfn-2 transfection can enhance the sensitivity of T47D cells to parthenolide.
2013, 20(1):43-47.
DOI: 10.3872/j.issn.1007-385X.2013.1.007
Abstract:
Objective:To investigate the inhibitory effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) combined with paclitaxel treatment on human glioma U87 cells and the possible mechanism. Methods: MTT assay was used to detect the proliferation inhibitory rates of U87 cells in the paclitaxel group, TRAIL group, and TRAIL/paclitaxel combination group, and flow cytometry was used to detect the effects of different treatments on apoptosis of U87 cells. The expression levels of TRAIL death receptor (DR) 4, DR5, caspase-8 and caspase-3 in U87 cells after different treatments were measured by Western blotting. Results: MTT results showed the TRAIL or paclitaxel used alone demonstrated a favorable inhibitory effect on proliferation of U87 cells in a concentration-dependent manner. Combined application of TRAIL (500 ng/ml) and paclitaxel (0.5 μmol/L) showed a synergistic inhibitory effect on the proliferation of U87 cells with the coefficient of drug interaction (CDI) being 0.59. The proliferation inhibitory rate of U87 cells in the combination group was significantly higher than that in the TRAIL or paclitaxel used alone groups (\[70.24±368\] % vs \[2701±2.36\] %, \[21.31±4.85\] %, P<0.01). The apoptotic rate of U87 cells in the TRAIL/paclitaxel combination group was significantly higher than that in the control group, TRAIL group, or paclitaxel group (\[67.67±2.46\] % vs \[1.80±1.13\] %, \[22.13±2.18\] %, \[35.90±2.53\]%, P<0.01). The up-regulation expressions DR4, caspase-8 and caspase-3 in U87 cells was more obvious in TRAIL/paclitaxel combination treatment group than that in the TRAIL or paclitaxel groups (P<0.05). However, no obvious change in DR5 xpression was observed (P>0.05). Conclusion: TRAIL combined with paclitaxel treatment can up-regulate DR4, caspase-8 and caspase-3 expressions, thereby inhibiting the proliferation and inducing the apoptosis of U87 cells.
Objective:To investigate the inhibitory effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) combined with paclitaxel treatment on human glioma U87 cells and the possible mechanism. Methods: MTT assay was used to detect the proliferation inhibitory rates of U87 cells in the paclitaxel group, TRAIL group, and TRAIL/paclitaxel combination group, and flow cytometry was used to detect the effects of different treatments on apoptosis of U87 cells. The expression levels of TRAIL death receptor (DR) 4, DR5, caspase-8 and caspase-3 in U87 cells after different treatments were measured by Western blotting. Results: MTT results showed the TRAIL or paclitaxel used alone demonstrated a favorable inhibitory effect on proliferation of U87 cells in a concentration-dependent manner. Combined application of TRAIL (500 ng/ml) and paclitaxel (0.5 μmol/L) showed a synergistic inhibitory effect on the proliferation of U87 cells with the coefficient of drug interaction (CDI) being 0.59. The proliferation inhibitory rate of U87 cells in the combination group was significantly higher than that in the TRAIL or paclitaxel used alone groups (\[70.24±368\] % vs \[2701±2.36\] %, \[21.31±4.85\] %, P<0.01). The apoptotic rate of U87 cells in the TRAIL/paclitaxel combination group was significantly higher than that in the control group, TRAIL group, or paclitaxel group (\[67.67±2.46\] % vs \[1.80±1.13\] %, \[22.13±2.18\] %, \[35.90±2.53\]%, P<0.01). The up-regulation expressions DR4, caspase-8 and caspase-3 in U87 cells was more obvious in TRAIL/paclitaxel combination treatment group than that in the TRAIL or paclitaxel groups (P<0.05). However, no obvious change in DR5 xpression was observed (P>0.05). Conclusion: TRAIL combined with paclitaxel treatment can up-regulate DR4, caspase-8 and caspase-3 expressions, thereby inhibiting the proliferation and inducing the apoptosis of U87 cells.
2013, 20(1):48-51.
DOI: 10.3872/j.issn.1007-385X.2013.1.008
Abstract:
Objective:To explore the effects of microRNA-100（miR-100）on expression of polo-like kinase 1 (Plk1) and the apoptosis of human hepatic carcinoma HepG2 cells. Methods: HepG2 cells were transfected with miR-100 mimics by oligofectamine. RT-PCR and immunofluorescence were used to analyze Plk1 mRNA and protein expressions in HepG2 cells, respectively. Moreover, the apoptosis of HepG2 cells was detected by AnnexinⅤ-FITC kit. Results: HepG2 cells were successfully transfected by miR-100 mimics and the transfection efficiency was (88.75±2.22) %. 48 h after transfection, the expression of Plk1 mRNA decreased significantly in the miR-100 mimics transfection group compared with the negative control, blank control, and liposome groups (\[0.71±0.01\] vs \[0.95±0.01\], \[0.92±0.02\], \[0.93±002\]，P<0.01). 72 h after transfection, Plk1 protein expression was almost undetectable in HepG2 cells transfected with miR-100 mimics. Meanwhile, the cell apoptosis rate in the miR-100 mimics group was significantly increased in comparison with those in the negative control, blank control, and liposome groups (\[26.95±6.72\]% vs \[15.03±512\]%, \[6.88±3.71\]%, \[9.00±3.37\]%, P<0.05). Conclusion: miR-100 can inhibit the expression of Plk1 gene, therefore promoting the apoptosis of hepatic carcinoma HepG2 cells.
Objective:To explore the effects of microRNA-100（miR-100）on expression of polo-like kinase 1 (Plk1) and the apoptosis of human hepatic carcinoma HepG2 cells. Methods: HepG2 cells were transfected with miR-100 mimics by oligofectamine. RT-PCR and immunofluorescence were used to analyze Plk1 mRNA and protein expressions in HepG2 cells, respectively. Moreover, the apoptosis of HepG2 cells was detected by AnnexinⅤ-FITC kit. Results: HepG2 cells were successfully transfected by miR-100 mimics and the transfection efficiency was (88.75±2.22) %. 48 h after transfection, the expression of Plk1 mRNA decreased significantly in the miR-100 mimics transfection group compared with the negative control, blank control, and liposome groups (\[0.71±0.01\] vs \[0.95±0.01\], \[0.92±0.02\], \[0.93±002\]，P<0.01). 72 h after transfection, Plk1 protein expression was almost undetectable in HepG2 cells transfected with miR-100 mimics. Meanwhile, the cell apoptosis rate in the miR-100 mimics group was significantly increased in comparison with those in the negative control, blank control, and liposome groups (\[26.95±6.72\]% vs \[15.03±512\]%, \[6.88±3.71\]%, \[9.00±3.37\]%, P<0.05). Conclusion: miR-100 can inhibit the expression of Plk1 gene, therefore promoting the apoptosis of hepatic carcinoma HepG2 cells.
9 Silencing MDR1 gene expression reverses resistance of drug-resistant leukemia HT9 cells to allicin
2013, 20(1):52-57.
DOI: 10.3872/j.issn.1007-385X.2013.1.009
Abstract:
Objective:To investigate the effects of short hairpin RNA (shRNA) expression plasmid on silencing of MDR1 gene in drug resistant H79 cells and thus reverse the drug resistance of human promyelocytic leukemia HT9 cells to allicin. Methods: shRNA fragment targeting MDR1 gene was designed and constructed to obtain pSilencer3.1-shMDR1 expression plasmid, which was then stably transfected into HT9 cells. The expression of MDR1 mRNA in HT9 cells was assayed by real-time PCR. The P-gp protein (encoded by the MDR1 gene) expression was assayed by Western blotting. The cell mortality rate of HT9 cells was determined by MTT method. After treated with allicin, the apoptosis of HT9 cells was observed by gel electrophorosis, cell ultrastructure changes were observed under a transmission electron microscope, and the cell cycle was detected by flow cytometry. Results: pSilencer3.1-shMDR1 expression plasmid targeting MDR1 was constructed successfully and then stably transfected into HT9 cells to form the HT9-shMDR1 cell line. The MDR1 mRNA (\[0.027±0.002\] vs \[0.110±0.005\]，P<0.01) and P-gp protein expressions (\[0.856±0.014\] vs \[1.454±0027\], P<005) were significantly decreased in HT9-shMDR1 cells. The IC50 of allicin to HT9-shMDR1 cells significantly decreased compared with that in untransfected HT9 cells (\[26.66±0.59\] vs \[52.75±0.64\] μg/ml，P<0.01). The relative drug resistance reversal rate of HT9-shMDR1 to allicin was (49.45±1.86)%. Compared with untransfected HT9 cells, DNA ladder was more obvious in HT9-shMDR1 cells treated with allicin, and the typical apoptotic body was found by electron microscopy. Allicin treatment did not affect the cell cycle distribution of untransfected HT9 cells and control plasmid transfected HT9 cells (HT9-neo cells). However, the ratios of allicin-treated HT9-shMDR1 cells were significantly decreased in the S phase (\[3140±2.13\]% vs \[53.80±1.87\]%, P<0.01\] and were significantly increased in the G2/M phase (\[35.62±206\]% vs \[9.37±2.09\]%, P<0.01\]. Conclusion: pSilencer3.1-MDR1 expression plasmid targeting MDR1 gene can inhibit the expression of MDR1 gene, therefore reversing drug resistance of HT9 cells to allicin.
Objective:To investigate the effects of short hairpin RNA (shRNA) expression plasmid on silencing of MDR1 gene in drug resistant H79 cells and thus reverse the drug resistance of human promyelocytic leukemia HT9 cells to allicin. Methods: shRNA fragment targeting MDR1 gene was designed and constructed to obtain pSilencer3.1-shMDR1 expression plasmid, which was then stably transfected into HT9 cells. The expression of MDR1 mRNA in HT9 cells was assayed by real-time PCR. The P-gp protein (encoded by the MDR1 gene) expression was assayed by Western blotting. The cell mortality rate of HT9 cells was determined by MTT method. After treated with allicin, the apoptosis of HT9 cells was observed by gel electrophorosis, cell ultrastructure changes were observed under a transmission electron microscope, and the cell cycle was detected by flow cytometry. Results: pSilencer3.1-shMDR1 expression plasmid targeting MDR1 was constructed successfully and then stably transfected into HT9 cells to form the HT9-shMDR1 cell line. The MDR1 mRNA (\[0.027±0.002\] vs \[0.110±0.005\]，P<0.01) and P-gp protein expressions (\[0.856±0.014\] vs \[1.454±0027\], P<005) were significantly decreased in HT9-shMDR1 cells. The IC50 of allicin to HT9-shMDR1 cells significantly decreased compared with that in untransfected HT9 cells (\[26.66±0.59\] vs \[52.75±0.64\] μg/ml，P<0.01). The relative drug resistance reversal rate of HT9-shMDR1 to allicin was (49.45±1.86)%. Compared with untransfected HT9 cells, DNA ladder was more obvious in HT9-shMDR1 cells treated with allicin, and the typical apoptotic body was found by electron microscopy. Allicin treatment did not affect the cell cycle distribution of untransfected HT9 cells and control plasmid transfected HT9 cells (HT9-neo cells). However, the ratios of allicin-treated HT9-shMDR1 cells were significantly decreased in the S phase (\[3140±2.13\]% vs \[53.80±1.87\]%, P<0.01\] and were significantly increased in the G2/M phase (\[35.62±206\]% vs \[9.37±2.09\]%, P<0.01\]. Conclusion: pSilencer3.1-MDR1 expression plasmid targeting MDR1 gene can inhibit the expression of MDR1 gene, therefore reversing drug resistance of HT9 cells to allicin.
2013, 20(1):63-69.
DOI: 10.3872/j.issn.1007-385X.2013.1.011
Abstract:
Objective: To evaluate the effect of rapamycin (RAPA) and cisplatin (DDP) used alone or in combination on the growth of human cervical carcinoma HeLa cell subcutaneous xenografts in nude mice and the possible mechanisms. Methods: Nude mice were subcutaneously inoculated with HeLa cells to establish a subcutaneous transplantation tumor model of cervical cancer. Mice were randomly divided into a control group, RAPA group, DDP group, and RAPA+DDP group. The tumor weight and volume were observed during therapeutic process. The mRNA and protein expressions of hypoxia-inducible factor-1alpha ( HIF-1α ) and vascular endothelial growth factor (VEGF) were detected by RT-PCR, immunohistochemistry and Western blotting. Results: Compared with the RAPA group, the tumor weight (\[0.42±0.04\] vs \[0.53±0.03\]g, P<0.05) and the tumor volume (\[568.70±36.12\] vs \[797.81±111.98\] mm3, P<0.01) were significantly reduced in the RAPA+DDP group. Moreover, in comparison with the DDP group, the tumor weight (\[0.42±0.04\] vs \[0.52±0.04\]g, P<0.05) and the tumor volume (\[568.70±36.12\] vs \[766.16±132.27\]mm3, P<0.01) were significantly reduced in the RAPA+DDP group. RT-PCR and Western blotting results showed that the expression of HIF-1α mRNA in transplanted tumors was down-regulated in the RAPA+DDP group compared with the RAPA and DDP groups (\[31.215±0.706\] vs \[50.58±1.25\], \[48.63±1.56\]，P<0.05), and the expression of HIF-1α protein was also down-regulated (\[38.07±0.09\] vs \[55.69±3.60\], \[59.50±1.54\], P<0.05). Moreover, the expression of VEGF mRNA in transplanted tumors was significantly decreased in the RAPA+DDP group compared with the RAPA and the DDP groups (\[46.64±0.60\] vs \[62.20±0.62\], \[61.64±1.21\], P<0.05), and decreased VEGF protein expression can be shown in the RAPA+DDP group (\[119.28±2.69\] vs \[150.31±4.77\], \[153.84±3.39\], P<0.05). Immunohistochemistry showed that the AOD value of HIF-1α in transplanted tumors was decreased in the RAPA+DDP group compared with the RAPA and the DDP groups (\[0.37±0.03\] vs \[0.57±0.06\], \[0.55±006\]，P<0.05), and decreased AOD value of VEGF can also be shown in the RAPA+DDP group (\[0.48±0.03\] vs \[0.62±0.04\], \[0.61±0.07\], P<0.05). Conclusion: RAPA combined with DDP shows an inhibitory effect on growth of human cervical carcinoma HeLa cell subcutaneous xenografts. The possible underlying mechanism is related to the down-regulation of HIF-1α and VEGF expressions.
Objective: To evaluate the effect of rapamycin (RAPA) and cisplatin (DDP) used alone or in combination on the growth of human cervical carcinoma HeLa cell subcutaneous xenografts in nude mice and the possible mechanisms. Methods: Nude mice were subcutaneously inoculated with HeLa cells to establish a subcutaneous transplantation tumor model of cervical cancer. Mice were randomly divided into a control group, RAPA group, DDP group, and RAPA+DDP group. The tumor weight and volume were observed during therapeutic process. The mRNA and protein expressions of hypoxia-inducible factor-1alpha ( HIF-1α ) and vascular endothelial growth factor (VEGF) were detected by RT-PCR, immunohistochemistry and Western blotting. Results: Compared with the RAPA group, the tumor weight (\[0.42±0.04\] vs \[0.53±0.03\]g, P<0.05) and the tumor volume (\[568.70±36.12\] vs \[797.81±111.98\] mm3, P<0.01) were significantly reduced in the RAPA+DDP group. Moreover, in comparison with the DDP group, the tumor weight (\[0.42±0.04\] vs \[0.52±0.04\]g, P<0.05) and the tumor volume (\[568.70±36.12\] vs \[766.16±132.27\]mm3, P<0.01) were significantly reduced in the RAPA+DDP group. RT-PCR and Western blotting results showed that the expression of HIF-1α mRNA in transplanted tumors was down-regulated in the RAPA+DDP group compared with the RAPA and DDP groups (\[31.215±0.706\] vs \[50.58±1.25\], \[48.63±1.56\]，P<0.05), and the expression of HIF-1α protein was also down-regulated (\[38.07±0.09\] vs \[55.69±3.60\], \[59.50±1.54\], P<0.05). Moreover, the expression of VEGF mRNA in transplanted tumors was significantly decreased in the RAPA+DDP group compared with the RAPA and the DDP groups (\[46.64±0.60\] vs \[62.20±0.62\], \[61.64±1.21\], P<0.05), and decreased VEGF protein expression can be shown in the RAPA+DDP group (\[119.28±2.69\] vs \[150.31±4.77\], \[153.84±3.39\], P<0.05). Immunohistochemistry showed that the AOD value of HIF-1α in transplanted tumors was decreased in the RAPA+DDP group compared with the RAPA and the DDP groups (\[0.37±0.03\] vs \[0.57±0.06\], \[0.55±006\]，P<0.05), and decreased AOD value of VEGF can also be shown in the RAPA+DDP group (\[0.48±0.03\] vs \[0.62±0.04\], \[0.61±0.07\], P<0.05). Conclusion: RAPA combined with DDP shows an inhibitory effect on growth of human cervical carcinoma HeLa cell subcutaneous xenografts. The possible underlying mechanism is related to the down-regulation of HIF-1α and VEGF expressions.
2013, 20(1):70-74.
DOI: 10.3872/j.issn.1007-385X.2013.1.012
Abstract:
Objective:To investigate the inhibitory effect of recombinant adenovirus (Ad-PTEN-GFP) containing phosphatase and tensin homologue deleted on chromosome 10 (PTEN) gene on the growth of human glioma U251 cell xenografts in nude mice. Methods: Human glioma U251 cells were injected under the skin of the dorsum to establish a nude mouse glioma model. The tumor-bearing nude mice were randomly divided into three groups: Ad-PTEN-GFP group，Ad-GFP group (empty vector group) and PBS group (blank group).The growth of tumor xenografts in nude mice of the three groups was observed. The tumor volumes were measured, the tumor growth curves were drawn and the survival time of the nude mice bearing tumors was observed. The cell apoptosis of tumor cells was detected by TUNEL assay, and the expressions of PTEN and P65 proteins in tumor tissues were detected by immunohistochemical assay. Results: Compared with the Ad-GFP group, the growth of glioma cell xenografts in the Ad-PTEN-GFP group was inhibited with tumor volume inhibition rate increasing significantly (\[82.5±12.7\]% vs \[72±1.3\]%, P<0.05), and the survival time of tumor-bearing nude mice was prolonged (\[103±10\] vs \[58±8\] d, P<0.01\]. TUNEL assay results showed that the apoptotic rate of glioma cells was significantly increased (\[46.4±8.3\]% vs \[4.6±1.0\]%, P<0.01\]. Immunohistochemical assay results showed that the positive expression rate of PTEN protein was increased in the Ad-PTEN-GFP group compared with the Ad-GFP group (83.3% vs 0, P<0.01), and the positive expression rate of P65 protein was decreased (16.7% vs 66.7%, P<0.01). Conclusion: The growth of human glioma U251 cell xenografts in nude mice is suppressed after Ad-PTEN-GFP infection, which may be related with the down-regulation of P65 protein expression.
Objective:To investigate the inhibitory effect of recombinant adenovirus (Ad-PTEN-GFP) containing phosphatase and tensin homologue deleted on chromosome 10 (PTEN) gene on the growth of human glioma U251 cell xenografts in nude mice. Methods: Human glioma U251 cells were injected under the skin of the dorsum to establish a nude mouse glioma model. The tumor-bearing nude mice were randomly divided into three groups: Ad-PTEN-GFP group，Ad-GFP group (empty vector group) and PBS group (blank group).The growth of tumor xenografts in nude mice of the three groups was observed. The tumor volumes were measured, the tumor growth curves were drawn and the survival time of the nude mice bearing tumors was observed. The cell apoptosis of tumor cells was detected by TUNEL assay, and the expressions of PTEN and P65 proteins in tumor tissues were detected by immunohistochemical assay. Results: Compared with the Ad-GFP group, the growth of glioma cell xenografts in the Ad-PTEN-GFP group was inhibited with tumor volume inhibition rate increasing significantly (\[82.5±12.7\]% vs \[72±1.3\]%, P<0.05), and the survival time of tumor-bearing nude mice was prolonged (\[103±10\] vs \[58±8\] d, P<0.01\]. TUNEL assay results showed that the apoptotic rate of glioma cells was significantly increased (\[46.4±8.3\]% vs \[4.6±1.0\]%, P<0.01\]. Immunohistochemical assay results showed that the positive expression rate of PTEN protein was increased in the Ad-PTEN-GFP group compared with the Ad-GFP group (83.3% vs 0, P<0.01), and the positive expression rate of P65 protein was decreased (16.7% vs 66.7%, P<0.01). Conclusion: The growth of human glioma U251 cell xenografts in nude mice is suppressed after Ad-PTEN-GFP infection, which may be related with the down-regulation of P65 protein expression.
2013, 20(1):75-81.
DOI: 10.3872/j.issn.1007-385X.2013.1.013
Abstract:
Objective:To explore the effect of miRNA eukaryotic expression vector targeting glucose regulated protein 78 ( GRP78 ) gene on proliferation of human esophageal carcinoma EC109 cells. Methods: Four pairs of specific miRNA interference sequences targeting GRP78 gene and one pair of negative control sequence were designed and synthesized. The recombined plasmids of miRNA targeting GRP78 , pcDNATM6.2-miR78-1, pcDNATM6.2-miR78-2, pcDNATM6.2-miR78-3 and pcDNATM6.2-miR78-4, were constructed using pcDNATM6.2-GW/EmGFP-miR eukaryotic expression vector, and were transfected into HEK293 cells by lipofectamine. After the treatment with blasticidin for two weeks, the stably transfected HEK293 cells were obtained. The transfection efficiency was observed by fluorescence microscopy. The best interference plasmid was then selected and transfected into EC109 cells. The expression of GRP78 mRNA and the proliferation of EC109 cells were detected by RT-PCR and CCK-8 assay, respectively. Results: Sequencing results indicated that four recombined plasmids of miRNA targeting GRP78 were successfully constructed. After transfection and screening, the expression of GFP was observed in all transfected HEK293 cells. The expression of GRP78 mRNA in HEK293 cells transfected with all the four interference plasmids was decreased (P<0.05), comparing with the untransfected or negative control group (pcDNATM6.2-Ctrl), in which the highest interference efficiency was observed in pcDNATM6.2-miR78-1 group. After pcDNATM6.2-miR78-1 transfection, the expression of GRP78 mRNA in EC109 cells was significantly decreased compared with the untransfected group and the pcDNATM6.2-Ctrl group (\[0.38±0.02\] vs \[1.03±004\], \[1.00±0.03\], P<0.05), respectively. CCK8 assay showed that EC109 cell proliferation was significantly suppressed after pcDNATM62-miR78-1 transfection in 12, 24, 48, 72 h (\[0.028±0.001\] vs \[0.086±0.010\], \[0.035±0.003\] vs \[0.155±0011\], \[0.112±0.009\] vs \[0.389±0.008\], \[0.169±0.013\] vs \[0.433±0.009\]; P<005). Conclusion: Four pairs of miRNA expression vectors targeting GRP78 gene are successfully constructed, in which pcDNATM62-miR78-1 shows the best gene silencing efficiency and efficiently inhibits the proliferation of EC109 cells.
Objective:To explore the effect of miRNA eukaryotic expression vector targeting glucose regulated protein 78 ( GRP78 ) gene on proliferation of human esophageal carcinoma EC109 cells. Methods: Four pairs of specific miRNA interference sequences targeting GRP78 gene and one pair of negative control sequence were designed and synthesized. The recombined plasmids of miRNA targeting GRP78 , pcDNATM6.2-miR78-1, pcDNATM6.2-miR78-2, pcDNATM6.2-miR78-3 and pcDNATM6.2-miR78-4, were constructed using pcDNATM6.2-GW/EmGFP-miR eukaryotic expression vector, and were transfected into HEK293 cells by lipofectamine. After the treatment with blasticidin for two weeks, the stably transfected HEK293 cells were obtained. The transfection efficiency was observed by fluorescence microscopy. The best interference plasmid was then selected and transfected into EC109 cells. The expression of GRP78 mRNA and the proliferation of EC109 cells were detected by RT-PCR and CCK-8 assay, respectively. Results: Sequencing results indicated that four recombined plasmids of miRNA targeting GRP78 were successfully constructed. After transfection and screening, the expression of GFP was observed in all transfected HEK293 cells. The expression of GRP78 mRNA in HEK293 cells transfected with all the four interference plasmids was decreased (P<0.05), comparing with the untransfected or negative control group (pcDNATM6.2-Ctrl), in which the highest interference efficiency was observed in pcDNATM6.2-miR78-1 group. After pcDNATM6.2-miR78-1 transfection, the expression of GRP78 mRNA in EC109 cells was significantly decreased compared with the untransfected group and the pcDNATM6.2-Ctrl group (\[0.38±0.02\] vs \[1.03±004\], \[1.00±0.03\], P<0.05), respectively. CCK8 assay showed that EC109 cell proliferation was significantly suppressed after pcDNATM62-miR78-1 transfection in 12, 24, 48, 72 h (\[0.028±0.001\] vs \[0.086±0.010\], \[0.035±0.003\] vs \[0.155±0011\], \[0.112±0.009\] vs \[0.389±0.008\], \[0.169±0.013\] vs \[0.433±0.009\]; P<005). Conclusion: Four pairs of miRNA expression vectors targeting GRP78 gene are successfully constructed, in which pcDNATM62-miR78-1 shows the best gene silencing efficiency and efficiently inhibits the proliferation of EC109 cells.
2013, 20(1):82-86.
DOI: 10.3872/j.issn.1007-385X.2013.1.014
Abstract:
Objective:To investigate the cytotoxicity of γδT cells combined with galectin-1 (Gal-1) monoclonal antibody against human cervical carcinoma cells. Methods: γδT cells were expanded in vitro using the solid-phase antibody coated method from tumor infiltrating lymphocyte (TILs) of the human cervical cancer specimens. The purity of γδT cells was measured by flow cytometry. The expressions of Gal-1 in cervical carcinoma SiHa and HeLa cells and in cell supernatants were detected by Western blotting and ELISA, respectively. The cytotoxicity of γδT cells combined with Gal-1 mAb against the human cervical cancer SiHa and HeLa cells was measured by lactate dehydrogenase (LDH) release assay. Tumor-bearing mouse model was established by subcutaneous injection of SiHa cells. Tumor-bearing mice were randomly divided into a control group (SiHa group), an isotype control group (SiHa+mouse IgG1 group), a γδT cell group (SiHa+γδT group), a Gal-1 mAb group (SiHa+γδT+Gal-1 mAb group) and a γδT cell + Gal-1 mAb group (γδT cell+Gal-1 mAb group). The tumor growth was observed in different groups. Results: The percentage of TCRγδ positive T cells expanded using the solid-phase antibody coated method in vitro was (91.2±1.2) %. Gal-1 was over-expressed in SiHa, HeLa cells and cell supernatants. Compared with the control group, the Gal-1 mAb group showed an increasing cytotoxicity efficiency on SiHa cells (\[68.1±3.0\] % vs \[48.7±3.8\]%, P<005) and HeLa cells (\[79.4±5.6\]% vs \[48.3±6.5\]%, P<0.05). The volume of implanted tumors in the γδT +Gal-1 mAb group was significantly smaller than that in the Gal-1 mAb group (\[31.3±9.1\] vs \[199.6±41.2\] mm3, P<0.01) and the γδT cell group (\[31.3±9.1\] vs \[85.6±45.1\] mm3, P<0.05) 30 days after tumor implantation in nude mice. Conclusion: Gal-1 mAb antibody can boost up the cytotoxicity of γδT cells against the human cervical carcinoma cells.
Objective:To investigate the cytotoxicity of γδT cells combined with galectin-1 (Gal-1) monoclonal antibody against human cervical carcinoma cells. Methods: γδT cells were expanded in vitro using the solid-phase antibody coated method from tumor infiltrating lymphocyte (TILs) of the human cervical cancer specimens. The purity of γδT cells was measured by flow cytometry. The expressions of Gal-1 in cervical carcinoma SiHa and HeLa cells and in cell supernatants were detected by Western blotting and ELISA, respectively. The cytotoxicity of γδT cells combined with Gal-1 mAb against the human cervical cancer SiHa and HeLa cells was measured by lactate dehydrogenase (LDH) release assay. Tumor-bearing mouse model was established by subcutaneous injection of SiHa cells. Tumor-bearing mice were randomly divided into a control group (SiHa group), an isotype control group (SiHa+mouse IgG1 group), a γδT cell group (SiHa+γδT group), a Gal-1 mAb group (SiHa+γδT+Gal-1 mAb group) and a γδT cell + Gal-1 mAb group (γδT cell+Gal-1 mAb group). The tumor growth was observed in different groups. Results: The percentage of TCRγδ positive T cells expanded using the solid-phase antibody coated method in vitro was (91.2±1.2) %. Gal-1 was over-expressed in SiHa, HeLa cells and cell supernatants. Compared with the control group, the Gal-1 mAb group showed an increasing cytotoxicity efficiency on SiHa cells (\[68.1±3.0\] % vs \[48.7±3.8\]%, P<005) and HeLa cells (\[79.4±5.6\]% vs \[48.3±6.5\]%, P<0.05). The volume of implanted tumors in the γδT +Gal-1 mAb group was significantly smaller than that in the Gal-1 mAb group (\[31.3±9.1\] vs \[199.6±41.2\] mm3, P<0.01) and the γδT cell group (\[31.3±9.1\] vs \[85.6±45.1\] mm3, P<0.05) 30 days after tumor implantation in nude mice. Conclusion: Gal-1 mAb antibody can boost up the cytotoxicity of γδT cells against the human cervical carcinoma cells.
2013, 20(1):87-92.
DOI: 10.3872/j.issn.1007-385X.2013.1.015
Abstract:
Objective:To prepare IL-2-loaded magnetic nanoparticle (IL-2-Fe3O4-PLGA) with Fe3O4 magnetic nano-particle (Fe3O4-MNP) and poly lactide-co-glycolide (PLGA), and to investigate its targeted accumulation function in tumor immunotherapy. Methods: The double emulsion method was employed for preparation of IL-2-Fe3O4-PLGA, its size and morphology were observed with a laser particle size analyzer and under a scanning electron microscope, respectively. In addition, the drug encapsulation efficiency and releasing characteristics of IL-2-Fe3O4-PLGA in vitro were measured with ELISA. The biological activity of the IL-2 released from the IL-2-Fe3O4-PLGA was evaluated by MTT assay. Sarcoma 180 cell-transplanted tumor mouse model was established to study the effect of IL-2-Fe3O4-PLGA in combination with external magnetic field on the growth of Sarcoma 180 tumors. The tumor, liver and kidney tissues were examined by Prussian blue staining to determine the accumulation and distribution of the IL-2-Fe3O4-PLGA in the tissue sections. Results: IL-2-Fe3O4-PLGA was constructed successfully, and was spherical with a mean diameter of (697±0.51) nm as well as a drug encapsulation efficiency of (83.76±1.24) %. In the drug release test in vitro, the mass concentration of released IL-2 was 100 ng/ml during the burst release phase and rose to 180 ng/ml at the end of 15 d. Moreover, the released IL-2 remained 85%-55% of its original activity during the releasing period. A strong IL-2-Fe3O4-PLGA positive reaction was observed in IL-2-Fe3O4-PLGA-treated mice combined with an external magnetic field, but only a weak reaction in mice injected with IL-2-Fe3O4-PLGA alone. Furthermore, in both groups, Prussian blue-stained liver showed occasionally light IL-2-Fe3O4-PLGA positive staining, while IL-2-Fe3O4-PLGA positive reaction was rarely detected in the kidneys. Conclusion: IL-2 loaded magnetic nanoparticle IL-2-Fe3O4-PLGA has controlled IL-2-release function and targeting accumulation capability in tumor tissues when combined with an external magnetic field.
Objective:To prepare IL-2-loaded magnetic nanoparticle (IL-2-Fe3O4-PLGA) with Fe3O4 magnetic nano-particle (Fe3O4-MNP) and poly lactide-co-glycolide (PLGA), and to investigate its targeted accumulation function in tumor immunotherapy. Methods: The double emulsion method was employed for preparation of IL-2-Fe3O4-PLGA, its size and morphology were observed with a laser particle size analyzer and under a scanning electron microscope, respectively. In addition, the drug encapsulation efficiency and releasing characteristics of IL-2-Fe3O4-PLGA in vitro were measured with ELISA. The biological activity of the IL-2 released from the IL-2-Fe3O4-PLGA was evaluated by MTT assay. Sarcoma 180 cell-transplanted tumor mouse model was established to study the effect of IL-2-Fe3O4-PLGA in combination with external magnetic field on the growth of Sarcoma 180 tumors. The tumor, liver and kidney tissues were examined by Prussian blue staining to determine the accumulation and distribution of the IL-2-Fe3O4-PLGA in the tissue sections. Results: IL-2-Fe3O4-PLGA was constructed successfully, and was spherical with a mean diameter of (697±0.51) nm as well as a drug encapsulation efficiency of (83.76±1.24) %. In the drug release test in vitro, the mass concentration of released IL-2 was 100 ng/ml during the burst release phase and rose to 180 ng/ml at the end of 15 d. Moreover, the released IL-2 remained 85%-55% of its original activity during the releasing period. A strong IL-2-Fe3O4-PLGA positive reaction was observed in IL-2-Fe3O4-PLGA-treated mice combined with an external magnetic field, but only a weak reaction in mice injected with IL-2-Fe3O4-PLGA alone. Furthermore, in both groups, Prussian blue-stained liver showed occasionally light IL-2-Fe3O4-PLGA positive staining, while IL-2-Fe3O4-PLGA positive reaction was rarely detected in the kidneys. Conclusion: IL-2 loaded magnetic nanoparticle IL-2-Fe3O4-PLGA has controlled IL-2-release function and targeting accumulation capability in tumor tissues when combined with an external magnetic field.
15 IL-12 plays anti-tumor effect by inducing NK cell activation in hepatic carcinoma microenvironment
2013, 20(1):93-98.
DOI: 10.3872/j.issn.1007-385X.2013.1.016
Abstract:
Objective:To explore the enhanced anti-tumor effect of IL-12 through inducing NK cell activition in hepatic carcinoma microenviroment. Methods: The hepatic cacinoma HepG2 cells were subcutaneously injected into NOD/SCID mice, and human peripheral blood lymphocytes (PBL) were introperitoneally injected after tumor formation to establish HCC-huPBL tumor-bearing mouse model. The tumor-bearing mice were randomized into IL-12 group and PBS control group. Mice were intratumoral injected with IL-12, and the changes of tumor volume and body weight as well as general conditions of tumor-bearing mice were observed. ELISA assay was performed to examine the expression levels of IL-12 and INF-γ in the microenvironment of hepatic carcinoma tissues in tumor-bearing mice, and the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in peripheral blood of mice 30 days after IL-12 intratumoral injection. Immunohistochemistry assay was used to analyze the expressions of NK-activating receptors: NKG2D, NKp44, NKp30, NKp46, and inhibitory NK receptors: KIR2DL3/CD158b and NKG2A/CD159a in hepatic carcinoma microenvironment after IL-12 treatment. Results: On day 12, 18, 24 and 30, the tumor volumes were smaller in the IL-12 group than those in the PBS group (\[594.47±205.51\] vs \[832.10±187.49\] mm3, \[963.61±427.95\] vs \[1 35087±468.23\] mm3, \[1 285.02±368.56\] vs \[1 975.49±655.54\] mm3, \[1 903.64±471.34\] vs \[2 568.77±784.68\] mm3, P<0.05). The expression levels of IL-12 and IFN-γ in the IL-12 group were significantly higher than those in the PBS group (\[2.96±1.02\] vs \[1.35±0.75\] pg/ml, \[12.26±4.11\] vs \[7.81±3.46\] pg/ml, P<005). The serum ALT level significantly increased in the IL-12 group compared to the PBS group on day 7 (\[7385±10.71\] vs \[41.73±13.13\] U/L, P<0.05), and reached a peak at day 14. The expressions of NK-activating receptors NKG2D, NKp44 and NKp30 were statistically higher in the IL-12 group than those in the PBS group (P<005), the expression level of NKp46 showed no significant up-regulation, while the expression levels of NK inhibitory receptors CD158b and CD159a were decreased compared to the PBS group (P<0.05). Conclusion: IL-12 intratumoral injection can up-regulate the expressions of NK-activating receptors, IL-12 and IFN-γ, and down-regulate the NK inhibitory receptors in the hepatic carcinoma mouse model, therefore effectively inhibiting the tumor growth in mouse model.
Objective:To explore the enhanced anti-tumor effect of IL-12 through inducing NK cell activition in hepatic carcinoma microenviroment. Methods: The hepatic cacinoma HepG2 cells were subcutaneously injected into NOD/SCID mice, and human peripheral blood lymphocytes (PBL) were introperitoneally injected after tumor formation to establish HCC-huPBL tumor-bearing mouse model. The tumor-bearing mice were randomized into IL-12 group and PBS control group. Mice were intratumoral injected with IL-12, and the changes of tumor volume and body weight as well as general conditions of tumor-bearing mice were observed. ELISA assay was performed to examine the expression levels of IL-12 and INF-γ in the microenvironment of hepatic carcinoma tissues in tumor-bearing mice, and the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in peripheral blood of mice 30 days after IL-12 intratumoral injection. Immunohistochemistry assay was used to analyze the expressions of NK-activating receptors: NKG2D, NKp44, NKp30, NKp46, and inhibitory NK receptors: KIR2DL3/CD158b and NKG2A/CD159a in hepatic carcinoma microenvironment after IL-12 treatment. Results: On day 12, 18, 24 and 30, the tumor volumes were smaller in the IL-12 group than those in the PBS group (\[594.47±205.51\] vs \[832.10±187.49\] mm3, \[963.61±427.95\] vs \[1 35087±468.23\] mm3, \[1 285.02±368.56\] vs \[1 975.49±655.54\] mm3, \[1 903.64±471.34\] vs \[2 568.77±784.68\] mm3, P<0.05). The expression levels of IL-12 and IFN-γ in the IL-12 group were significantly higher than those in the PBS group (\[2.96±1.02\] vs \[1.35±0.75\] pg/ml, \[12.26±4.11\] vs \[7.81±3.46\] pg/ml, P<005). The serum ALT level significantly increased in the IL-12 group compared to the PBS group on day 7 (\[7385±10.71\] vs \[41.73±13.13\] U/L, P<0.05), and reached a peak at day 14. The expressions of NK-activating receptors NKG2D, NKp44 and NKp30 were statistically higher in the IL-12 group than those in the PBS group (P<005), the expression level of NKp46 showed no significant up-regulation, while the expression levels of NK inhibitory receptors CD158b and CD159a were decreased compared to the PBS group (P<0.05). Conclusion: IL-12 intratumoral injection can up-regulate the expressions of NK-activating receptors, IL-12 and IFN-γ, and down-regulate the NK inhibitory receptors in the hepatic carcinoma mouse model, therefore effectively inhibiting the tumor growth in mouse model.
2013, 20(1):99-104.
DOI: 10.3872/j.issn.1007-385X.2013.1.017
Abstract:
Objective:To study the expression and gene mutation of epidermal growth factor receptor (EGFR) in endometrial carcinoma tissues. Methods: One hundred and four paraffin-embedded endometrial tissues were obtained from Tumor Hospital of Jiangxi Province and the Second Affiliated Hospital of Nanchang University from January 2007 to April 2011, including 56 endometrial carcinoma tissues, 18 endometrial atypical hyperplasia tissues, and 30 normal endometrial tissues. Immunohistochemistry was used to detect the expression of EGFR protein in the above tissues. Exon 19 and exon 21 of EGFR gene in endometrial carcinoma or normal endometrial tissues were amplified by PCR assay, and the mutations were detected by sequencing. Results: The positive expression rate of EGFR in the endometrial carcinoma tissues was higher than that in the normal endometrial tissues (73.2% \[41/56\] vs 30.0% \[9/30\], P<0.01) and that in atypical hyperplasia tissues (73.2% \[41/56\] vs 44.4% \[8/18\], P<0.05). Further analysis indicated that, the positive expression rate of EGFR in the G3 endometrial carcinoma tissues was significantly higher than that in G1 (81.8% \[9/11\] vs 66.7% \[12/18\], P<0.01) and G2 tissues (81.8% \[9/11\] vs 74.1% \[20/27\], P<0.05), and the positive expression rate of EGFR in the >1/2 myometrial invasion group was higher than that in the ≤1/2 myometrial invasion group (86.8% \[33/38\] vs 44.4% \[8/18\], P<0.01). However, the expression of EGFR had no correlation with the FIGO stage and the lymph node metastasis of endometrial carcinoma. One endometrial carcinoma case showed the mutation of exon 19 (G2281A) in EGFR gene, whereas, no mutation was found in exon 21. Conclusion: The positive expression rate of EGFR in endometrial carcinoma is correlated with the histological grade and the infiltration depth of muscular layer, and some endometrial carcinoma tissues show the mutation of exon 19 (G2281A) in EGFR gene.
Objective:To study the expression and gene mutation of epidermal growth factor receptor (EGFR) in endometrial carcinoma tissues. Methods: One hundred and four paraffin-embedded endometrial tissues were obtained from Tumor Hospital of Jiangxi Province and the Second Affiliated Hospital of Nanchang University from January 2007 to April 2011, including 56 endometrial carcinoma tissues, 18 endometrial atypical hyperplasia tissues, and 30 normal endometrial tissues. Immunohistochemistry was used to detect the expression of EGFR protein in the above tissues. Exon 19 and exon 21 of EGFR gene in endometrial carcinoma or normal endometrial tissues were amplified by PCR assay, and the mutations were detected by sequencing. Results: The positive expression rate of EGFR in the endometrial carcinoma tissues was higher than that in the normal endometrial tissues (73.2% \[41/56\] vs 30.0% \[9/30\], P<0.01) and that in atypical hyperplasia tissues (73.2% \[41/56\] vs 44.4% \[8/18\], P<0.05). Further analysis indicated that, the positive expression rate of EGFR in the G3 endometrial carcinoma tissues was significantly higher than that in G1 (81.8% \[9/11\] vs 66.7% \[12/18\], P<0.01) and G2 tissues (81.8% \[9/11\] vs 74.1% \[20/27\], P<0.05), and the positive expression rate of EGFR in the >1/2 myometrial invasion group was higher than that in the ≤1/2 myometrial invasion group (86.8% \[33/38\] vs 44.4% \[8/18\], P<0.01). However, the expression of EGFR had no correlation with the FIGO stage and the lymph node metastasis of endometrial carcinoma. One endometrial carcinoma case showed the mutation of exon 19 (G2281A) in EGFR gene, whereas, no mutation was found in exon 21. Conclusion: The positive expression rate of EGFR in endometrial carcinoma is correlated with the histological grade and the infiltration depth of muscular layer, and some endometrial carcinoma tissues show the mutation of exon 19 (G2281A) in EGFR gene.
2013, 20(1):105-109.
DOI: 10.3872/j.issn.1007-385X.2013.1.018
Abstract:
EML4-ALK 为棘皮动物微管相关蛋白样4（echinoderm microtubule associated protein-like4， EML4 ）和间变性淋巴瘤激酶（anaplastic lymphoma kinase，ALK）的融合基因，自在非小细胞肺癌 (non-small cell lung cancer，NSCLC) 首次被发现以来受到越来越多的关注， EML4-ALK 最常见于从不/轻度吸烟的肺腺癌患者， EML4-ALK 阳性NSCLC代表了NSCLC患者一个独特的亚型。所有 EML4-ALK 变体（variant）均具有生物学功能，其表达产物为嵌合酪氨酸激酶，可持续性促进细胞增殖，导致肿瘤的发生和转移。以 EML4-ALK 为靶点的ALK抑制剂克里唑蒂尼（crizotinib）治疗该亚型晚期NSCLC效果较佳。未来的挑战是寻找最佳的EML4-ALK检测方法，能简单、快速、灵敏和准确地鉴定出EML4-ALK阳性晚期NSCLC患者，以促使克里唑蒂尼早日成为晚期NSCLC患者的一线标准治疗。本研究对 EML4-ALK 在NSCLC患者中的突变情况、EML4-ALK的检测方法及克里唑蒂尼在治疗NSCLC中的潜在价值与临床应用进展作一综述。
EML4-ALK 为棘皮动物微管相关蛋白样4（echinoderm microtubule associated protein-like4， EML4 ）和间变性淋巴瘤激酶（anaplastic lymphoma kinase，ALK）的融合基因，自在非小细胞肺癌 (non-small cell lung cancer，NSCLC) 首次被发现以来受到越来越多的关注， EML4-ALK 最常见于从不/轻度吸烟的肺腺癌患者， EML4-ALK 阳性NSCLC代表了NSCLC患者一个独特的亚型。所有 EML4-ALK 变体（variant）均具有生物学功能，其表达产物为嵌合酪氨酸激酶，可持续性促进细胞增殖，导致肿瘤的发生和转移。以 EML4-ALK 为靶点的ALK抑制剂克里唑蒂尼（crizotinib）治疗该亚型晚期NSCLC效果较佳。未来的挑战是寻找最佳的EML4-ALK检测方法，能简单、快速、灵敏和准确地鉴定出EML4-ALK阳性晚期NSCLC患者，以促使克里唑蒂尼早日成为晚期NSCLC患者的一线标准治疗。本研究对 EML4-ALK 在NSCLC患者中的突变情况、EML4-ALK的检测方法及克里唑蒂尼在治疗NSCLC中的潜在价值与临床应用进展作一综述。
2013, 20(1):110-114.
DOI: 10.3872/j.issn.1007-385X.2013.1.019
Abstract:
随着分子生物学技术的进步和在分子水平上对恶性肿瘤发病机制认识的不断加深，以细胞受体、表面抗原、关键基因和细胞内调控分子为靶点的分子靶向治疗已成为辅助传统治疗的合理选择。抗体导向治疗是一种新颖的分子靶向治疗手段。目前已研发多种抗体导向类抗癌药物，这类药物由特异性识别肿瘤细胞的靶向部分和杀伤肿瘤的效应部分组成，因而具有较强的靶向杀伤肿瘤细胞作用，同时可有效降低药物对局部正常组织和全身的系统性毒性作用。利用基因工程技术将人类血清可溶性肿瘤坏死因子相关凋亡诱导配体（serum-soluble TNF-related apoptosis-inducing ligand，sTRAIL）与抗肿瘤单链抗体（single chain antibody fragment, scFv）融合，构建scFv-sTRAIL融合蛋白，是一种理想的抗体导向治疗策略。通过scFv与肿瘤细胞所特有的细胞表面抗原的特异结合，加强sTRAIL在肿瘤病灶的富集，靶向诱导肿瘤细胞凋亡，从而获得增强抗体的疗效和更高的药物安全性。
随着分子生物学技术的进步和在分子水平上对恶性肿瘤发病机制认识的不断加深，以细胞受体、表面抗原、关键基因和细胞内调控分子为靶点的分子靶向治疗已成为辅助传统治疗的合理选择。抗体导向治疗是一种新颖的分子靶向治疗手段。目前已研发多种抗体导向类抗癌药物，这类药物由特异性识别肿瘤细胞的靶向部分和杀伤肿瘤的效应部分组成，因而具有较强的靶向杀伤肿瘤细胞作用，同时可有效降低药物对局部正常组织和全身的系统性毒性作用。利用基因工程技术将人类血清可溶性肿瘤坏死因子相关凋亡诱导配体（serum-soluble TNF-related apoptosis-inducing ligand，sTRAIL）与抗肿瘤单链抗体（single chain antibody fragment, scFv）融合，构建scFv-sTRAIL融合蛋白，是一种理想的抗体导向治疗策略。通过scFv与肿瘤细胞所特有的细胞表面抗原的特异结合，加强sTRAIL在肿瘤病灶的富集，靶向诱导肿瘤细胞凋亡，从而获得增强抗体的疗效和更高的药物安全性。
2013, 20(1):115-119.
DOI: 10.3872/j.issn.1007-385X.2013.1.020
Abstract:
尽管弥漫大B细胞淋巴瘤（diffuse large B-cell lymphoma，DLBCL）患者的总体预后有所改善，但仍有大约三分之一患者属于难治性/复发性DLBCL，这是导致其发病率和病死率增加的主要原因。临床上治疗难治性/复发性DLBCL的方法仍以大剂量化疗以及对无并发症的患者进行大剂量化疗-自体干细胞移植（high-dose chemotherapy-autologous stem cell transplant，HD-ASCT）为主。但对于给予利妥昔单抗联合CHOP化疗（rituximab-CHOP，R-CHOP）治疗后无反应的难治性DLBCL患者进行HD-ASCT，预后极差。因此，如何提高难治性/复发性DLBCL患者总生存期成了新的研究热点。本文主要从治疗方面对难治性/复发性DLBCL进行综述。
尽管弥漫大B细胞淋巴瘤（diffuse large B-cell lymphoma，DLBCL）患者的总体预后有所改善，但仍有大约三分之一患者属于难治性/复发性DLBCL，这是导致其发病率和病死率增加的主要原因。临床上治疗难治性/复发性DLBCL的方法仍以大剂量化疗以及对无并发症的患者进行大剂量化疗-自体干细胞移植（high-dose chemotherapy-autologous stem cell transplant，HD-ASCT）为主。但对于给予利妥昔单抗联合CHOP化疗（rituximab-CHOP，R-CHOP）治疗后无反应的难治性DLBCL患者进行HD-ASCT，预后极差。因此，如何提高难治性/复发性DLBCL患者总生存期成了新的研究热点。本文主要从治疗方面对难治性/复发性DLBCL进行综述。
2013, 20(1):120-123.
DOI: 10.3872/j.issn.1007-385X.2013.1.021
Abstract:
肾细胞癌（renal cell cancer，RCC）起源于肾小管细胞，早期RCC预后良好，晚期和转移性RCC对放疗、化疗均不敏感。免疫治疗可通过刺激机体自身免疫系统达到抑制肿瘤生长和治疗肿瘤的目的。生物治疗作为肿瘤综合治疗的第4大模式被认为是目前已知的有望完全消灭癌细胞的治疗手段。以往研究发现，IFN、IL-2等细胞因子以及过继细胞等治疗RCC均有一定的疗效。近年来随着对RCC研究的不断深入，对细胞因子突变体、细胞因子与其他药物联合用药、细胞因子基因、细胞因子诱导外周血单个核细胞产生的DC-CIK、RCC细胞疫苗、DC疫苗、自杀基因等治疗RCC开展了一系列的临床试验，并取得了较好的临床效果。通过下调CD4+CD25+调节性T细胞数量治疗RCC有望取得良好疗效。虽然这些方法已应用于临床，但仍存在不足，有待进一步的研究提高其临床疗效。
肾细胞癌（renal cell cancer，RCC）起源于肾小管细胞，早期RCC预后良好，晚期和转移性RCC对放疗、化疗均不敏感。免疫治疗可通过刺激机体自身免疫系统达到抑制肿瘤生长和治疗肿瘤的目的。生物治疗作为肿瘤综合治疗的第4大模式被认为是目前已知的有望完全消灭癌细胞的治疗手段。以往研究发现，IFN、IL-2等细胞因子以及过继细胞等治疗RCC均有一定的疗效。近年来随着对RCC研究的不断深入，对细胞因子突变体、细胞因子与其他药物联合用药、细胞因子基因、细胞因子诱导外周血单个核细胞产生的DC-CIK、RCC细胞疫苗、DC疫苗、自杀基因等治疗RCC开展了一系列的临床试验，并取得了较好的临床效果。通过下调CD4+CD25+调节性T细胞数量治疗RCC有望取得良好疗效。虽然这些方法已应用于临床，但仍存在不足，有待进一步的研究提高其临床疗效。
2013, 20(1):124-127.
DOI: 10.3872/j.issn.1007-385X.2013.1.022
Abstract:
Twist基因属于碱性螺旋-环-螺旋（basic helix-loop-helix，bHLH）转录因子家族，具有高度保守性，是肿瘤细胞发生间质-上皮转化（epithelial-mesenchymal transition，EMT）并获得迁徙、侵袭和转移能力的主要诱导因子之一，它的信号通路是一个复杂、多途径的网络系统。Twist可通过AKT磷酸化影响口腔鳞癌和膀胱癌的发生和侵袭，通过调控AKT信号途径使鼻咽癌细胞和乳腺癌细胞株产生对紫杉醇的耐药性；STAT3、Twist1和AKT2形成一个功能信号轴参与乳腺癌细胞的侵袭和转移、参与胃癌的发生和发展，异常的p-STAT3/Twist/E-cadherin信号轴可能介导肝癌的侵袭与转移。随着Twist相关信号通路与肿瘤关系的深入研究，其在肿瘤的诊断、治疗和预后预测中的重要作用逐步显现，本文就近年来关于Twist癌基因的结构、功能及其相关信号通路与肿瘤的关系的研究做一综述。
Twist基因属于碱性螺旋-环-螺旋（basic helix-loop-helix，bHLH）转录因子家族，具有高度保守性，是肿瘤细胞发生间质-上皮转化（epithelial-mesenchymal transition，EMT）并获得迁徙、侵袭和转移能力的主要诱导因子之一，它的信号通路是一个复杂、多途径的网络系统。Twist可通过AKT磷酸化影响口腔鳞癌和膀胱癌的发生和侵袭，通过调控AKT信号途径使鼻咽癌细胞和乳腺癌细胞株产生对紫杉醇的耐药性；STAT3、Twist1和AKT2形成一个功能信号轴参与乳腺癌细胞的侵袭和转移、参与胃癌的发生和发展，异常的p-STAT3/Twist/E-cadherin信号轴可能介导肝癌的侵袭与转移。随着Twist相关信号通路与肿瘤关系的深入研究，其在肿瘤的诊断、治疗和预后预测中的重要作用逐步显现，本文就近年来关于Twist癌基因的结构、功能及其相关信号通路与肿瘤的关系的研究做一综述。
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.