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Volume 20,Issue 5,2013 Table of Contents
2013, 20(5):507-514.
DOI: 10.3872/j.issn.1007-385X.2013.05.001
Abstract:
Interferon (IFN) was early discovered as a family of antiviral cytokines which includes three main classes: type I IFNs, type Ⅱ and type Ⅲ . IFN plays critical roles in multiple biological activities such as anti-virus infection, regulation of cell proliferation and immunological response through JAK-STAT dependent or independent signaling pathways. In addition, IFNs also take an important role in tumor immunology. Type Ⅰ IFNs can activate DC cells to relase tumor necrosis factor-related apoptosis inducing ligand (TRAIL), which can enhance the cytotoxicity of NK cells or kill tumor cellls directly. Meanwhile, type Ⅱ IFNs can activate CTL to kill tumor cells, and can enhance identification of CTL to tumors by increasing the expression of major histocompatibility complex (MHC) or indirectly inhibit tumor progression through regulating cell metabolism. However, under certain conditions, IFN-γ was found to increase the numbers of Treg and Th17 cells and induce MDSC infiltrating in the tumor microenvironment that can suppress the function of immune system and “help” tumor cells escape from immunosurveillance. Therefore, IFNs participate in tumor immunology as a “double-edged sword”. Further studies on the function and mechanism of IFNs in tumor immunolgy, and exploration of new IFN-based cancer treatment (such as IFN-based immuno-chemotherapy) are significant guides to improve tumor treatment.
Interferon (IFN) was early discovered as a family of antiviral cytokines which includes three main classes: type I IFNs, type Ⅱ and type Ⅲ . IFN plays critical roles in multiple biological activities such as anti-virus infection, regulation of cell proliferation and immunological response through JAK-STAT dependent or independent signaling pathways. In addition, IFNs also take an important role in tumor immunology. Type Ⅰ IFNs can activate DC cells to relase tumor necrosis factor-related apoptosis inducing ligand (TRAIL), which can enhance the cytotoxicity of NK cells or kill tumor cellls directly. Meanwhile, type Ⅱ IFNs can activate CTL to kill tumor cells, and can enhance identification of CTL to tumors by increasing the expression of major histocompatibility complex (MHC) or indirectly inhibit tumor progression through regulating cell metabolism. However, under certain conditions, IFN-γ was found to increase the numbers of Treg and Th17 cells and induce MDSC infiltrating in the tumor microenvironment that can suppress the function of immune system and “help” tumor cells escape from immunosurveillance. Therefore, IFNs participate in tumor immunology as a “double-edged sword”. Further studies on the function and mechanism of IFNs in tumor immunolgy, and exploration of new IFN-based cancer treatment (such as IFN-based immuno-chemotherapy) are significant guides to improve tumor treatment.
2013, 20(5):515-521.
DOI: 10.3872/j.issn.1007-385X.2013.05.002
Abstract:
Objective: To screen a peptide strongly binding to the bladder cancer BIU-87 cells from phage 7-mer cyclic peptide library in vitro in Chinese, and identify its specificity. Methods: 3 rounds subtraction screening was performed on a phage 7-mer cyclic peptide library, with the BIU-87 cells as the target cells and normal human bladder epithelial cells as the absorber cells. Phage clones which can positively bind to BIU-87 cells were identified by ELISA, and those coding DNA were sequenced to analyze the homology. Strongly positive peptide was chemically synthetized to prepare fluorescent probe by taging fluorescein isothiocyanate (FITC). The affinity between the fluorescent probes and BIU- 87 bladder cancer cells, human normal bladder epithelial cells, human prostate carcinoma PC3M cells, human hepatocellular carcinoma SMMC-7721 cells and human colon cancer HCT116 cells were identified by flow cytometry and fluorescence microscopy.Results:The c7c phage-display peptides library was enriched for 25 times through 3 rounds of subtraction screening and the positive rate was 76%. 10 strongly positive clones were obtained. For the 10 strongly positive clones, the amino acid sequences of SISSLTH, MARYMSA and TVRTSAD were consensus sequence. The binding rate of small molecular fluorescent probe FITC-SISSLATH binding to the BIU- 87 bladder cancer cells (80.06±8.78)% was obviouly higher than of FITC-MARYMSA(52.93±7.28)%, FITC-TVRTSAD(38.04±7.47)%, FITC -EDRKETA(1.91±1.37)% and FITC(9.85±2.9)% (all P<0.01). The binding rate of the BIU- 87 bladder cancer cells binding to FITC-SISSLATH(80.06±8.78)% was higher than that of the normal human bladder epithelial cells (13.89±197)%, human prostate carcinoma PC3M cells(8.13±2.85)%, human hepatocellular carcinoma SMMC-7721 cells (27±2.87)% and human colon cancer HCT116 cells(2.33±1.75)%(all P<0.01). Conclusion: A targeting peptide SISSLTH binding to BIU-87 cells with high specificity and efficiency was obtained from the phage 7-mer cyclic peptide library through 3 rounds of subtraction screening in vitro.
Objective: To screen a peptide strongly binding to the bladder cancer BIU-87 cells from phage 7-mer cyclic peptide library in vitro in Chinese, and identify its specificity. Methods: 3 rounds subtraction screening was performed on a phage 7-mer cyclic peptide library, with the BIU-87 cells as the target cells and normal human bladder epithelial cells as the absorber cells. Phage clones which can positively bind to BIU-87 cells were identified by ELISA, and those coding DNA were sequenced to analyze the homology. Strongly positive peptide was chemically synthetized to prepare fluorescent probe by taging fluorescein isothiocyanate (FITC). The affinity between the fluorescent probes and BIU- 87 bladder cancer cells, human normal bladder epithelial cells, human prostate carcinoma PC3M cells, human hepatocellular carcinoma SMMC-7721 cells and human colon cancer HCT116 cells were identified by flow cytometry and fluorescence microscopy.Results:The c7c phage-display peptides library was enriched for 25 times through 3 rounds of subtraction screening and the positive rate was 76%. 10 strongly positive clones were obtained. For the 10 strongly positive clones, the amino acid sequences of SISSLTH, MARYMSA and TVRTSAD were consensus sequence. The binding rate of small molecular fluorescent probe FITC-SISSLATH binding to the BIU- 87 bladder cancer cells (80.06±8.78)% was obviouly higher than of FITC-MARYMSA(52.93±7.28)%, FITC-TVRTSAD(38.04±7.47)%, FITC -EDRKETA(1.91±1.37)% and FITC(9.85±2.9)% (all P<0.01). The binding rate of the BIU- 87 bladder cancer cells binding to FITC-SISSLATH(80.06±8.78)% was higher than that of the normal human bladder epithelial cells (13.89±197)%, human prostate carcinoma PC3M cells(8.13±2.85)%, human hepatocellular carcinoma SMMC-7721 cells (27±2.87)% and human colon cancer HCT116 cells(2.33±1.75)%(all P<0.01). Conclusion: A targeting peptide SISSLTH binding to BIU-87 cells with high specificity and efficiency was obtained from the phage 7-mer cyclic peptide library through 3 rounds of subtraction screening in vitro.
Kong Xianglin
,
Cheng Xianshuo
,
Li Furong
,
Xiao Youchuan
,
Yang Zhibin
,
Huang Youguang
,
Xia Cuifeng
,
Yu Kun
,
Li Yunfeng
2013, 20(5):522-528.
DOI: 10.3872/j.issn.1007-385X.2013.05.003
Abstract:
Objective: To investigate the effect of soluble Tie 2 (sTie2) on the vascular mimicry (VM) formation, proliferation, migration and invasion of colonic cancer HCT116 cells. Methods: The recombinant plasmid pBLAST49-hsTie2 or control plasmid pBLAST49 was transfected into HCT116 cells by liposome to form hsTie2-HCT116 or Ctrl-HCT116 cells. 3D model culture, SRB, scratch and Transwell assay were conducted respectively to detect the VM formation, proliferation, migration and invasion of HCT116 cells. Western blotting was used to detect the expression of VE-cadherin protein. Results: The recombinant plasmid pBLAST49-hsTie2 was transfected into colorectal cancer HCT116 cells successfully. Compared with that in the Ctrl-HCT116 cells, the formation of VM (\[0.75±0.45\] vs \[7.50±052\], P<0.01) and the expression of VE-cadherin protein (\[1.23±0.08\] vs \[1.73±0.02\], P<0.01) in hsTie2-HCT116 cells was significantly decreased, and the survival rate was also significantly decreased (\[32.57±4.57\]% vs \[88.24±2194\]%, P<0.01). The migration (\[0.37±0.07\] vs \[0.80±0.03\] mm, P<0.01) and invasion capacity (\[5725±3.17\] vs \[127.25±6.25\], P<0.01) of HCT116 cells were inhibited significantly. Conclusion: sTie2 inhibits the proliferation, migration and invasion of colorectal cancer cells through suppression of the VM formation, which provides a good basis for the development of new drugs for the treatment of colorectal cancer by targeting both angiogenesis and VM formation.
Objective: To investigate the effect of soluble Tie 2 (sTie2) on the vascular mimicry (VM) formation, proliferation, migration and invasion of colonic cancer HCT116 cells. Methods: The recombinant plasmid pBLAST49-hsTie2 or control plasmid pBLAST49 was transfected into HCT116 cells by liposome to form hsTie2-HCT116 or Ctrl-HCT116 cells. 3D model culture, SRB, scratch and Transwell assay were conducted respectively to detect the VM formation, proliferation, migration and invasion of HCT116 cells. Western blotting was used to detect the expression of VE-cadherin protein. Results: The recombinant plasmid pBLAST49-hsTie2 was transfected into colorectal cancer HCT116 cells successfully. Compared with that in the Ctrl-HCT116 cells, the formation of VM (\[0.75±0.45\] vs \[7.50±052\], P<0.01) and the expression of VE-cadherin protein (\[1.23±0.08\] vs \[1.73±0.02\], P<0.01) in hsTie2-HCT116 cells was significantly decreased, and the survival rate was also significantly decreased (\[32.57±4.57\]% vs \[88.24±2194\]%, P<0.01). The migration (\[0.37±0.07\] vs \[0.80±0.03\] mm, P<0.01) and invasion capacity (\[5725±3.17\] vs \[127.25±6.25\], P<0.01) of HCT116 cells were inhibited significantly. Conclusion: sTie2 inhibits the proliferation, migration and invasion of colorectal cancer cells through suppression of the VM formation, which provides a good basis for the development of new drugs for the treatment of colorectal cancer by targeting both angiogenesis and VM formation.
2013, 20(5):529-534.
DOI: 10.3872/j.issn.1007-385X.2013.05.004
Abstract:
Objective : To prepare streptavidin-tagged interleukin-7 (SA-IL-7) fusion protein and to value its therapeutic effect on mouse superficial bladder cancer. Methods: Recombinant pET24a-SA-IL-7 plasmid was constructed and then SA-IL-7 fusion protein was prepared. The binding of SA-IL-7 to biotinylated bladder cancer MB49 cells was examined by flow cytometry and the bioactivity of SA-IL-7 fusion protein was examined by thymocyte proliferation assay. The mouse superficial bladder cancer model was used to evaluate the therapeutic effect of intravesical instillation of SA-IL-7. Mice were monitored for survival and the tumor-infiltrated CD8+T cells were examined by immunohistochemistry assay. Results: SA-IL-7 fusion protein efficiently anchored on the biotinylated MB49 cell surface, with a binding ratio of (96.6±13)%; SA-IL-7 promoted the proliferation of thymocytes. The SA-IL-7 fusion protein anchored on biotinylated bladder mucosal surface for more than 7 d. Whereas, IL-7 was no longer detected in the first day in the unbiotinylation group. After being attacked by bladder cancer MB49 cells for 80 d, 90% mice in the IL-7 group, while 60% mice survived without tumors in the SA-IL-7 group (P<0.05). Moreover, tumor-infiltrated CD8+ T cells in the SA-IL-7 group was significantly higher than those in the control group (P<0.01). Importantly, 11 out of 15 SA-IL-7-cured mice resisted to a second intravesical MB49 cell challenge, whereas only 1 of 15 control mice survived the challenge (P<0.01). Conclusion: Intravesical instillation of SA-IL-7 fusion protein can induce anticancer immunity, which may represent a promising immunotherapy for superficial bladder cancer.
Objective : To prepare streptavidin-tagged interleukin-7 (SA-IL-7) fusion protein and to value its therapeutic effect on mouse superficial bladder cancer. Methods: Recombinant pET24a-SA-IL-7 plasmid was constructed and then SA-IL-7 fusion protein was prepared. The binding of SA-IL-7 to biotinylated bladder cancer MB49 cells was examined by flow cytometry and the bioactivity of SA-IL-7 fusion protein was examined by thymocyte proliferation assay. The mouse superficial bladder cancer model was used to evaluate the therapeutic effect of intravesical instillation of SA-IL-7. Mice were monitored for survival and the tumor-infiltrated CD8+T cells were examined by immunohistochemistry assay. Results: SA-IL-7 fusion protein efficiently anchored on the biotinylated MB49 cell surface, with a binding ratio of (96.6±13)%; SA-IL-7 promoted the proliferation of thymocytes. The SA-IL-7 fusion protein anchored on biotinylated bladder mucosal surface for more than 7 d. Whereas, IL-7 was no longer detected in the first day in the unbiotinylation group. After being attacked by bladder cancer MB49 cells for 80 d, 90% mice in the IL-7 group, while 60% mice survived without tumors in the SA-IL-7 group (P<0.05). Moreover, tumor-infiltrated CD8+ T cells in the SA-IL-7 group was significantly higher than those in the control group (P<0.01). Importantly, 11 out of 15 SA-IL-7-cured mice resisted to a second intravesical MB49 cell challenge, whereas only 1 of 15 control mice survived the challenge (P<0.01). Conclusion: Intravesical instillation of SA-IL-7 fusion protein can induce anticancer immunity, which may represent a promising immunotherapy for superficial bladder cancer.
2013, 20(5):535-539.
DOI: 10.3872/j.issn.1007-385X.2013.05.005
Abstract:
Objective : To explore the effect of melanoma-associated antigen (MAGE)-A9 on the transcriptional activity and the function of P53. Methods: Plasmids pcDNA3.0, pcDNA3.0-p53 and pCMV6-MAGE-A9 were transfeced in vitro into human breast cancer MDA-MB-231 cells using LipofectamineTM2000. The expressions of p21WAF1 mRNA and protein in MDA-MB-231 cells were analyzed by RT-PCR and Western blotting. Luciferase reporter assay was performed to determine the luciferase activity induced by p21WAF1 promoter in MDA-MB-231 cells. MTT assay were adopted to explore the effect of different plasmids on the cell proliferation. Results: The expression of p21WAF1 both in mRNA and protein levels was decreased in pcDNA3.0-p53/MAGE-A9 group, compared with pcDNA3.0-p53 group (\[0.15±0.01\] vs \[0.18±002\], \[0.03±0.00\] vs \[0.06±0.01\], P<0.05). The luciferase activity induced by p21WAF1 promoter was remarkably increased in MDA-MB-231 cells transfected with plasmid pcDNA3.0-p53 (\[58.56±3.47\] vs \[1.00±012\], P<001). After transfected with pcDNA3.0-p53/MAGE-A9, the luciferase activity induced by p21WAF1 promoter in MDA-MB-231 cells was significantly reduced compared with that in pcDNA3.0-p53 group (\[22.02±4.91\] vs \[58.56±3.47\], P<005). Compared with pcDNA3.0 group, the proliferation rate were significantly decreased in pcDNA3.0-p53 group (\[228.89±22.39\]% vs \[337.23±23.67\]%, P<0.05), while in pcDNA3.0-p53/MAGE-A9 group, it was significantly higher than that in pcDNA3.0-p53 group (\[291.51±5.91\]% vs \[228.89±22.39\]%, P<0.05). Conclusion: MAGE-A9 can inhibit the transcriptional activity of P53 and proliferation of MDA-MB-231 cells.
Objective : To explore the effect of melanoma-associated antigen (MAGE)-A9 on the transcriptional activity and the function of P53. Methods: Plasmids pcDNA3.0, pcDNA3.0-p53 and pCMV6-MAGE-A9 were transfeced in vitro into human breast cancer MDA-MB-231 cells using LipofectamineTM2000. The expressions of p21WAF1 mRNA and protein in MDA-MB-231 cells were analyzed by RT-PCR and Western blotting. Luciferase reporter assay was performed to determine the luciferase activity induced by p21WAF1 promoter in MDA-MB-231 cells. MTT assay were adopted to explore the effect of different plasmids on the cell proliferation. Results: The expression of p21WAF1 both in mRNA and protein levels was decreased in pcDNA3.0-p53/MAGE-A9 group, compared with pcDNA3.0-p53 group (\[0.15±0.01\] vs \[0.18±002\], \[0.03±0.00\] vs \[0.06±0.01\], P<0.05). The luciferase activity induced by p21WAF1 promoter was remarkably increased in MDA-MB-231 cells transfected with plasmid pcDNA3.0-p53 (\[58.56±3.47\] vs \[1.00±012\], P<001). After transfected with pcDNA3.0-p53/MAGE-A9, the luciferase activity induced by p21WAF1 promoter in MDA-MB-231 cells was significantly reduced compared with that in pcDNA3.0-p53 group (\[22.02±4.91\] vs \[58.56±3.47\], P<005). Compared with pcDNA3.0 group, the proliferation rate were significantly decreased in pcDNA3.0-p53 group (\[228.89±22.39\]% vs \[337.23±23.67\]%, P<0.05), while in pcDNA3.0-p53/MAGE-A9 group, it was significantly higher than that in pcDNA3.0-p53 group (\[291.51±5.91\]% vs \[228.89±22.39\]%, P<0.05). Conclusion: MAGE-A9 can inhibit the transcriptional activity of P53 and proliferation of MDA-MB-231 cells.
2013, 20(5):540-546.
DOI: 10.3872/j.issn.1007-385X.2013.05.006
Abstract:
Objective : To investigate the effect of melatonin (MT) and cisplatin (DDP) used alone or in combination on proliferation and apoptosis of human glioma cells U251 and SHG-44. Methods: U251 and SHG-44 cells were treated with various concentrations of MT and DDP alone or in combination, and the control group (without adding any drug) and ethanol group (adding the ethanol) were included. CCK-8 assay was used to detect cell proliferation, and flow cytometry was used to detect cell apoptosis and cell cycle. The coefficient of drug interaction (CDI) was used to evaluate whether MT could affect the sensitivity of U251 and SHG-44 cells to DDP. Results: CCK-8 results showed that MT or DDP used alone inhibited the proliferation of U251 and SHG-44 cells in a concentration dependent manner, and 0.5 mmol/L MT synergistically enhanced the proliferation inhibitory effect of DDP on U251 and SHG-44 cells (CDI<1). Flow cytometry results showed that MT promoted U251 and SHG-44 cell apoptosis, and MT enhanced the apoptosis inducing effect of DDP on U251 and SHG-44 cells. The apoptotic rate of U251 and SHG-44 cells in the 0.5 mmol/L MT and 20 μg/ml DDP combination group was significantly higher than that in 20 μg/ml DDP group (\[66.3±1.0\]% vs \[45.9±1.7\]%, \[355±0.8\]% vs \[15.5±08\]%, P<0.01). And the percentage of U251 and SHG-44 cells in sub-G1 phase in the 0.5 mmol/L MT and 20 μg/ml DDP combination group was significantly higher than that in 20 μg/ml DDP group (\[524±2.1\]% vs \[27.9±1.5\]%, \[39.7±1.5\]% vs \[27.7±1.3\]%, P<0.01\]. Conclusion: MT can enhance the apoptosis inducing effect of DDP on human glioma cells U251 and SHG-44, thereby synergistically enhancing the suppressive effects of DDP on human glioma cell proliferation, which may be used as a complementary drug in human glioma chemotherapy.
Objective : To investigate the effect of melatonin (MT) and cisplatin (DDP) used alone or in combination on proliferation and apoptosis of human glioma cells U251 and SHG-44. Methods: U251 and SHG-44 cells were treated with various concentrations of MT and DDP alone or in combination, and the control group (without adding any drug) and ethanol group (adding the ethanol) were included. CCK-8 assay was used to detect cell proliferation, and flow cytometry was used to detect cell apoptosis and cell cycle. The coefficient of drug interaction (CDI) was used to evaluate whether MT could affect the sensitivity of U251 and SHG-44 cells to DDP. Results: CCK-8 results showed that MT or DDP used alone inhibited the proliferation of U251 and SHG-44 cells in a concentration dependent manner, and 0.5 mmol/L MT synergistically enhanced the proliferation inhibitory effect of DDP on U251 and SHG-44 cells (CDI<1). Flow cytometry results showed that MT promoted U251 and SHG-44 cell apoptosis, and MT enhanced the apoptosis inducing effect of DDP on U251 and SHG-44 cells. The apoptotic rate of U251 and SHG-44 cells in the 0.5 mmol/L MT and 20 μg/ml DDP combination group was significantly higher than that in 20 μg/ml DDP group (\[66.3±1.0\]% vs \[45.9±1.7\]%, \[355±0.8\]% vs \[15.5±08\]%, P<0.01). And the percentage of U251 and SHG-44 cells in sub-G1 phase in the 0.5 mmol/L MT and 20 μg/ml DDP combination group was significantly higher than that in 20 μg/ml DDP group (\[524±2.1\]% vs \[27.9±1.5\]%, \[39.7±1.5\]% vs \[27.7±1.3\]%, P<0.01\]. Conclusion: MT can enhance the apoptosis inducing effect of DDP on human glioma cells U251 and SHG-44, thereby synergistically enhancing the suppressive effects of DDP on human glioma cell proliferation, which may be used as a complementary drug in human glioma chemotherapy.
Xu Yipeng
,
Zhu Shaoxing
,
Liu Weihui
,
Li Yongsheng
,
Chen Guiping
,
Wang Hua
,
Zhao Yang
,
Li Fangyin
,
Wang Zongping
2013, 20(5):547-551.
DOI: 10.3872/j.issn.1007-385X.2013.05.007
Abstract:
Objective : To explore the influence of IL-2, IFN-α and IFN-γ on the expression of B7-H4 in clear cell renal cell carcinoma 786-0 cells． Methods: Clear cell renal cell carcinoma 786-0 cells were stimulated by IL-2, IFN-α and IFN-γ for 24 h. The expression of B7-H4 mRNA was detected by RT-PCR. The expression of B7-H4 protein was detected by ELISA assay, cytoimmunochemistry assay and flow cytometry. Results: RT-PCR result showed that the expression of B7-H4 mRNA in IL-2 (0.75±0.06), IFN-α (0.68±0.05) and IFN-γ (0.95±0.08) group cells was significantly higher than that in the untreated group (0.30±0.03) (P<0.05). Immunocytochemistry showed that the expression of B7-H4 protein was detected both in the cell membrane and cytoplasm, and the expression of B7-H4 protein was up-regulated after stimulated by IL-2, IFN-α and IFN-γ. ELISA result showed that the expression of soluble B7-H4 protein in the supernatants of IL-2 (44.89±0.97) ng/ml, IFN-α (46.74±2.25) ng/ml and IFN-γ group cells (47.31±1.12) ng/ml was significantly higher than that in the untreated group (34.42±1.69) ng/ml (P<0.05). Flow cytometry assay result showed that the positive expression rate of B7-H4 in IL-2 (44.89±0.94)%, IFN-α (46.41±0.55)% and IFN-γ (54.18±1.42)% group cells were significantly higher than that in the untreated group (30.45±0.96)% (P<005). Conclusion: IL-2, IFN-α and IFN-γ can up-regulate the expression of B7-H4, in which IFN-γ has the highest capacity.
Objective : To explore the influence of IL-2, IFN-α and IFN-γ on the expression of B7-H4 in clear cell renal cell carcinoma 786-0 cells． Methods: Clear cell renal cell carcinoma 786-0 cells were stimulated by IL-2, IFN-α and IFN-γ for 24 h. The expression of B7-H4 mRNA was detected by RT-PCR. The expression of B7-H4 protein was detected by ELISA assay, cytoimmunochemistry assay and flow cytometry. Results: RT-PCR result showed that the expression of B7-H4 mRNA in IL-2 (0.75±0.06), IFN-α (0.68±0.05) and IFN-γ (0.95±0.08) group cells was significantly higher than that in the untreated group (0.30±0.03) (P<0.05). Immunocytochemistry showed that the expression of B7-H4 protein was detected both in the cell membrane and cytoplasm, and the expression of B7-H4 protein was up-regulated after stimulated by IL-2, IFN-α and IFN-γ. ELISA result showed that the expression of soluble B7-H4 protein in the supernatants of IL-2 (44.89±0.97) ng/ml, IFN-α (46.74±2.25) ng/ml and IFN-γ group cells (47.31±1.12) ng/ml was significantly higher than that in the untreated group (34.42±1.69) ng/ml (P<0.05). Flow cytometry assay result showed that the positive expression rate of B7-H4 in IL-2 (44.89±0.94)%, IFN-α (46.41±0.55)% and IFN-γ (54.18±1.42)% group cells were significantly higher than that in the untreated group (30.45±0.96)% (P<005). Conclusion: IL-2, IFN-α and IFN-γ can up-regulate the expression of B7-H4, in which IFN-γ has the highest capacity.
2013, 20(5):552-558.
DOI: 10.3872/j.issn.1007-385X.2013.05.008
Abstract:
Objective:A human CD40 monoclonal antibody, 5C11, was coupled with three kinds of polymeric micelles (PMs) containing different chemical groups respectively to construct the corresponding 5C11 coupled polymeric micelles (PM-5C11). The optimal PM for coupling with 5C11 was selected among them, and evaluate its biological activity. Methods: Three kinds of copolymers with different chemical composition were synthesized respectively to prepare the corresponding PM and PM-5C11 after coupled with 5C11. The PM-5C11 which exhibited both higher coupling efficiency and greatest biological safety was selected to be the optimal one among them. The optimal PM-5C11 was then interacted with Daudi cell line, derived from human Burkitt lymphoma, to evaluate its biological activity and the cellular uptake by Daudi cells. Results: Three kinds of PMs containing tosyl group, allyl group or carboxyl group had been prepared successfully. The coupling efficiencies of 5C11 to PMs containing tosyl group or allyl group were both higher than that to PM containing carboxyl group (\[28.08±2.24\]%, \[29.06±137\]% vs \[21.26±1.04\]%, P<0.05). The PM containing tosyl group was chosen to be the optimal one because cytotoxic reagent remained during the preparation of the PM containing allyl group. The corresponding optimal PM coupled with 5C11 to form PM-5C11, which exhibited the original bioactivity of 5C11 and could be uptaken by Daudi cells. Conclusion: Among the three kinds of PMs, the PM containing tosyl group exhibits the best biological safety and the relative higher coupling efficiency; its corresponding PM-5C11 shows the best biological activity. This PM could be the best choice for constructing the nano-drug carrier.
Objective:A human CD40 monoclonal antibody, 5C11, was coupled with three kinds of polymeric micelles (PMs) containing different chemical groups respectively to construct the corresponding 5C11 coupled polymeric micelles (PM-5C11). The optimal PM for coupling with 5C11 was selected among them, and evaluate its biological activity. Methods: Three kinds of copolymers with different chemical composition were synthesized respectively to prepare the corresponding PM and PM-5C11 after coupled with 5C11. The PM-5C11 which exhibited both higher coupling efficiency and greatest biological safety was selected to be the optimal one among them. The optimal PM-5C11 was then interacted with Daudi cell line, derived from human Burkitt lymphoma, to evaluate its biological activity and the cellular uptake by Daudi cells. Results: Three kinds of PMs containing tosyl group, allyl group or carboxyl group had been prepared successfully. The coupling efficiencies of 5C11 to PMs containing tosyl group or allyl group were both higher than that to PM containing carboxyl group (\[28.08±2.24\]%, \[29.06±137\]% vs \[21.26±1.04\]%, P<0.05). The PM containing tosyl group was chosen to be the optimal one because cytotoxic reagent remained during the preparation of the PM containing allyl group. The corresponding optimal PM coupled with 5C11 to form PM-5C11, which exhibited the original bioactivity of 5C11 and could be uptaken by Daudi cells. Conclusion: Among the three kinds of PMs, the PM containing tosyl group exhibits the best biological safety and the relative higher coupling efficiency; its corresponding PM-5C11 shows the best biological activity. This PM could be the best choice for constructing the nano-drug carrier.
2013, 20(5):559-564.
DOI: 10.3872/j.issn.1007-385X.2013.05.009
Abstract:
Objective:To construct Jurkat cells expressing chimeric antigen receptor (CAR) of vascular endothelial growth factor receptor 1 (VEGFR1/FLT1) using lentivirus and to investigate their chemotaxis to VEGF. Methods: The CAR targeting VEGFR1 was synthetized, and the recombinant lentiviral vector named LV-gn-FLT1-CAR was constructed and infected into Jurkat cells. Stable infected cell stains were obtained through G418 screening. The expressions of FLT1-CAR mRNA and protein in the stable infected cell stains were detected by PCR and flow cytometry. The chemotaxis of selected cell strains toward VEGF was detected by Transwell assay. Results: Recombinant lentivirus LV-gn-FLT1-CAR was successfully constructed. FLT1-CAR was successfully integrated into Jurkat cells and FLT1-CAR protein was stably expressed. The selected cell strains, Jurkat-gn-FLT1CAR-1 and Jurkat-gn-FLT1CAR-2, showed obvious chemotaxis to VEGF. In a VEGF concentration of 100 ng/ml, Jurkat-gn-FLT1-CAR-1 cell chemotactic number was (62±8)，which was significantly higher than that of the control group Jurkat cells (18±5) (P<0.01). Conclusion: Jurkat cell strains stably express FLT1-CAR are successfully obtained, which have a distinct chemotaxis to VEGF.
Objective:To construct Jurkat cells expressing chimeric antigen receptor (CAR) of vascular endothelial growth factor receptor 1 (VEGFR1/FLT1) using lentivirus and to investigate their chemotaxis to VEGF. Methods: The CAR targeting VEGFR1 was synthetized, and the recombinant lentiviral vector named LV-gn-FLT1-CAR was constructed and infected into Jurkat cells. Stable infected cell stains were obtained through G418 screening. The expressions of FLT1-CAR mRNA and protein in the stable infected cell stains were detected by PCR and flow cytometry. The chemotaxis of selected cell strains toward VEGF was detected by Transwell assay. Results: Recombinant lentivirus LV-gn-FLT1-CAR was successfully constructed. FLT1-CAR was successfully integrated into Jurkat cells and FLT1-CAR protein was stably expressed. The selected cell strains, Jurkat-gn-FLT1CAR-1 and Jurkat-gn-FLT1CAR-2, showed obvious chemotaxis to VEGF. In a VEGF concentration of 100 ng/ml, Jurkat-gn-FLT1-CAR-1 cell chemotactic number was (62±8)，which was significantly higher than that of the control group Jurkat cells (18±5) (P<0.01). Conclusion: Jurkat cell strains stably express FLT1-CAR are successfully obtained, which have a distinct chemotaxis to VEGF.
2013, 20(5):565-568.
DOI: 10.3872/j.issn.1007-385X.2013.05.010
Abstract:
Objective:To explore the effect of midkine (MK), a heparin-binding cytokine, on the anoikis resistance in hepatocellular carcinoma Hep3B cells. Methods: Human hepatoma-derived cell line Hep3B was kept in suspension to induce an anoikis model and treated with different concentrations of MK (0, 10, 50 and 100 ng/ml) or PBS (control group). The apoptosis of Hep3B cells was detected by flow cytometry and the expressions of apoptosis-related proteins Bcl-2 and caspase-3 were determined by Western blotting. Results:As incubation time was prolonged, the apoptotic rate of hepatocellular carcinoma Hep3B cells cultured in suspension was gradually increased. After incubation for 72 h, the apoptotic rate of Hep3B cells cultured in suspension was significantly higher than that in the adherent-cultured cells (\[38.76±423\]% vs \[6.76±1.43\]%，P<0.01). After exposure of different concentrations of MK for 24 h, the apoptotic rate of the suspension-cultured Hep3B cells was significantly lower than that of the control group and negatively correlated with the concentration of MK (r=0.951, P=0.049). Simultaneously, the expression of intracellular anti-apoptotic protein Bcl-2 was significantly increased, whereas the expression of pro-apoptotic protein caspase-3 was significantly decreased. Conclusion: MK confers enhanced anoikis resistance in hepatocellular carcinoma Hep3B cells cultured in suspension, which may be associated with the up-regulation of Bcl-2 protein and down-regulation of caspase-3 protein.
Objective:To explore the effect of midkine (MK), a heparin-binding cytokine, on the anoikis resistance in hepatocellular carcinoma Hep3B cells. Methods: Human hepatoma-derived cell line Hep3B was kept in suspension to induce an anoikis model and treated with different concentrations of MK (0, 10, 50 and 100 ng/ml) or PBS (control group). The apoptosis of Hep3B cells was detected by flow cytometry and the expressions of apoptosis-related proteins Bcl-2 and caspase-3 were determined by Western blotting. Results:As incubation time was prolonged, the apoptotic rate of hepatocellular carcinoma Hep3B cells cultured in suspension was gradually increased. After incubation for 72 h, the apoptotic rate of Hep3B cells cultured in suspension was significantly higher than that in the adherent-cultured cells (\[38.76±423\]% vs \[6.76±1.43\]%，P<0.01). After exposure of different concentrations of MK for 24 h, the apoptotic rate of the suspension-cultured Hep3B cells was significantly lower than that of the control group and negatively correlated with the concentration of MK (r=0.951, P=0.049). Simultaneously, the expression of intracellular anti-apoptotic protein Bcl-2 was significantly increased, whereas the expression of pro-apoptotic protein caspase-3 was significantly decreased. Conclusion: MK confers enhanced anoikis resistance in hepatocellular carcinoma Hep3B cells cultured in suspension, which may be associated with the up-regulation of Bcl-2 protein and down-regulation of caspase-3 protein.
2013, 20(5):569-574.
DOI: 10.3872/j.issn.1007-385X.2013.05.011
Abstract:
Objective: To investigate the effect of silencing ATP-binding cassette protein E1 ( ABCE1) gene expression by electroporation on apoptosis, proliferation, migration and invasion of human esophageal squamous carcinoma EC-109 cells. Methods: The siRNA sequence (ABCE1-siRNA) targeting ABCE1 and the negative control sequence (NC-siRNA) were constructed and transfected into EC109 cells to obtain ABCE1-EC109 and NC-siRNA-EC109 cells, respectively. The expressions of ABCE1 mRNA and protein were detected by RT-PCR and Western blotting, respectively. Flow cytometry was used to detect the cell cycle and apoptosis of EC109 cells. The proliferation, migration and invasion of EC109 cells were evaluated by CCK-8 assay, wound closure assay and Transwell assay, respectively. Results: Compared with the NC-siRNA-EC109 cells, the expression levels of ABCE1 mRNA and protein were significantly decreased in the ABCE1-EC109 cells (\[0.47±0.04\] vs \[0.67±0.05\], \[0.63±0.09\] vs \[0.86±0.11\], both P<0.05). Compared with the NC-siRNA-EC109 cells, the proliferation of ABCE1-EC109 cells was significantly decreased (\[2.20±0.10\] vs \[2.91±0.13\]，P<0.05), the number of ABCE1-EC109 cells arrested at G0/G1 phase was increased (\[76.5±3.1\] vs \[56.1±2.7\], P<0.05), the cell apoptotic rate was increased (\[15.46±3.12\] vs \[0.54±0.24\], P<0.01), and the migration and invasion abilities were significantly decreased (migration: \[8.12±0.23\] vs \[1.91±0.11\], P<005; invasion: \[42.56±4.68\] vs \[68.78±6.98\]，P<0.01). Conclusion: ABCE1 gene silencing by electroporation promotes the apoptosis of esophageal squamous carcinoma EC109 cells, and inhibits their proliferation, migration and invasion in vitro.
Objective: To investigate the effect of silencing ATP-binding cassette protein E1 ( ABCE1) gene expression by electroporation on apoptosis, proliferation, migration and invasion of human esophageal squamous carcinoma EC-109 cells. Methods: The siRNA sequence (ABCE1-siRNA) targeting ABCE1 and the negative control sequence (NC-siRNA) were constructed and transfected into EC109 cells to obtain ABCE1-EC109 and NC-siRNA-EC109 cells, respectively. The expressions of ABCE1 mRNA and protein were detected by RT-PCR and Western blotting, respectively. Flow cytometry was used to detect the cell cycle and apoptosis of EC109 cells. The proliferation, migration and invasion of EC109 cells were evaluated by CCK-8 assay, wound closure assay and Transwell assay, respectively. Results: Compared with the NC-siRNA-EC109 cells, the expression levels of ABCE1 mRNA and protein were significantly decreased in the ABCE1-EC109 cells (\[0.47±0.04\] vs \[0.67±0.05\], \[0.63±0.09\] vs \[0.86±0.11\], both P<0.05). Compared with the NC-siRNA-EC109 cells, the proliferation of ABCE1-EC109 cells was significantly decreased (\[2.20±0.10\] vs \[2.91±0.13\]，P<0.05), the number of ABCE1-EC109 cells arrested at G0/G1 phase was increased (\[76.5±3.1\] vs \[56.1±2.7\], P<0.05), the cell apoptotic rate was increased (\[15.46±3.12\] vs \[0.54±0.24\], P<0.01), and the migration and invasion abilities were significantly decreased (migration: \[8.12±0.23\] vs \[1.91±0.11\], P<005; invasion: \[42.56±4.68\] vs \[68.78±6.98\]，P<0.01). Conclusion: ABCE1 gene silencing by electroporation promotes the apoptosis of esophageal squamous carcinoma EC109 cells, and inhibits their proliferation, migration and invasion in vitro.
2013, 20(5):575-579.
DOI: 10.3872/j.issn.1007-385X.2013.05.012
Abstract:
Objective:To investigate the effect of metformin on the proliferation and apoptosis of acute promyelocytic leukemia (APL) NB4 cells and the possible mechanism. Methods: After the treatment of metformin alone or in combination with daunorubicin, MTT assay and flow cytometry were used to detect the proliferation and apoptosis of NB4 cells, respectively. The activation of apoptosis-related proteins caspase-3 and caspase-9 was determined by Western blotting. Results: Metformin inhibited proliferation of NB4 cells in a dose-dependent (r=0.952, P<0.01) and time-dependent (r=0.967, P<0.01) manner. After metformin treatment for 72 h, its IC50 value to NB4 cells was (6.39±0.37) mmol/L. Metformin increased the chemosensitivity of NB4 cells to daunorubicin. The inhibitory rate of NB4 cell proliferation in 0.625 mmol/L metformin and 0.02 μmol/L daunorubicin combination group was significantly higher than that in daunorubicin used alone group (\[29.84±0.21\]% vs \[10.68±0.45\]%, P<0.05). After the treatment of 5 mmol/L metformin for 48 h, the apoptotic rate of NB4 cells was significantly increased compared with that of the control group (\[43.95±0.29\]% vs \[7.12±0.29\]%，P<0.01). The expressions of activated caspase-3 and caspase-9 fragments were up-regulated after metformin treatment.Conclusion: Metformin can efficiently inhibit the proliferation, promote the apoptosis of NB4 cells, and enhance the chemosensitivity of NB4 cells to daunorubicin. Caspase-3 and caspase-9 may be involved in the NB4 cell apoptosis induced by metformin.
Objective:To investigate the effect of metformin on the proliferation and apoptosis of acute promyelocytic leukemia (APL) NB4 cells and the possible mechanism. Methods: After the treatment of metformin alone or in combination with daunorubicin, MTT assay and flow cytometry were used to detect the proliferation and apoptosis of NB4 cells, respectively. The activation of apoptosis-related proteins caspase-3 and caspase-9 was determined by Western blotting. Results: Metformin inhibited proliferation of NB4 cells in a dose-dependent (r=0.952, P<0.01) and time-dependent (r=0.967, P<0.01) manner. After metformin treatment for 72 h, its IC50 value to NB4 cells was (6.39±0.37) mmol/L. Metformin increased the chemosensitivity of NB4 cells to daunorubicin. The inhibitory rate of NB4 cell proliferation in 0.625 mmol/L metformin and 0.02 μmol/L daunorubicin combination group was significantly higher than that in daunorubicin used alone group (\[29.84±0.21\]% vs \[10.68±0.45\]%, P<0.05). After the treatment of 5 mmol/L metformin for 48 h, the apoptotic rate of NB4 cells was significantly increased compared with that of the control group (\[43.95±0.29\]% vs \[7.12±0.29\]%，P<0.01). The expressions of activated caspase-3 and caspase-9 fragments were up-regulated after metformin treatment.Conclusion: Metformin can efficiently inhibit the proliferation, promote the apoptosis of NB4 cells, and enhance the chemosensitivity of NB4 cells to daunorubicin. Caspase-3 and caspase-9 may be involved in the NB4 cell apoptosis induced by metformin.
2013, 20(5):580-585.
DOI: 10.3872/j.issn.1007-385X.2013.05.013
Abstract:
Objective: To explore whether norcantharidin (NCTD) can enhance the cytotoxicity of IL-15 activated peripheral blood mononuclear cells (PBMCs) on human acute myeloblastic leukemic KG1a cells and its underlying mechanism. Methods: The effect of NCTD on the proliferation of KG1a cells was detected by typan blue assay and CCK-8 assay. The effect of NCTD on the cell cycle of KG1a cells was examined by flow cytometry. The cytotoxicity of IL-15 activated PBMCs (IL-15-PBMCs) against NCTD treated-KG1a cells was detected by LDH releasing assay. The expressions of NKG2D (natural killer group 2 member D) ligands on KG1a cells were detected by flow cytometry. Results: NCTD effectively inhibited the proliferation of leukemic KG1a cells, in a time- (r=0.398，P=0.000) and dose-dependent manner (r=0.861，P=0.000), and arrested KG1a cell cycle at G2/M phase. NCTD within a concentration of 4.00 μg/ml has no obvious cytotoxicity on the IL-15 activated PBMCs (IL-15-PBMCs) (P>0.05). Compared with the control group, the cytotoxic rate of IL-15-PBMCs on 0.125 μg/ml NCTD treated-KG1a cells was significantly increased (donor A: \[37.44±5.78\]% vs \[9.33±1.69\]%, \[38.33±3.07\]% vs \[16.75±1.20\]%, P<0.05). NCTD treatment showed no effect on expressions level of NKG2D ligands on KG1a cell surface (P>0.05). Conclusion: NCTD can enhance the cytotoxicity of IL-15-PBMCs on leukemic KG1a cells, which is possibly related to the inhibition of proliferation of KG1a cells and cell cycle arrest in G2/M phase.
Objective: To explore whether norcantharidin (NCTD) can enhance the cytotoxicity of IL-15 activated peripheral blood mononuclear cells (PBMCs) on human acute myeloblastic leukemic KG1a cells and its underlying mechanism. Methods: The effect of NCTD on the proliferation of KG1a cells was detected by typan blue assay and CCK-8 assay. The effect of NCTD on the cell cycle of KG1a cells was examined by flow cytometry. The cytotoxicity of IL-15 activated PBMCs (IL-15-PBMCs) against NCTD treated-KG1a cells was detected by LDH releasing assay. The expressions of NKG2D (natural killer group 2 member D) ligands on KG1a cells were detected by flow cytometry. Results: NCTD effectively inhibited the proliferation of leukemic KG1a cells, in a time- (r=0.398，P=0.000) and dose-dependent manner (r=0.861，P=0.000), and arrested KG1a cell cycle at G2/M phase. NCTD within a concentration of 4.00 μg/ml has no obvious cytotoxicity on the IL-15 activated PBMCs (IL-15-PBMCs) (P>0.05). Compared with the control group, the cytotoxic rate of IL-15-PBMCs on 0.125 μg/ml NCTD treated-KG1a cells was significantly increased (donor A: \[37.44±5.78\]% vs \[9.33±1.69\]%, \[38.33±3.07\]% vs \[16.75±1.20\]%, P<0.05). NCTD treatment showed no effect on expressions level of NKG2D ligands on KG1a cell surface (P>0.05). Conclusion: NCTD can enhance the cytotoxicity of IL-15-PBMCs on leukemic KG1a cells, which is possibly related to the inhibition of proliferation of KG1a cells and cell cycle arrest in G2/M phase.
2013, 20(5):586-589.
DOI: 10.3872/j.issn.1007-385X.2013.05.014
Abstract:
Objective : To explore the relationship of K-Ras and P53 gene mutation with development and postoperative recurrence of hysteromyoma. Methods: Fifty-six patients with hysteromyoma undergoing myomectomy were selected from First People Hospital of Xining during June 2008 to June 2010. The hysteromyoma tissues and normal uterus tissues were obtained from patients. K-Ras and P53 gene mutations were analyzed by polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP) and gene sequencing. The postoperative recurrence rate after uterine fibroid surgery in patients with K-Ras and P53 gene mutation was analyzed. Results: K-Ras gene mutation was focused on exon 1 and 2. P53 gene mutation was focused on exon 7 and 8. The mutation rate of K-Ras and P53 and the double mutation rate in the hysteromyoma tissues were significantly increased when compared with those in the normal uterus tissues (73.21% vs 7.14%, 8393% vs 10.71%, 32.14% vs 1.79%, P<0.05). Postoperative 3-year cumulative recurrence rates were 14.28% (6/42) and 8.51% (4/47) in the hysteromyoma patients with K-Ras or P53 mutation. Postoperative 3-year cumulative recurrence rate in the patients with double mutation was 66.67% (12/18), which was significantly higher than that in patients with single mutation (P<0.05). Conclusion: K-Ras and P53 gene mutations may be one of the main reasons leading to the occurrence and postoperative recurrence of hysteromyoma, which can be indicators for clinical diagnosis and prognosis.
Objective : To explore the relationship of K-Ras and P53 gene mutation with development and postoperative recurrence of hysteromyoma. Methods: Fifty-six patients with hysteromyoma undergoing myomectomy were selected from First People Hospital of Xining during June 2008 to June 2010. The hysteromyoma tissues and normal uterus tissues were obtained from patients. K-Ras and P53 gene mutations were analyzed by polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP) and gene sequencing. The postoperative recurrence rate after uterine fibroid surgery in patients with K-Ras and P53 gene mutation was analyzed. Results: K-Ras gene mutation was focused on exon 1 and 2. P53 gene mutation was focused on exon 7 and 8. The mutation rate of K-Ras and P53 and the double mutation rate in the hysteromyoma tissues were significantly increased when compared with those in the normal uterus tissues (73.21% vs 7.14%, 8393% vs 10.71%, 32.14% vs 1.79%, P<0.05). Postoperative 3-year cumulative recurrence rates were 14.28% (6/42) and 8.51% (4/47) in the hysteromyoma patients with K-Ras or P53 mutation. Postoperative 3-year cumulative recurrence rate in the patients with double mutation was 66.67% (12/18), which was significantly higher than that in patients with single mutation (P<0.05). Conclusion: K-Ras and P53 gene mutations may be one of the main reasons leading to the occurrence and postoperative recurrence of hysteromyoma, which can be indicators for clinical diagnosis and prognosis.
15 High methylation level of CpG island in HHIP gene participates in the occurrence of gastric cancer
2013, 20(5):590-596.
DOI: 10.3872/j.issn.1007-385X.2013.05.015
Abstract:
Objective : To Observe the methylation level of CpG island in human hedgehog interacting protein (HHIP) gene in gastric cancer tissues, peritumoral tissues and AGS cells, and to explore the relationship between the methylation level of CpG island and the tumorigenesis of gastric cancer. Methods: The expressions of HHIP mRNA in 30 human gastric carcinnoma tissues, peritumoral tissues and gastric carcinoma AGS cells were detected by RT-PCR. The expressions of HHIP and the methylation level of promoter region of HHIP gene in gastric carcinnoma tissues and peritumoral tissues were detected by immunohistochemical staining and methylation specific PCR (MSP), respectively. Before and after the treatment of methyl transferase inhibitor 5-Aza-2′-deoxycitydine (5-Aza-dc), the HHIP mRNA expression, methylation level of promoter region and methylation sites locus on CpG island in AGS cells were detected by RT-PCR, MSP and bisulfite sequencing PCR(BSP), respectively. The correlation between the methylation status of CpG island at HHIP promoter region and HHIP mRNA expression level was analyzed. Results: The expression of HHIP mRNA (0.82±0.38 vs 1.60±0.26, P=0.000) and HHIP (0.5±0.03 vs 0.8±0.27, P<0.05) in gastric carcinoma was significantly lower than that in the peritumoral tissues. No significant correlation was observed between the expression of HHIP mRNA or protein and age, sex, TNM stage, differentiation degree and lymph node metastasis (P>0.05). The methylation level of HHIP gene promtor in the peritumoral tissues was significantly higher than that in the gastric cancer tissues and AGS cells (\[17.7±359\]% vs \[62.9±6.14\]%, \[99.7±0.67\]%; all P<0.05). Compared with that before 5-Aza-dc treatment, the HHIP mRNA expression in AGS cells was significantly increased after treatment (4.68±0.22 vs 0.21±0.12, P<001), while the methylation level of AGS cells decreased significantly (\[10.1±0.21\]% vs \[90.2±0.67\]%, P<001), and the methylation sites in CpG islands were significantly reduced. The degree of HHIP methylation showed a negative correlation with the mRNA expression(r=-0.693, P=0.00). Conclusion: High methylation level of CpG island in HHIP gene promoter may increase the expression of HHIP gene, and then be involved in the carcinogenesis of gastric cancer.
Objective : To Observe the methylation level of CpG island in human hedgehog interacting protein (HHIP) gene in gastric cancer tissues, peritumoral tissues and AGS cells, and to explore the relationship between the methylation level of CpG island and the tumorigenesis of gastric cancer. Methods: The expressions of HHIP mRNA in 30 human gastric carcinnoma tissues, peritumoral tissues and gastric carcinoma AGS cells were detected by RT-PCR. The expressions of HHIP and the methylation level of promoter region of HHIP gene in gastric carcinnoma tissues and peritumoral tissues were detected by immunohistochemical staining and methylation specific PCR (MSP), respectively. Before and after the treatment of methyl transferase inhibitor 5-Aza-2′-deoxycitydine (5-Aza-dc), the HHIP mRNA expression, methylation level of promoter region and methylation sites locus on CpG island in AGS cells were detected by RT-PCR, MSP and bisulfite sequencing PCR(BSP), respectively. The correlation between the methylation status of CpG island at HHIP promoter region and HHIP mRNA expression level was analyzed. Results: The expression of HHIP mRNA (0.82±0.38 vs 1.60±0.26, P=0.000) and HHIP (0.5±0.03 vs 0.8±0.27, P<0.05) in gastric carcinoma was significantly lower than that in the peritumoral tissues. No significant correlation was observed between the expression of HHIP mRNA or protein and age, sex, TNM stage, differentiation degree and lymph node metastasis (P>0.05). The methylation level of HHIP gene promtor in the peritumoral tissues was significantly higher than that in the gastric cancer tissues and AGS cells (\[17.7±359\]% vs \[62.9±6.14\]%, \[99.7±0.67\]%; all P<0.05). Compared with that before 5-Aza-dc treatment, the HHIP mRNA expression in AGS cells was significantly increased after treatment (4.68±0.22 vs 0.21±0.12, P<001), while the methylation level of AGS cells decreased significantly (\[10.1±0.21\]% vs \[90.2±0.67\]%, P<001), and the methylation sites in CpG islands were significantly reduced. The degree of HHIP methylation showed a negative correlation with the mRNA expression(r=-0.693, P=0.00). Conclusion: High methylation level of CpG island in HHIP gene promoter may increase the expression of HHIP gene, and then be involved in the carcinogenesis of gastric cancer.
Yang Xiaogang
,
Sang Meixiang
,
Fan Xiaojie
,
Zhou Xinliang
,
Ding Chunyan
,
Chen Ping
,
He Cuiying
,
Shan Baoen
2013, 20(5):597-602.
DOI: 10.3872/j.issn.1007-385X.2013.05.016
Abstract:
Objective : To investigate the expressions of melanoma-associated antigen (MAGE)-A4 and tumor suppressor gene P73 in breast cancer tissues and paracancerous tissues and their clinical significance. Methods: Sixty patients with breast cancer from September 2007 to December 2007 in Fourth Hospital of Hebei Medical University were enrolled in the study. The expressions of MAGE-A4 and P73 proteins in breast cancer tissues and paracancerous tissues were detected by immunohistochemisty assay, and their correlations with the clinicopathological features of breast cancer were analyzed. Results: The positive expression rates of MAGE-A4 and P73 in breast cancer tissues were 63.3% (38/60) and 43.3% (26/60), respectively, while in adjacent tumor tissues were 0 (0/60) and 85% (51/60), respectively (P<001). In the breast cancer tissues, the expression of MAGE-A4 protein was positively correlated with P73 (r=0.316, P=0.014). MAGE-A4 protein expression was not correlated with patients’ age, pathological type, histological grade, clinical stage, tumor size, metastatic state of lymph node, estrogen receptor (ER), as well as progestrogen receptor (PR) status. However, MAGE-A4 protein expression was negatively correlated with expression of C-erbB2 protein (r=-0.259, P=0046). P73 protein expression was not correlated with patients’ age, pathological type, histological grade, clinical stage, tumor size, metastatic state of lymph node, and C-erbB2 expression. Whereas, the expression of P73 protein was positively correlated with the expression of ER (r=0.274, P=0.034) and PR (r=0.262, P=0.043). Conclusion: Breast cancer tissues highly express MAGE-A4 and P73 protein, and there exists an important regulatory mechanism between MAGE-A4 and P73 protein.
Objective : To investigate the expressions of melanoma-associated antigen (MAGE)-A4 and tumor suppressor gene P73 in breast cancer tissues and paracancerous tissues and their clinical significance. Methods: Sixty patients with breast cancer from September 2007 to December 2007 in Fourth Hospital of Hebei Medical University were enrolled in the study. The expressions of MAGE-A4 and P73 proteins in breast cancer tissues and paracancerous tissues were detected by immunohistochemisty assay, and their correlations with the clinicopathological features of breast cancer were analyzed. Results: The positive expression rates of MAGE-A4 and P73 in breast cancer tissues were 63.3% (38/60) and 43.3% (26/60), respectively, while in adjacent tumor tissues were 0 (0/60) and 85% (51/60), respectively (P<001). In the breast cancer tissues, the expression of MAGE-A4 protein was positively correlated with P73 (r=0.316, P=0.014). MAGE-A4 protein expression was not correlated with patients’ age, pathological type, histological grade, clinical stage, tumor size, metastatic state of lymph node, estrogen receptor (ER), as well as progestrogen receptor (PR) status. However, MAGE-A4 protein expression was negatively correlated with expression of C-erbB2 protein (r=-0.259, P=0046). P73 protein expression was not correlated with patients’ age, pathological type, histological grade, clinical stage, tumor size, metastatic state of lymph node, and C-erbB2 expression. Whereas, the expression of P73 protein was positively correlated with the expression of ER (r=0.274, P=0.034) and PR (r=0.262, P=0.043). Conclusion: Breast cancer tissues highly express MAGE-A4 and P73 protein, and there exists an important regulatory mechanism between MAGE-A4 and P73 protein.
2013, 20(5):609-617.
DOI: 10.3872/j.issn.1007-385X.2013.05.018
Abstract:
树突状细胞（dendritic cell，DC）免疫治疗作为一种无严重毒性反应、前景广阔的主动免疫治疗策略，自从1998年以来，已经在转移性肾细胞癌（metastatic renal cell carcinoma，mRCC）的治疗中进行了广泛研究，并在2007年5月韩国FDA批准了一种称为CreaVax RCC的自体DC疫苗用于mRCC的治疗。目前已经有31项非随机对照的Ⅰ/Ⅱ期临床试验研究了DC免疫治疗用于mRCC患者的安全性和有效性，虽然这些试验结果已经证实DC免疫治疗的安全性和对一小部分mRCC患者的有效性，但这些临床试验在受试者选择、DC疫苗的标准化、免疫学终点和临床终点的选择和评价等方面难以统一标准，这导致DC免疫治疗疗效的发挥受限且不同试验结果无法进行直接比较；另外，所开展的试验均为非随机对照小样本临床研究。因此，期待临床试验设计方案的进一步优化，以客观评估DC免疫治疗对mRCC患者的临床治疗效果，并应在临床试验中加强对DC免疫治疗联合mRCC一线治疗方案疗效的评价，以进一步开发对mRCC更加有效的联合治疗策略。
树突状细胞（dendritic cell，DC）免疫治疗作为一种无严重毒性反应、前景广阔的主动免疫治疗策略，自从1998年以来，已经在转移性肾细胞癌（metastatic renal cell carcinoma，mRCC）的治疗中进行了广泛研究，并在2007年5月韩国FDA批准了一种称为CreaVax RCC的自体DC疫苗用于mRCC的治疗。目前已经有31项非随机对照的Ⅰ/Ⅱ期临床试验研究了DC免疫治疗用于mRCC患者的安全性和有效性，虽然这些试验结果已经证实DC免疫治疗的安全性和对一小部分mRCC患者的有效性，但这些临床试验在受试者选择、DC疫苗的标准化、免疫学终点和临床终点的选择和评价等方面难以统一标准，这导致DC免疫治疗疗效的发挥受限且不同试验结果无法进行直接比较；另外，所开展的试验均为非随机对照小样本临床研究。因此，期待临床试验设计方案的进一步优化，以客观评估DC免疫治疗对mRCC患者的临床治疗效果，并应在临床试验中加强对DC免疫治疗联合mRCC一线治疗方案疗效的评价，以进一步开发对mRCC更加有效的联合治疗策略。
2013, 20(5):618-622.
DOI: 10.3872/j.issn.1007-385X.2013.05.019
Abstract:
DC是目前已知人体内功能最强的免疫提呈细胞（antigen presenting cell, APC），成熟的DC通过诱导细胞免疫、参与体液免疫以及分泌多种细胞因子等多种途径，在抗原的捕获、加工、提呈和激活T细胞产生抗肿瘤免疫效应中发挥重要作用。恶性肿瘤患者往往存在一定的DC功能缺陷，基因修饰的DC可为机体免疫系统提供多个可供识别的抗原表位，是加强抗肿瘤免疫的重要手段之一。基因修饰的DC疫苗种类较多，如肿瘤相关抗原基因、细胞因子基因、共刺激分子基因、黏附分子基因、免疫调控分子基因、凋亡相关基因、癌基因以及多种基因共同修饰的DC疫苗等，这些经基因修饰后的DC疫苗在抗肿瘤免疫的基础研究方面已经证实了其免疫增强作用，而且部分DC疫苗在临床试验中也取得了一定的抗肿瘤疗效，这些都将为基因修饰的DC在临床中的应用提供重要的理论基础。本文将从DC的生物学特征、DC的抗肿瘤机制以及基因修饰的DC疫苗在抗肿瘤免疫中的基础研究和相关临床试验等几个方面做一综述。
DC是目前已知人体内功能最强的免疫提呈细胞（antigen presenting cell, APC），成熟的DC通过诱导细胞免疫、参与体液免疫以及分泌多种细胞因子等多种途径，在抗原的捕获、加工、提呈和激活T细胞产生抗肿瘤免疫效应中发挥重要作用。恶性肿瘤患者往往存在一定的DC功能缺陷，基因修饰的DC可为机体免疫系统提供多个可供识别的抗原表位，是加强抗肿瘤免疫的重要手段之一。基因修饰的DC疫苗种类较多，如肿瘤相关抗原基因、细胞因子基因、共刺激分子基因、黏附分子基因、免疫调控分子基因、凋亡相关基因、癌基因以及多种基因共同修饰的DC疫苗等，这些经基因修饰后的DC疫苗在抗肿瘤免疫的基础研究方面已经证实了其免疫增强作用，而且部分DC疫苗在临床试验中也取得了一定的抗肿瘤疗效，这些都将为基因修饰的DC在临床中的应用提供重要的理论基础。本文将从DC的生物学特征、DC的抗肿瘤机制以及基因修饰的DC疫苗在抗肿瘤免疫中的基础研究和相关临床试验等几个方面做一综述。
2013, 20(5):623-628.
DOI: 10.3872/j.issn.1007-385X.2013.05.020
Abstract:
Shp2（SH2 domain-containing protein-tyrosine phosphatase-2）分子由 PTPN11 基因编码，是一个在体内广泛存在的非受体型蛋白酪氨酸磷酸酶，既可以通过磷酸酶的催化活性来正向调控下游信号转导通路，也可以作为磷酸酶非依赖性的接头蛋白发挥正向调控作用，在特定的条件下亦可发挥负向调控作用，从而广泛参与细胞的分化、迁移等生物学功能的调控及相关的信号转导过程。 PTPN11 突变被认为是青少年粒单细胞白血病（juvenile myelomonocytic leukemia，JMML）的高危因素，同时，因其在不同类型白血病中均存在着 Shp2 的异常活化和突变而被认为是白血病的原癌基因；在前列腺癌、乳腺癌、胰腺癌、胃癌和神经胶质瘤中，Shp2也被报道呈过度活化状态；在肺癌中 Shp2 作为癌基因通过调控多种机制促进肿瘤的发生、发展。但在肝癌发生过程中， Shp2 却在特定环境的影响下发挥抑癌基因的作用。总之，作为重要的节点分子，Shp2在肿瘤发生、发展的过程中发挥着重要的调控作用，是潜在的治疗靶点。
Shp2（SH2 domain-containing protein-tyrosine phosphatase-2）分子由 PTPN11 基因编码，是一个在体内广泛存在的非受体型蛋白酪氨酸磷酸酶，既可以通过磷酸酶的催化活性来正向调控下游信号转导通路，也可以作为磷酸酶非依赖性的接头蛋白发挥正向调控作用，在特定的条件下亦可发挥负向调控作用，从而广泛参与细胞的分化、迁移等生物学功能的调控及相关的信号转导过程。 PTPN11 突变被认为是青少年粒单细胞白血病（juvenile myelomonocytic leukemia，JMML）的高危因素，同时，因其在不同类型白血病中均存在着 Shp2 的异常活化和突变而被认为是白血病的原癌基因；在前列腺癌、乳腺癌、胰腺癌、胃癌和神经胶质瘤中，Shp2也被报道呈过度活化状态；在肺癌中 Shp2 作为癌基因通过调控多种机制促进肿瘤的发生、发展。但在肝癌发生过程中， Shp2 却在特定环境的影响下发挥抑癌基因的作用。总之，作为重要的节点分子，Shp2在肿瘤发生、发展的过程中发挥着重要的调控作用，是潜在的治疗靶点。
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.