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Volume 14,Issue 2,2007 Table of Contents
2007, 14(2):101-104.
DOI: 10.3872/j.issn.1007-385X.2007.2.001
Abstract:
2 EGFR mutations and targeted therapy with tyrosine kinases inhibitors in non-small cell lung cancer
2007, 14(2):105-109.
DOI: 10.3872/j.issn.1007-385X.2007.2.002
Abstract:
肺癌的高发病率和高病死率，以及化疗对晚期肺癌疗效的十分有限，使之成为世界性医学难题。最近发现，表皮生长因子受体（epidermal growth factor receptor,EGFR）酪氨酸激酶抑制剂（tyrosine kinases inhibitors,TKIs）的分子靶向治疗可以使晚期非小细胞肺癌瘤体缩小；然而，以Gefitinib和Erlotinib为代表的TKIs治疗敏感性与EGFR基因突变显著相关。研究证实，EGFR最常见发生的19区缺失突变和21区点突变导致相应氨基酸序列和EGFR结构的改变，是其增加药物敏感性的主要机制。此外，EGFR基因扩增和CA重复序列的多态性、EGFR通路下游信号（如p-AKT等）激活、HER2和（或）HER3表达的增加、K-RAS基因突变等因素皆影响其对TKIs的治疗敏感性。鉴于此，进一步研究EGFR基因不同突变的功能，寻找能预测TKIs治疗敏感性的因素，并作为TKIs敏感患者的筛选指标，对于提高晚期肺癌患者TKIs靶向治疗疗效具有重要意义。
肺癌的高发病率和高病死率，以及化疗对晚期肺癌疗效的十分有限，使之成为世界性医学难题。最近发现，表皮生长因子受体（epidermal growth factor receptor,EGFR）酪氨酸激酶抑制剂（tyrosine kinases inhibitors,TKIs）的分子靶向治疗可以使晚期非小细胞肺癌瘤体缩小；然而，以Gefitinib和Erlotinib为代表的TKIs治疗敏感性与EGFR基因突变显著相关。研究证实，EGFR最常见发生的19区缺失突变和21区点突变导致相应氨基酸序列和EGFR结构的改变，是其增加药物敏感性的主要机制。此外，EGFR基因扩增和CA重复序列的多态性、EGFR通路下游信号（如p-AKT等）激活、HER2和（或）HER3表达的增加、K-RAS基因突变等因素皆影响其对TKIs的治疗敏感性。鉴于此，进一步研究EGFR基因不同突变的功能，寻找能预测TKIs治疗敏感性的因素，并作为TKIs敏感患者的筛选指标，对于提高晚期肺癌患者TKIs靶向治疗疗效具有重要意义。
2007, 14(2):110-114.
DOI: 10.3872/j.issn.1007-385X.2007.2.003
Abstract:
To identify a functional monoclonal antibody 1E2 against lung cancer and its antigen, so as to provide a candidate antibody drug and molecule target for the anti-lung cancer therapy. Methods: The inhibition of the lung cancer cells, binding with tumor cells, isotypes, and inhibition of implanted tumor growth of 1E2 monoclonal antibody were studied by immunofluorescence, ELISA, cell proliferation assay, and in vivo tumor treatment experiments. The corresponding antigen of 1E2 was identified by Western blotting and MALDI-TOF mass spectrometry. Results: The 1E2 antibody could recognize cell surface protein of lung cancer cell line GLC82 and HCI-H520 and obviously inhibited the lung cancer cell growth in vivo. Animal experiment revealed that 1E2 antibody significantly inhibited lung tumor growth by 49%. Western blotting revealed that the molecular weight of the antigen of 1E2 antibody was about 110 000. Mass fingerprint revealed this protein was carbamoyl-phosphate synthetase 1(CPS1). Conclusion: 1E2 monoclonal antibody can suppress lung tumor growth in vitro and in vivo; it might become a candidate drug for targeted lung cancer treatment. CPS1, the antigen of 1E2, locates on the membrane of lung cancer cells and it may become a novel molecule target for lung caner therapy.
To identify a functional monoclonal antibody 1E2 against lung cancer and its antigen, so as to provide a candidate antibody drug and molecule target for the anti-lung cancer therapy. Methods: The inhibition of the lung cancer cells, binding with tumor cells, isotypes, and inhibition of implanted tumor growth of 1E2 monoclonal antibody were studied by immunofluorescence, ELISA, cell proliferation assay, and in vivo tumor treatment experiments. The corresponding antigen of 1E2 was identified by Western blotting and MALDI-TOF mass spectrometry. Results: The 1E2 antibody could recognize cell surface protein of lung cancer cell line GLC82 and HCI-H520 and obviously inhibited the lung cancer cell growth in vivo. Animal experiment revealed that 1E2 antibody significantly inhibited lung tumor growth by 49%. Western blotting revealed that the molecular weight of the antigen of 1E2 antibody was about 110 000. Mass fingerprint revealed this protein was carbamoyl-phosphate synthetase 1(CPS1). Conclusion: 1E2 monoclonal antibody can suppress lung tumor growth in vitro and in vivo; it might become a candidate drug for targeted lung cancer treatment. CPS1, the antigen of 1E2, locates on the membrane of lung cancer cells and it may become a novel molecule target for lung caner therapy.
2007, 14(2):115-119.
DOI: 10.3872/j.issn.1007-385X.2007.2.004
Abstract:
To investigate the effects of recombinant adiponectin (RA) on the invasive ability of human breast cancer cell line MDAMB231. Methods: MDAMB231 cells were divided into control group and RA(2.5, 5.0, 10, 20, and 30 μg/ml) treatment groups. The effect of RA on proliferation of MDAMB231 cells was measured by MTT assay; the invasive and migration abilities of MDAMB231 cell were assayed in Transwell cell culture chamber; cell adhesion assay was carried out in a microculture well precoated with fibronectin．Gelatinolytic activities of both MMP2 and MMP9 secreted by cancer cells were measured by zymogrophy analysis. Results: The growth and invasive ability of MDAMB231 cells were obviously inhibited when the concentration of RA was higher than 5.0 μg/ml (P<0.01); the inhibition of migration was in a concentrationdependent manner, with the inhibitory rate ranging from 22.64％77.84％. When the concentration of RA was higher than 2.5 μg/ml, the migration of tumor and the secretion of MMP2 and MMP9 were obviously inhibited (P<0.05,P<0.01); the secretion of MMP9 decreased with the increase of RA concentration. RA was more sensitive to FN and than to ECM. Conclusion: These results indicate that adiponectin is effective in suppressing tumor cell invasion when its concentration is >2.5 μg/ml; the mechanism might be the downregulation of MMPs.
To investigate the effects of recombinant adiponectin (RA) on the invasive ability of human breast cancer cell line MDAMB231. Methods: MDAMB231 cells were divided into control group and RA(2.5, 5.0, 10, 20, and 30 μg/ml) treatment groups. The effect of RA on proliferation of MDAMB231 cells was measured by MTT assay; the invasive and migration abilities of MDAMB231 cell were assayed in Transwell cell culture chamber; cell adhesion assay was carried out in a microculture well precoated with fibronectin．Gelatinolytic activities of both MMP2 and MMP9 secreted by cancer cells were measured by zymogrophy analysis. Results: The growth and invasive ability of MDAMB231 cells were obviously inhibited when the concentration of RA was higher than 5.0 μg/ml (P<0.01); the inhibition of migration was in a concentrationdependent manner, with the inhibitory rate ranging from 22.64％77.84％. When the concentration of RA was higher than 2.5 μg/ml, the migration of tumor and the secretion of MMP2 and MMP9 were obviously inhibited (P<0.05,P<0.01); the secretion of MMP9 decreased with the increase of RA concentration. RA was more sensitive to FN and than to ECM. Conclusion: These results indicate that adiponectin is effective in suppressing tumor cell invasion when its concentration is >2.5 μg/ml; the mechanism might be the downregulation of MMPs.
2007, 14(2):120-125.
DOI: 10.3872/j.issn.1007-385X.2007.2.005
Abstract:
To investigate the targeting and antitumor ability of the tumor vesselspecific antibody ScFvH1 selected from phageScFv library, and to discuss the application of the antibody in clinical diagnosis and therapy of cancer. Methods: The ScFvH1 gene was inserted into pET28a(+)/EGFP vector containing green fluorescent protein(GFP) gene and pTIGTrx vector containing thioredoxin gene; the products were then expressed in E.coli and purified by using NiNTA. Tumorbearing mice model was established by subcutanuous injection of cervical cancer cell line HeLa. The mice were injected with purified ScFvEGFP fusion protein through vena caudalis and the GFP signals were observed by fluorescent microscope to evaluate the targeting ability of the antibody. Meanwhile, the mice model also received intratumoral injection of purified ScFvEGFP fusion protein to evaluate the antitumor effect of the antibody. Results: Soluble ScFvH1 gene and ScFvH1EGFP protein were successfully expressed in E.coli; a single band was showed in SDSPAGE after the purification by NiNTA. We found that ScFvH1EGFP fusion protein was enriched to tumor tissues, but there was only weak fluorescent signal when EGFP protein was injected. No EGFP signal was observed in the lung of tumorbearing mice. Tumor inhibition experiment showed that the tumor growth in the antibody treatment group was similar to that of the PBS control group. Conclusion: The tumor vesselspecific antibody ScFvH1 selected from phageScFv library can specifically target tumor vessels, but it has no obvious inhibitory effect on tumor growth. Our findings pave a way for antibody in cancer diagnosis and treatment.
To investigate the targeting and antitumor ability of the tumor vesselspecific antibody ScFvH1 selected from phageScFv library, and to discuss the application of the antibody in clinical diagnosis and therapy of cancer. Methods: The ScFvH1 gene was inserted into pET28a(+)/EGFP vector containing green fluorescent protein(GFP) gene and pTIGTrx vector containing thioredoxin gene; the products were then expressed in E.coli and purified by using NiNTA. Tumorbearing mice model was established by subcutanuous injection of cervical cancer cell line HeLa. The mice were injected with purified ScFvEGFP fusion protein through vena caudalis and the GFP signals were observed by fluorescent microscope to evaluate the targeting ability of the antibody. Meanwhile, the mice model also received intratumoral injection of purified ScFvEGFP fusion protein to evaluate the antitumor effect of the antibody. Results: Soluble ScFvH1 gene and ScFvH1EGFP protein were successfully expressed in E.coli; a single band was showed in SDSPAGE after the purification by NiNTA. We found that ScFvH1EGFP fusion protein was enriched to tumor tissues, but there was only weak fluorescent signal when EGFP protein was injected. No EGFP signal was observed in the lung of tumorbearing mice. Tumor inhibition experiment showed that the tumor growth in the antibody treatment group was similar to that of the PBS control group. Conclusion: The tumor vesselspecific antibody ScFvH1 selected from phageScFv library can specifically target tumor vessels, but it has no obvious inhibitory effect on tumor growth. Our findings pave a way for antibody in cancer diagnosis and treatment.
2007, 14(2):126-131.
DOI: 10.3872/j.issn.1007-385X.2007.2.006
Abstract:
To explore the therapeutic efficacy and mechanism of shRNA in treatment of subcutaneously implanted SHSY5Y cells in nude mice by interfering human etherαgogorelated gene （HERG）potassium channel expression. Methods: Eukaryotic vector mU6pro was used to construct herg genetargeting small hairpin interfering RNA plasmid: shRNAherg. Eighteen BALB/c nude mice were inoculated with SHSY5Y cells subcutaneously to establish neuroblastoma model. When the implanted tumors grew to about 100 mm3, the mice were divided into randomly 3 groups: Intratumor injection of normal saline (group Ⅰ), shRNAcontrol plasmid (group Ⅱ), and shRNAherg plasmid (group Ⅲ). The injections were given twice at an interval of 7 d after the first injection. The tumor growth was observed; the expression of mRNA and protein of HERG potassium was determined by RTPCR and the immunohistochemical method, respectively. Results: The tumor growth was obviously inhibited in group Ⅲ; the inhibitory rates in groupⅡand group Ⅲ were 3.04% and 29.78%, respectively (P< 0.05). Compared with groupⅠandⅡ, group Ⅲ had lower levels of HERG mRNA and protein after treatment. Conclusion: The constructed shRNA can depress the growth of neuroblastoma by specifically silencing the expression of HERG potassium protein.
To explore the therapeutic efficacy and mechanism of shRNA in treatment of subcutaneously implanted SHSY5Y cells in nude mice by interfering human etherαgogorelated gene （HERG）potassium channel expression. Methods: Eukaryotic vector mU6pro was used to construct herg genetargeting small hairpin interfering RNA plasmid: shRNAherg. Eighteen BALB/c nude mice were inoculated with SHSY5Y cells subcutaneously to establish neuroblastoma model. When the implanted tumors grew to about 100 mm3, the mice were divided into randomly 3 groups: Intratumor injection of normal saline (group Ⅰ), shRNAcontrol plasmid (group Ⅱ), and shRNAherg plasmid (group Ⅲ). The injections were given twice at an interval of 7 d after the first injection. The tumor growth was observed; the expression of mRNA and protein of HERG potassium was determined by RTPCR and the immunohistochemical method, respectively. Results: The tumor growth was obviously inhibited in group Ⅲ; the inhibitory rates in groupⅡand group Ⅲ were 3.04% and 29.78%, respectively (P< 0.05). Compared with groupⅠandⅡ, group Ⅲ had lower levels of HERG mRNA and protein after treatment. Conclusion: The constructed shRNA can depress the growth of neuroblastoma by specifically silencing the expression of HERG potassium protein.
WANG Zhenfa
,
WANG Lie
,
WANG Yu
,
CHEN Shaoquan
,
LIN Chen
,
WEI Lixin
,
QIN Linhua
,
ZHANG Changsong
2007, 14(2):132-136.
DOI: 10.3872/j.issn.1007-385X.2007.2.007
Abstract:
To establish a rat breast cancer cell line stably coexpressing mouse MIP1α and B71，and to assay its in vitro biological activity．Methods: mMIP1αcDNA was cloned into retrovirus vector pBabe puro to construct pBahe puro/mMIP1α, then pBabe puro/mMIP1α was used to transfect packaging cells and the antipuromycin PA317 packaging cells were proliferated. Meanwhile, pLXSN/mB71 was constructed and the antiG418 cells were proliferated. Finally, the two supernatants were used to infect SHZ88 together and the cotransfected cells were selected with 1 mg/ml G418 and 2 μg/ml puromycin together. Expression of mM1Plα mRNA and protein in SHZ88 and SHZ88/mB71+mM1P1α cells were analyzed by RTPCR and immunocytochemistry, respectively. Expression of mB71mRNA and protein was analyzed by RTPCR and flow cytometry, respectively. Lymphocyte proliferation activity of SHZ88/B71+mM1P1α was detected by MTT assay; chemotactic activity of MIP1α was measured by chemotaxis assay. Results：A titer of 4.6×107 CFU/L was obtained after transfection with recombinant retroviral vector. The growth curve of cells showed that the recombinant retroviral had no effect on the growth of rat breast cancer cells. There was expression of B71 and MIP1αmRNA/protein in SHZ88/mMIP1α+mB71 cells. The proliferation indices(PI) in mMIP1α+mB71 group (1.95±0.31) was significantly higher than that in SHZ88/PLXSN group (0.76±0.25)(P<0.01). Chemotaxis assay showed that chemotactic activity of lymphocytes in mMIP1α+mB71 group (3.88±0.33) was significantly higher than that in SHZ88/pBabe puro group (0.99±0.19)(P<0.001).Conclusion: A new rat breast cancer cel1 line SHZ88/mMIP1α+mB71 has been established, which can stably coexpress MIP1α and B71 gene and possesses biologic activity in vitro.
To establish a rat breast cancer cell line stably coexpressing mouse MIP1α and B71，and to assay its in vitro biological activity．Methods: mMIP1αcDNA was cloned into retrovirus vector pBabe puro to construct pBahe puro/mMIP1α, then pBabe puro/mMIP1α was used to transfect packaging cells and the antipuromycin PA317 packaging cells were proliferated. Meanwhile, pLXSN/mB71 was constructed and the antiG418 cells were proliferated. Finally, the two supernatants were used to infect SHZ88 together and the cotransfected cells were selected with 1 mg/ml G418 and 2 μg/ml puromycin together. Expression of mM1Plα mRNA and protein in SHZ88 and SHZ88/mB71+mM1P1α cells were analyzed by RTPCR and immunocytochemistry, respectively. Expression of mB71mRNA and protein was analyzed by RTPCR and flow cytometry, respectively. Lymphocyte proliferation activity of SHZ88/B71+mM1P1α was detected by MTT assay; chemotactic activity of MIP1α was measured by chemotaxis assay. Results：A titer of 4.6×107 CFU/L was obtained after transfection with recombinant retroviral vector. The growth curve of cells showed that the recombinant retroviral had no effect on the growth of rat breast cancer cells. There was expression of B71 and MIP1αmRNA/protein in SHZ88/mMIP1α+mB71 cells. The proliferation indices(PI) in mMIP1α+mB71 group (1.95±0.31) was significantly higher than that in SHZ88/PLXSN group (0.76±0.25)(P<0.01). Chemotaxis assay showed that chemotactic activity of lymphocytes in mMIP1α+mB71 group (3.88±0.33) was significantly higher than that in SHZ88/pBabe puro group (0.99±0.19)(P<0.001).Conclusion: A new rat breast cancer cel1 line SHZ88/mMIP1α+mB71 has been established, which can stably coexpress MIP1α and B71 gene and possesses biologic activity in vitro.
2007, 14(2):137-142.
DOI: 10.3872/j.issn.1007-385X.2007.2.008
Abstract:
To investigate the in vivo antitumor effects of Icarrin (ICA) on H22bearing mice and the related antitumor mechanisms. Methods: C57BL/6 mice were subcutaneously inoculated with H22 hepatocarcinoma cell line to establish H22bearing mice model. 24 h after the inoculation, H22bearing mice were treated with different doses of ICA (9.0 mg/ml, 4.5 mg/ml, and 2.25 mg/ml) and cyclophosphamide (30 mg/kg). When the tumors in H22bearing mice grew to 1 g, the mice were weighed, sacrificed and their spleens and tumors were harvested. The mice sera were also collected for determination of TNFα. Half of the spleens and tumors were made into singlecell suspensions and the other half was fixed by 10% formaldehyde solution for pathology analysis and TUNEL detection. Flow cytometry technique was used to detect the expression of surface antigens CD3, CD4, CD8, and DX5 in the spleen. Annexin V/PI double staining method was used to analyze the early and late apoptosis of tumor singlecell suspension. 1.0×106 cells were used to analyze the cell cycle of the solid tumor cells by flow cytometry. Results: The tumor inhibition rate of cyclophosphamide was 67.16%(P<0.001), and those of high, medium and lowdose ICA groups were 24.63% （P<0.05）, 32.84% （P<0.01）and 5.22%, respectively. Annexin V/PI double staining results indicated that the early apoptosis rate of tumor cells in cyclophosphamide group and mediumdose ICA group were higher than those in H22bearing group \[Cy (911±1.584)%, ICA (7.14±1.376)%, H22bearing group (3.6±2.38)%\]. TUNEL result showed that apoptosis index in ICAmedium group was significantly higher than that in H22bearing group\[(5.35±0.73)% vs (1.03±117)%, P<0.01\]. No TNFα was detected in normal control mice and only very low level of TNFα was found in ICA treatment group and Cy group. Pathology study showed that there were severe necrosis in tumors of medium and highdose ICA groups, with the necrosis found in the capsule and the margin of tumors. A small number of inflammatory cells (lymphocytes and monocytes) were observed in the capsule of tumor and tumor tissues.Conclusion: ICA can effectively inhibit tumor growth, which is probably through inducing early apoptosis of solid tumor cells and the subsequent tumor tissues necrosis.
To investigate the in vivo antitumor effects of Icarrin (ICA) on H22bearing mice and the related antitumor mechanisms. Methods: C57BL/6 mice were subcutaneously inoculated with H22 hepatocarcinoma cell line to establish H22bearing mice model. 24 h after the inoculation, H22bearing mice were treated with different doses of ICA (9.0 mg/ml, 4.5 mg/ml, and 2.25 mg/ml) and cyclophosphamide (30 mg/kg). When the tumors in H22bearing mice grew to 1 g, the mice were weighed, sacrificed and their spleens and tumors were harvested. The mice sera were also collected for determination of TNFα. Half of the spleens and tumors were made into singlecell suspensions and the other half was fixed by 10% formaldehyde solution for pathology analysis and TUNEL detection. Flow cytometry technique was used to detect the expression of surface antigens CD3, CD4, CD8, and DX5 in the spleen. Annexin V/PI double staining method was used to analyze the early and late apoptosis of tumor singlecell suspension. 1.0×106 cells were used to analyze the cell cycle of the solid tumor cells by flow cytometry. Results: The tumor inhibition rate of cyclophosphamide was 67.16%(P<0.001), and those of high, medium and lowdose ICA groups were 24.63% （P<0.05）, 32.84% （P<0.01）and 5.22%, respectively. Annexin V/PI double staining results indicated that the early apoptosis rate of tumor cells in cyclophosphamide group and mediumdose ICA group were higher than those in H22bearing group \[Cy (911±1.584)%, ICA (7.14±1.376)%, H22bearing group (3.6±2.38)%\]. TUNEL result showed that apoptosis index in ICAmedium group was significantly higher than that in H22bearing group\[(5.35±0.73)% vs (1.03±117)%, P<0.01\]. No TNFα was detected in normal control mice and only very low level of TNFα was found in ICA treatment group and Cy group. Pathology study showed that there were severe necrosis in tumors of medium and highdose ICA groups, with the necrosis found in the capsule and the margin of tumors. A small number of inflammatory cells (lymphocytes and monocytes) were observed in the capsule of tumor and tumor tissues.Conclusion: ICA can effectively inhibit tumor growth, which is probably through inducing early apoptosis of solid tumor cells and the subsequent tumor tissues necrosis.
2007, 14(2):143-147.
DOI: 10.3872/j.issn.1007-385X.2007.2.009
Abstract:
To study the inhibitory effect of retrovirusmediated antisense human telomerase RNA (hTR) gene on hepatocelluar carcinoma, so as to explore an effective way to inhibit telomrerase activity in the treatment of hepatocellular carcinoma. Methods: Sense and antisense hTR gene were transfected into the packaging cell line PT67 by electroporation, and the stablely transfected cells were selected with G418. The recombinant retroviral supernatant was collected and transfected into HepG2 cells. After G418 selection, PCR was used to verify the integration of the hTR gene. Cell growth curves were drawn using MTT assay and the expression of PCNA was determined by immunofluorescence. TRAPPCRELISA was adopted to detect the telomerase activity; cell cycle and apoptosis were evaluated by flow cytometry (FCM).Results: The expression of hTR gene could be amplified in HepG2hTREcoRⅠ and HepG2hTRBamHⅠ cells, but not in untransfected HepG2 cells. The antisense hTR complementary to the template region of telomerase inhibited growth and proliferation of HepG2 cells. The expression of neutrophil proliferating cell nuclear antigen (PCNA) was decreased. Telomerase activity in the antisense hTRtreated group was (2.31±0.16),which was significantly lower than those of the other 2 groups(P<001). FCM showed that the apoptosis rate of the experimental group was (958±138)%, which was significantly different from those of the other 2 groups(P<0.01). G2/M phase blockage was detected in the HepG2hTRBamHⅠ cells. Conclusion: Telomerase RNA is an important component of telomerase; downregulation of its expression through antitechnology can lead to the growth inhibition and apoptosis of hepatocellular carcinoma cells.
To study the inhibitory effect of retrovirusmediated antisense human telomerase RNA (hTR) gene on hepatocelluar carcinoma, so as to explore an effective way to inhibit telomrerase activity in the treatment of hepatocellular carcinoma. Methods: Sense and antisense hTR gene were transfected into the packaging cell line PT67 by electroporation, and the stablely transfected cells were selected with G418. The recombinant retroviral supernatant was collected and transfected into HepG2 cells. After G418 selection, PCR was used to verify the integration of the hTR gene. Cell growth curves were drawn using MTT assay and the expression of PCNA was determined by immunofluorescence. TRAPPCRELISA was adopted to detect the telomerase activity; cell cycle and apoptosis were evaluated by flow cytometry (FCM).Results: The expression of hTR gene could be amplified in HepG2hTREcoRⅠ and HepG2hTRBamHⅠ cells, but not in untransfected HepG2 cells. The antisense hTR complementary to the template region of telomerase inhibited growth and proliferation of HepG2 cells. The expression of neutrophil proliferating cell nuclear antigen (PCNA) was decreased. Telomerase activity in the antisense hTRtreated group was (2.31±0.16),which was significantly lower than those of the other 2 groups(P<001). FCM showed that the apoptosis rate of the experimental group was (958±138)%, which was significantly different from those of the other 2 groups(P<0.01). G2/M phase blockage was detected in the HepG2hTRBamHⅠ cells. Conclusion: Telomerase RNA is an important component of telomerase; downregulation of its expression through antitechnology can lead to the growth inhibition and apoptosis of hepatocellular carcinoma cells.
2007, 14(2):148-152.
DOI: 10.3872/j.issn.1007-385X.2007.2.010
Abstract:
To establish a gefitinibresistant human colon carcinoma HT29/ZD cell line and to preliminarily study its drug resistance mechanisms. Methods: Gefitinibresistant HT29/ZD was induced by stepwise selection after exposure to increasing doses of gefitinib. IC50were determined by MTT assay and the resistance index (RI) was calculated. Cell growth curves were plotted and the double times were calculated by cell counting assay. Distribution of cell cycles were detected by flow cytometry. Crossresistance profiles of HT29/ZD to 5Fu, DDP, LOHP, and CPT11 were tested by MTT assay. Expression levels of IGFRa, PIGFR and Pgp were determined by immunocytochemistry method. Results: A genfibinibresistant human colon carcinoma cell line HT29/ZD has been established successfully, with the RI being 2667. The doubling times of HT29/ZD and HT29 cells were 33.25 and 37.7 h, respectively. Flow cytometry demonstrated that HT29/ZD cells of phase S and G2M were increased, but those of G0/G1 phase were reduced. We also found that, compared to HT29 cells, HT29/ZD cells had an increased sensitivity to 5Fu, DDP （P>005）, and showed certain resistance to CPT11（P>0.05）, but had a significantly increased sensitivity to LOHP. Immunocytochemical analysis demonstrated that HT29 and HT29/ZD cells had a similar expression of Pgp （P>0.05）. HT29/ZD cells had a lower expression of IGF1Ra (P<0.01) than HI29 cells but a higher expression of phosphoIGF1R (P<001). Conclusion:HT29/ZD has no multidrug resistance like traditional anticancer drugs. PhosphoIGF1R of HT29/ZD has an increased activity, which might be one of the mechanisms for the acquired resistance of HT29/ZD. Pgp has no relationship with acquired resistance to gefitinib.
To establish a gefitinibresistant human colon carcinoma HT29/ZD cell line and to preliminarily study its drug resistance mechanisms. Methods: Gefitinibresistant HT29/ZD was induced by stepwise selection after exposure to increasing doses of gefitinib. IC50were determined by MTT assay and the resistance index (RI) was calculated. Cell growth curves were plotted and the double times were calculated by cell counting assay. Distribution of cell cycles were detected by flow cytometry. Crossresistance profiles of HT29/ZD to 5Fu, DDP, LOHP, and CPT11 were tested by MTT assay. Expression levels of IGFRa, PIGFR and Pgp were determined by immunocytochemistry method. Results: A genfibinibresistant human colon carcinoma cell line HT29/ZD has been established successfully, with the RI being 2667. The doubling times of HT29/ZD and HT29 cells were 33.25 and 37.7 h, respectively. Flow cytometry demonstrated that HT29/ZD cells of phase S and G2M were increased, but those of G0/G1 phase were reduced. We also found that, compared to HT29 cells, HT29/ZD cells had an increased sensitivity to 5Fu, DDP （P>005）, and showed certain resistance to CPT11（P>0.05）, but had a significantly increased sensitivity to LOHP. Immunocytochemical analysis demonstrated that HT29 and HT29/ZD cells had a similar expression of Pgp （P>0.05）. HT29/ZD cells had a lower expression of IGF1Ra (P<0.01) than HI29 cells but a higher expression of phosphoIGF1R (P<001). Conclusion:HT29/ZD has no multidrug resistance like traditional anticancer drugs. PhosphoIGF1R of HT29/ZD has an increased activity, which might be one of the mechanisms for the acquired resistance of HT29/ZD. Pgp has no relationship with acquired resistance to gefitinib.
11 Expression of histone deacetyase 4 in human liver carcinoma cell line Bel7402 and its significance
2007, 14(2):153-157.
DOI: 10.3872/j.issn.1007-385X.2007.2.011
Abstract:
To investigate the expression of histone deacetyase4 (HDAC4) in human liver carcinoma cell line Bel7402 and to explore the regulatory effects of HDAC4 on the proliferation and differentiation of Bel7402. Methods: Carcinoma cells Bel7402 was treated with different concentrations of sodium phenylbutyrate (SPB), an inhibitor of HDAC4. Expression of HDAC4 mRNA in Bel7402 cells was analyzed by RTPCR before and after SPB treatment. Reverse microscope was used to observe the morphological changes of Bel7402 cells. MTT assay and flow cytometry were adopted to describe the proliferation and cell cycle of Bel7402 cells. Expression of P27 protein was determined by immunohistochemical method. The statistical analysis was performed using oneway ANOVA and student t test. Results: SPB significantly decreased the expression of HDAC4 in Bel7402\[(0.88±0.13) vs (0.12±0.04), P<0.05\]. It also inhibited the Bel7402 cell growth in a timeand dosedependent manner. Fibrous changes of Bel7402 cells was observed after SPB treatment. SPB treatment arrested cell at G1 phase \[(50.6±4.0)% vs (78.8±3.6)%, P<0.05\] and enhanced the expression of P27 in Bel7402 \[（23±11) vs (61±7),P<0.05\].Conclusion: SPB treatment can decrease the expression of HDAC4 in human liver cancer cell Bel7402 and subsequently inhibits proliferation of Bel7402 cells, which might be associated with the change of P27 protein expression.
To investigate the expression of histone deacetyase4 (HDAC4) in human liver carcinoma cell line Bel7402 and to explore the regulatory effects of HDAC4 on the proliferation and differentiation of Bel7402. Methods: Carcinoma cells Bel7402 was treated with different concentrations of sodium phenylbutyrate (SPB), an inhibitor of HDAC4. Expression of HDAC4 mRNA in Bel7402 cells was analyzed by RTPCR before and after SPB treatment. Reverse microscope was used to observe the morphological changes of Bel7402 cells. MTT assay and flow cytometry were adopted to describe the proliferation and cell cycle of Bel7402 cells. Expression of P27 protein was determined by immunohistochemical method. The statistical analysis was performed using oneway ANOVA and student t test. Results: SPB significantly decreased the expression of HDAC4 in Bel7402\[(0.88±0.13) vs (0.12±0.04), P<0.05\]. It also inhibited the Bel7402 cell growth in a timeand dosedependent manner. Fibrous changes of Bel7402 cells was observed after SPB treatment. SPB treatment arrested cell at G1 phase \[(50.6±4.0)% vs (78.8±3.6)%, P<0.05\] and enhanced the expression of P27 in Bel7402 \[（23±11) vs (61±7),P<0.05\].Conclusion: SPB treatment can decrease the expression of HDAC4 in human liver cancer cell Bel7402 and subsequently inhibits proliferation of Bel7402 cells, which might be associated with the change of P27 protein expression.
LIU Jiangqiu
,
LING Lei
,
LIU Yanqiu
,
LI Jieying
,
ZHAO Wei
,
ZHU Liqing
,
ZHU Xiaohong
,
WU Caizhong
,
XU Lu
,
LI Zhongyi
2007, 14(2):158-162.
DOI: 10.3872/j.issn.1007-385X.2007.2.012
Abstract:
To investigate the in vivo pharmacokinetics of recombinant human Endostatin (rhEndostatin) in Wistar rats after i.v. injection, so as to provide pharmacokinetic data for clinical application. Methods:Radioactive isotopic (125I) tracing combined with trichloroacetic acid (TCA) precipitation was used to measure the residual isotope in different tissues at different time points after i.v. injection of 125IrhEndostatin to the rats. Plasma drug concentrationtime data were analyzed by computer fitting. Compartment model and the pharmacokinetic parameters were also established. Results: The distribution halflife time and elimination halflife time of rhEndostatin in rats were (0.154+0.024) h and (4.03+0.58) h, respectively. The AUC were positively correlated with dosages of rhEndostatin (r=0.9940). The mean of CLs value was (0.165±0.024)L/h; the CLs values for high, middle or low dosages were basically the same. The liver, lung, spleen, stomach and thyroid were the major organs for deposition of 125IrhEndostatin. Urinary excretion was the major pathway of elimination. Conclusion: The pharmacokinetics of 125IrhEndostatin in rats basically has a linear character, and the plasma drug concentrationtime curve consists to a twocompartment model.
To investigate the in vivo pharmacokinetics of recombinant human Endostatin (rhEndostatin) in Wistar rats after i.v. injection, so as to provide pharmacokinetic data for clinical application. Methods:Radioactive isotopic (125I) tracing combined with trichloroacetic acid (TCA) precipitation was used to measure the residual isotope in different tissues at different time points after i.v. injection of 125IrhEndostatin to the rats. Plasma drug concentrationtime data were analyzed by computer fitting. Compartment model and the pharmacokinetic parameters were also established. Results: The distribution halflife time and elimination halflife time of rhEndostatin in rats were (0.154+0.024) h and (4.03+0.58) h, respectively. The AUC were positively correlated with dosages of rhEndostatin (r=0.9940). The mean of CLs value was (0.165±0.024)L/h; the CLs values for high, middle or low dosages were basically the same. The liver, lung, spleen, stomach and thyroid were the major organs for deposition of 125IrhEndostatin. Urinary excretion was the major pathway of elimination. Conclusion: The pharmacokinetics of 125IrhEndostatin in rats basically has a linear character, and the plasma drug concentrationtime curve consists to a twocompartment model.
2007, 14(2):163-168.
DOI: 10.3872/j.issn.1007-385X.2007.2.013
Abstract:
To investigate the pathological changes of human hepatocellular carcinoma (HCC) after continuous passaging in nude mice. Methods: The mice model of HCC SMMULTMN were continuously observed for 20 years (19872007) . The subcutaneously transplanted carcinoma had been passaged for 228 generations. The pathological data of abdominally transplanted HCC, orthotopically transplanted HCC in nude mice, and orthotopically transplanted HCC in NODSCID mice were recorded. The pathological studies were conducted by light microscope, electron microscope, image analysis, chromosomal analysis, and measurement of alphafetoprotein (AFP) in peripheral blood. Results: (1) The local invasion and metastasis of of tumors were present in all the above 4 models for a long time. The local invasion rate and the pulmonary metastasis rate of subcutaneously transplanted tumors were 59.70% (40/67) and 37.10% (23/62), respectively. The pulmonary metastasis rate of abdominally transplanted tumors was 59.02%(36/61). The intrahepatic and pulmonary metastasis rate of the othotopically transplanted tumors were 18.18%(4/22) and 31.82% (7/22), respectively. The pulmonary metastasis rate of HCC in NODSCID mice was 53.85%. (2) The tissue structure and the differentiation of the 10th generation tumor cells was similar to those of primary HCC, with grade 2 differentiation and coarse trabecular pattern as the main characteristics. From the 11th generation to the 228th generation, the main characteristics of tumor cells were grade 3 differentiation and lump pattern. Electron microscope also showed worse differentiation. (3)The AFP level was 92 500 μg/L in cells before the 32th generation; it decreased to 6 729μg/L from the 33th130th generation cells; and the level of the 220th generation was 1 0005 000 μg/L.（4）The DNA contents had a wide distribution (from 2c to 6c) in abdominally transplanted tumors and the pulmonary metastatic tumors; the mean DNA index in the former tumors (2.60±0.20) was wider than the that in the latter (2.10±0.26) . (5)From the 55th generation to 206th generation, it was found that tumor cells had integrated into the chromosome of the nude mice. Conclusion: The subcutaneously transplanted HCC in nude mice can be stably expressed for 20 years, with no change in the local invasion and metastasis ability of HCC. The differentiation of the tumor cells worsenes and the AFP level is decreased in the blood; some chromosome of tumor cells integrate into the chromosome of nude mice, which may be related to the internal environment of nude mice and the multipotential differentiation of the tumor cells.
To investigate the pathological changes of human hepatocellular carcinoma (HCC) after continuous passaging in nude mice. Methods: The mice model of HCC SMMULTMN were continuously observed for 20 years (19872007) . The subcutaneously transplanted carcinoma had been passaged for 228 generations. The pathological data of abdominally transplanted HCC, orthotopically transplanted HCC in nude mice, and orthotopically transplanted HCC in NODSCID mice were recorded. The pathological studies were conducted by light microscope, electron microscope, image analysis, chromosomal analysis, and measurement of alphafetoprotein (AFP) in peripheral blood. Results: (1) The local invasion and metastasis of of tumors were present in all the above 4 models for a long time. The local invasion rate and the pulmonary metastasis rate of subcutaneously transplanted tumors were 59.70% (40/67) and 37.10% (23/62), respectively. The pulmonary metastasis rate of abdominally transplanted tumors was 59.02%(36/61). The intrahepatic and pulmonary metastasis rate of the othotopically transplanted tumors were 18.18%(4/22) and 31.82% (7/22), respectively. The pulmonary metastasis rate of HCC in NODSCID mice was 53.85%. (2) The tissue structure and the differentiation of the 10th generation tumor cells was similar to those of primary HCC, with grade 2 differentiation and coarse trabecular pattern as the main characteristics. From the 11th generation to the 228th generation, the main characteristics of tumor cells were grade 3 differentiation and lump pattern. Electron microscope also showed worse differentiation. (3)The AFP level was 92 500 μg/L in cells before the 32th generation; it decreased to 6 729μg/L from the 33th130th generation cells; and the level of the 220th generation was 1 0005 000 μg/L.（4）The DNA contents had a wide distribution (from 2c to 6c) in abdominally transplanted tumors and the pulmonary metastatic tumors; the mean DNA index in the former tumors (2.60±0.20) was wider than the that in the latter (2.10±0.26) . (5)From the 55th generation to 206th generation, it was found that tumor cells had integrated into the chromosome of the nude mice. Conclusion: The subcutaneously transplanted HCC in nude mice can be stably expressed for 20 years, with no change in the local invasion and metastasis ability of HCC. The differentiation of the tumor cells worsenes and the AFP level is decreased in the blood; some chromosome of tumor cells integrate into the chromosome of nude mice, which may be related to the internal environment of nude mice and the multipotential differentiation of the tumor cells.
2007, 14(2):169-172.
DOI: 10.3872/j.issn.1007-385X.2007.2.014
Abstract:
To observe the expression of hMLH1 protein in esophagus squamous cell carcinoma, atypical hyperplasia tissue and normal esophagus tissue, so as to discuss the relationship between hMLH1 expression and esophagus carcinogenesis. Methods: The specimens of esophagus squamous cell carcinoma, atypical hyperplasia and normal esophagus tissue were obtained from 92 esophagus carcinoma patients. Immunohistochemistry (IHC) staining technique was used to detect expression of hMLH1 protein. The relationship between hMLH1 expression with clinical parameters, such as gender, age, cancer differentiation level, infiltration depth, tumor stage, lymphatic metastasis, was analyzed. Results: The positive rates of hMLH1 protein in esophagus squamous cell carcinoma, and atypical hyperplasia tissue and normal esophagus tissue were 36.96%，56.52%, and 84.78%,respectively,with the former 2 significantly lower than the latter (P<001). The average age of patients negative of hMLH1 protein in tumor tissues was older than that of patients positive of hMLH1 (P<0.05). There was significant correlation between the expression of hMLH1 with tumor staging and lymphatic metastasis (P<0.05). Conclusion: Deletion of hMLH1 may participate in the carcinogenesis of esophageal squamous cell carcinoma at an early stage.hMLH1 gene may delay and suppress the development and progression of esophageal squamous cell carcinoma.
To observe the expression of hMLH1 protein in esophagus squamous cell carcinoma, atypical hyperplasia tissue and normal esophagus tissue, so as to discuss the relationship between hMLH1 expression and esophagus carcinogenesis. Methods: The specimens of esophagus squamous cell carcinoma, atypical hyperplasia and normal esophagus tissue were obtained from 92 esophagus carcinoma patients. Immunohistochemistry (IHC) staining technique was used to detect expression of hMLH1 protein. The relationship between hMLH1 expression with clinical parameters, such as gender, age, cancer differentiation level, infiltration depth, tumor stage, lymphatic metastasis, was analyzed. Results: The positive rates of hMLH1 protein in esophagus squamous cell carcinoma, and atypical hyperplasia tissue and normal esophagus tissue were 36.96%，56.52%, and 84.78%,respectively,with the former 2 significantly lower than the latter (P<001). The average age of patients negative of hMLH1 protein in tumor tissues was older than that of patients positive of hMLH1 (P<0.05). There was significant correlation between the expression of hMLH1 with tumor staging and lymphatic metastasis (P<0.05). Conclusion: Deletion of hMLH1 may participate in the carcinogenesis of esophageal squamous cell carcinoma at an early stage.hMLH1 gene may delay and suppress the development and progression of esophageal squamous cell carcinoma.
ZHANG Haifeng
,
ZHANG Chunhua
,
SHENG Weihua
,
WANG Jinzhi
,
XIE Yufeng
,
MIAO Jingcheng
,
YANG Jicheng
2007, 14(2):173-178.
DOI: 10.3872/j.issn.1007-385X.2007.2.015
Abstract:
To study the inhibitory effect of adenovirus mediated (phosphatase and tensin homologuedeleted chromosome ten gene) PTEN gene on the growth of A549 cells in vitro and in vivo. Methods: The PTEN gene was amplified by RTPCR from human peripheral blood and was inserted into pAdTrackCMV to construct pAdTrackCMVPTEN. The growth inhibition effect of pAdTrackCMVPTEN on A549 cells was detected by MTT; flow cytometry was used to detect the cell cycle and apoptosis of the tumor cells. The tumor inhibitory effect of pAdTrackCMVPTEN on A549 cells was studied with tumorbearing mice model. The microvessel density (MVD) of the implanted tumor tissue was determined immunohistochemically. Results: The sequencing result of cloned PTEN accorded well to that in GeneBank. The apoptosis rate of A549 cells was 10.5% 48 h after infection with pAdTrackCMVPTEN, significantly higher than that in the control group (0). Four days after infection, the growth of the A549 was inhibited by 57%. At the end of tumor inhibition experiment, the average tumor weight was (0.58±0.29) g in pAdTrackCMVPTEN treated group and (1.42±0.24) g in the control group, with a inhibitory rate of 59%（P<0.05）; pAdTrackCMVPTEN also decreased MVD by 49%（P<005）. Conclusion: We have sucessfully constructed pAdTrackCMVPTEN, which can inhibit the growth of A549 cells in vitro and in vivo.
To study the inhibitory effect of adenovirus mediated (phosphatase and tensin homologuedeleted chromosome ten gene) PTEN gene on the growth of A549 cells in vitro and in vivo. Methods: The PTEN gene was amplified by RTPCR from human peripheral blood and was inserted into pAdTrackCMV to construct pAdTrackCMVPTEN. The growth inhibition effect of pAdTrackCMVPTEN on A549 cells was detected by MTT; flow cytometry was used to detect the cell cycle and apoptosis of the tumor cells. The tumor inhibitory effect of pAdTrackCMVPTEN on A549 cells was studied with tumorbearing mice model. The microvessel density (MVD) of the implanted tumor tissue was determined immunohistochemically. Results: The sequencing result of cloned PTEN accorded well to that in GeneBank. The apoptosis rate of A549 cells was 10.5% 48 h after infection with pAdTrackCMVPTEN, significantly higher than that in the control group (0). Four days after infection, the growth of the A549 was inhibited by 57%. At the end of tumor inhibition experiment, the average tumor weight was (0.58±0.29) g in pAdTrackCMVPTEN treated group and (1.42±0.24) g in the control group, with a inhibitory rate of 59%（P<0.05）; pAdTrackCMVPTEN also decreased MVD by 49%（P<005）. Conclusion: We have sucessfully constructed pAdTrackCMVPTEN, which can inhibit the growth of A549 cells in vitro and in vivo.
2007, 14(2):179-183.
DOI: 10.3872/j.issn.1007-385X.2007.2.016
Abstract:
To investigate the apoptosisinducing effect of arsenic trioxide (As2O3) on gastric carcinoma cell line AGS in vitro and to assess the influence of As2O3 on the expression of signal transducers and activators of transcription 3 (STAT3) and vascular endothelial growth factor (VEGF). Methods: AGS cells were treated with different concentrations of As2O3 (1, 5, and 10 μmol/L) for 24,48, and 72 h. The cell proliferation was detected by MTT assay, cell apoptosis and cell cycle distribution were measured by flow cytometry and TUNEL, and the expression of STAT3 and VEGF was investigated by ELISA, immunohistochemistry and realtime PCR. Results: (1) As2O3 inhibited AGS cell proliferation in a time and dosedependent manner; (2) FCM results showed a typical subdiploid peak before G0/ G1 phase and cell cycle analysis showed G2/M phase arrest; (3) TUNEL analysis revealed the DNA fragmentation; (4) During the As2O3induced apoptosis of AGS cells, the expression of STAT3 and VEGF was downregulated, especially when As2O3 was at 10 mol/L. Conclusion: As2O3 can inhibit the proliferation of AGS cells and induce AGS cell apoptosis, which might be related with cell cycle block and downregulation of STAT3 and VEGF expression.
To investigate the apoptosisinducing effect of arsenic trioxide (As2O3) on gastric carcinoma cell line AGS in vitro and to assess the influence of As2O3 on the expression of signal transducers and activators of transcription 3 (STAT3) and vascular endothelial growth factor (VEGF). Methods: AGS cells were treated with different concentrations of As2O3 (1, 5, and 10 μmol/L) for 24,48, and 72 h. The cell proliferation was detected by MTT assay, cell apoptosis and cell cycle distribution were measured by flow cytometry and TUNEL, and the expression of STAT3 and VEGF was investigated by ELISA, immunohistochemistry and realtime PCR. Results: (1) As2O3 inhibited AGS cell proliferation in a time and dosedependent manner; (2) FCM results showed a typical subdiploid peak before G0/ G1 phase and cell cycle analysis showed G2/M phase arrest; (3) TUNEL analysis revealed the DNA fragmentation; (4) During the As2O3induced apoptosis of AGS cells, the expression of STAT3 and VEGF was downregulated, especially when As2O3 was at 10 mol/L. Conclusion: As2O3 can inhibit the proliferation of AGS cells and induce AGS cell apoptosis, which might be related with cell cycle block and downregulation of STAT3 and VEGF expression.
2007, 14(2):184-186.
DOI: 10.3872/j.issn.1007-385X.2007.2.017
Abstract:
构建针对ATM基因的RNA干扰表达质粒pRiATM，并观察其抑制SPCA1人肺癌细胞ATM表达的效果。方法: 构建针对ATM基因的短发夹状小干扰RNA真核表达载体pRiATM，并短暂转染SPCA1细胞，采用荧光定量RTPCR和Western blotting观察其抑制SPCA1细胞ATM表达的效果。结果：pRiATM1和pRiATM2转染SPCA1细胞后，与转染pRiGFP非特异性对照组相比，ATM mRNA水平分别下降86.4%和77.6%（两组均P<0.01）。与转染pRiGFP对照组相比，pRiATM1组和pRiATM2组ATM蛋白表达水平分别是对照组的4.3%和10.6%（均P＜0.01），以pRiATM1抑制ATM表达效果最显著。结论: pRiATM质粒构建成功，转染SPCA1细胞后可以明显抑制ATM基因的表达。
构建针对ATM基因的RNA干扰表达质粒pRiATM，并观察其抑制SPCA1人肺癌细胞ATM表达的效果。方法: 构建针对ATM基因的短发夹状小干扰RNA真核表达载体pRiATM，并短暂转染SPCA1细胞，采用荧光定量RTPCR和Western blotting观察其抑制SPCA1细胞ATM表达的效果。结果：pRiATM1和pRiATM2转染SPCA1细胞后，与转染pRiGFP非特异性对照组相比，ATM mRNA水平分别下降86.4%和77.6%（两组均P<0.01）。与转染pRiGFP对照组相比，pRiATM1组和pRiATM2组ATM蛋白表达水平分别是对照组的4.3%和10.6%（均P＜0.01），以pRiATM1抑制ATM表达效果最显著。结论: pRiATM质粒构建成功，转染SPCA1细胞后可以明显抑制ATM基因的表达。
2007, 14(2):187-189.
DOI: 10.3872/j.issn.1007-385X.2007.2.018
Abstract:
设计并构建Survivin靶向的microRNA表达载体，观察其对Survivin基因的抑制作用。方法： 以Survivin为靶基因，根据pPRIME载体要求，设计针对Survivin的microRNA序列，PCR扩增获得目的片段并克隆到载体中，序列正确的载体脂质体转染Hela细胞，RTPCR和Western blotting检测其对Survivin的抑制作用。结果： 用XhoⅠ和EcoRⅠ双酶切鉴定、DNA测序证实构建的 microRNA表达载体序列正确，后者在mRNA和蛋白水平上均能明显抑制Hela细胞中Survivin的表达。结论：Survivin靶向microRNA表达载体构建成功，并能抑制靶基因的表达，为其在肿瘤基因治疗中的应用奠定了基础。
设计并构建Survivin靶向的microRNA表达载体，观察其对Survivin基因的抑制作用。方法： 以Survivin为靶基因，根据pPRIME载体要求，设计针对Survivin的microRNA序列，PCR扩增获得目的片段并克隆到载体中，序列正确的载体脂质体转染Hela细胞，RTPCR和Western blotting检测其对Survivin的抑制作用。结果： 用XhoⅠ和EcoRⅠ双酶切鉴定、DNA测序证实构建的 microRNA表达载体序列正确，后者在mRNA和蛋白水平上均能明显抑制Hela细胞中Survivin的表达。结论：Survivin靶向microRNA表达载体构建成功，并能抑制靶基因的表达，为其在肿瘤基因治疗中的应用奠定了基础。
2007, 14(2):190-193.
DOI: 10.3872/j.issn.1007-385X.2007.2.019
Abstract:
碳酸酐酶9(carbonic anhydrase IX，CA IX)是新发现的碳酸酐酶家族异构体之一，是由酸性氨基酸组成的跨膜糖蛋白，在调控细胞增殖、转化方面有重要作用。它能催化CO2 水解为碳酸和水,参与机体的酸碱平衡,调节细胞内外pH值，有利于肿瘤的生长和转移。CA IX位于VHL肿瘤抑制基因的下游，由HIF1途径激活，在正常组织中表达极低，在肾细胞癌中高度表达，是其特异性抗原。CA IX的表达水平可预测肾癌患者对白介素2治疗的反应和生存期，CA IX低表达是不良预后因素。近年来对CA IX相应抗体的研究取得了很大进展，131I标记的MAbG250在肾癌组织中具有高摄取率和高蓄积率，可对肾癌进行放射性核素显像，用来诊断肾癌；131I标记的cG250MAb用于治疗晚期肾癌，已显示其安全性和有效性。肾细胞癌CA IX的高度特异性表达使其成为肿瘤疫苗潜在的靶抗原和肾癌治疗的重要靶位，在肾癌靶向治疗方面的应用前景广阔。
碳酸酐酶9(carbonic anhydrase IX，CA IX)是新发现的碳酸酐酶家族异构体之一，是由酸性氨基酸组成的跨膜糖蛋白，在调控细胞增殖、转化方面有重要作用。它能催化CO2 水解为碳酸和水,参与机体的酸碱平衡,调节细胞内外pH值，有利于肿瘤的生长和转移。CA IX位于VHL肿瘤抑制基因的下游，由HIF1途径激活，在正常组织中表达极低，在肾细胞癌中高度表达，是其特异性抗原。CA IX的表达水平可预测肾癌患者对白介素2治疗的反应和生存期，CA IX低表达是不良预后因素。近年来对CA IX相应抗体的研究取得了很大进展，131I标记的MAbG250在肾癌组织中具有高摄取率和高蓄积率，可对肾癌进行放射性核素显像，用来诊断肾癌；131I标记的cG250MAb用于治疗晚期肾癌，已显示其安全性和有效性。肾细胞癌CA IX的高度特异性表达使其成为肿瘤疫苗潜在的靶抗原和肾癌治疗的重要靶位，在肾癌靶向治疗方面的应用前景广阔。
2007, 14(2):194-196.
DOI: 10.3872/j.issn.1007-385X.2007.2.020
Abstract:
Flt3配体(Flt3 ligand，FL)是一种与造血调控有关的早期造血生长因子。FL与造血干细胞表面的Fm样酪氨酸激酶受体3结合，激活造血前体细胞，促进其增殖;与其他细胞因子联合应用，可显著提高造血效应。研究还证实，FL作为一种免疫佐剂，单独或者联合其他细胞因子能在体内外扩增树突状细胞(dendritic cells, DCs) ，促进其发育、成熟。树突状细胞是免疫系统中一类重要的专职抗原提呈细胞，能够摄取外来抗原，与自身的MHC 形成复合物，提呈给T 细胞，显著刺激抗原特异性T细胞反应，在肿瘤免疫治疗中发挥重要作用。和T细胞不同，NK细胞不需要抗原提呈细胞的作用，通过分泌干扰素等细胞因子能够直接杀伤肿瘤细胞；FL 联合其他细胞因子能够诱导NK前体细胞（proNK cell）的扩增和增殖，进而增强机体的抗肿瘤能力。目前FL已经被应用于造血系统疾病、乳腺肿瘤、前列腺肿瘤、肝脏肿瘤、神经系统肿瘤等疾病的基础或临床研究。此外,在抗感染、抗宿主移植以及自身免疫反应疾病等方面FL也有较好的作用。随着研究的不断深入，FL显示出了优越的生物学效应，展示了其在生物治疗领域中的广阔前景。
Flt3配体(Flt3 ligand，FL)是一种与造血调控有关的早期造血生长因子。FL与造血干细胞表面的Fm样酪氨酸激酶受体3结合，激活造血前体细胞，促进其增殖;与其他细胞因子联合应用，可显著提高造血效应。研究还证实，FL作为一种免疫佐剂，单独或者联合其他细胞因子能在体内外扩增树突状细胞(dendritic cells, DCs) ，促进其发育、成熟。树突状细胞是免疫系统中一类重要的专职抗原提呈细胞，能够摄取外来抗原，与自身的MHC 形成复合物，提呈给T 细胞，显著刺激抗原特异性T细胞反应，在肿瘤免疫治疗中发挥重要作用。和T细胞不同，NK细胞不需要抗原提呈细胞的作用，通过分泌干扰素等细胞因子能够直接杀伤肿瘤细胞；FL 联合其他细胞因子能够诱导NK前体细胞（proNK cell）的扩增和增殖，进而增强机体的抗肿瘤能力。目前FL已经被应用于造血系统疾病、乳腺肿瘤、前列腺肿瘤、肝脏肿瘤、神经系统肿瘤等疾病的基础或临床研究。此外,在抗感染、抗宿主移植以及自身免疫反应疾病等方面FL也有较好的作用。随着研究的不断深入，FL显示出了优越的生物学效应，展示了其在生物治疗领域中的广阔前景。
2007, 14(2):197-200.
DOI: 10.3872/j.issn.1007-385X.2007.2.021
Abstract:
树突状细胞(dendritic cell, DC)是体内功能最强的专职抗原提呈细胞,具有很强的抗原提呈能力,并能有效激发T细胞应答。DC在免疫学及肿瘤学乃至其他各学科中的作用日益受到重视，DC表面表达的共刺激分子及其生物学意义已成为研究的热点。共刺激分子的作用不仅限于对T细胞的激发、赋予T细胞活化的第二信号，并且参与Th细胞的极化。OX40/OX40L和ICOS/ICOSL信号介导Th2细胞分化；ICAM1/LFA1以及人41BB/4IBBL信号介导Th1细胞分化；CD40/CD40L信号在Th1/Th2型免疫应答中均发挥重要作用；PDL1/ PD1可能与Th1细胞极化有关，而人PDL2/ PD1共刺激信号下调Th1应答；人B7H3信号参与Th1型免疫反应，鼠B7H3信号负向调控Th1细胞分化；B71分子选择性的促进Th1反应，而B72分子参与调节Th2反应。深入探讨DC表达的共刺激分子与Th细胞分化的关系，调整Th1或Th2优势应答类型，可能为肿瘤等疾病的治疗提供新的思路。
树突状细胞(dendritic cell, DC)是体内功能最强的专职抗原提呈细胞,具有很强的抗原提呈能力,并能有效激发T细胞应答。DC在免疫学及肿瘤学乃至其他各学科中的作用日益受到重视，DC表面表达的共刺激分子及其生物学意义已成为研究的热点。共刺激分子的作用不仅限于对T细胞的激发、赋予T细胞活化的第二信号，并且参与Th细胞的极化。OX40/OX40L和ICOS/ICOSL信号介导Th2细胞分化；ICAM1/LFA1以及人41BB/4IBBL信号介导Th1细胞分化；CD40/CD40L信号在Th1/Th2型免疫应答中均发挥重要作用；PDL1/ PD1可能与Th1细胞极化有关，而人PDL2/ PD1共刺激信号下调Th1应答；人B7H3信号参与Th1型免疫反应，鼠B7H3信号负向调控Th1细胞分化；B71分子选择性的促进Th1反应，而B72分子参与调节Th2反应。深入探讨DC表达的共刺激分子与Th细胞分化的关系，调整Th1或Th2优势应答类型，可能为肿瘤等疾病的治疗提供新的思路。
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.