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目的探讨外源性FHIT基因的表达对多西紫杉醇(DOC)诱导人胃癌细胞株(MGC-803)凋亡的影响及其分子机制。方法用脂质体将包含有外源性FHIT基因的重组真核表达质粒转染胃癌细胞MGC-803,获得稳定表达重组质粒pRcCMV-FHIT的细胞株(已由山东大学附属省立医院中心实验室成功构建),Western blot法检测外源性FHIT蛋白的表达;采用MTT法检测不同浓度(1.0、5.0、10.0、20.0、40.0 mg/L)及不同时间(24、48、72 h)下多西紫杉醇对MGC-803细胞的抑制率并选择最佳浓度和作用时间;采用流式细胞术检测FHIT转染和DOC单独及联合作用后的细胞凋亡率;蛋白免疫印迹法检测干预前后cleaved-Caspase3的蛋白表达。结果获得稳定表达FHIT基因的胃癌细胞株;DOC对MGC-803细胞具有抑制作用,且浓度为20 mg/L、时间为48 h时最明显;DOC+pRcCMV-FHIT组细胞的凋亡水平(65.54%)较阴性对照组(3.27%)、pRcCMV组(3.55%)、pRcCMV-FHIT组(13.94%)、DOC组(44.13%)、DOC+pRcCMV(45.29%)组明显增高,且差异具有统计学意义(P<0.05);DOC+pRcCMV-FHIT组细胞的cleaved-Caspase3蛋白表达较其它各组明显增强。结论FHIT基因表达与DOC能够协同促进胃癌细胞的凋亡,这可能与二者能够协同上调Caspase-3蛋白表达相关。
Objective To investigate the effect of exogenous FHIT gene on the apoptosis of human gastric cancer cell line MGC-803 induced by docetaxel and its molecular mechanism. Methods The recombinant eukaryotic expression plasmid containing exogenous FHIT gene was transfected into gastric cancer cell line MGC-803 by lipofectamine to obtain the cell line stably expressing the recombinant plasmid pRcCMV-FHIT (which was obtained from the Laboratory of Provincial Hospital of Shandong University Western blot was used to detect the expression of exogenous FHIT protein. MTT assay was used to detect the expression of FHIT at different concentrations (1.0, 5.0, 10.0, 20.0 and 40.0 mg / L) and at different times (24, 48 and 72 h) The inhibition rate of paclitaxel on MGC-803 cells and the optimal concentration and duration of action were determined. Flow cytometry was used to detect the apoptosis rate of FHIT transfected cells and DOC alone and in combination. Western blotting was used to detect cleaved-Caspase3 Protein expression. Results The gastric cancer cell lines stably expressing FHIT gene were obtained. DOC had inhibitory effect on MGC-803 cells at the concentration of 20 mg / L for 48 h, and the apoptotic rate in DOC + pRcCMV-FHIT group was 65.54 %) Was significantly higher than that of negative control group (3.27%), pRcCMV group (3.55%), pRcCMV-FHIT group (13.94%), DOC group (44.13%) and DOC + pRcCMV (45.29% (P <0.05). The cleaved-Caspase3 protein expression in DOC + pRcCMV-FHIT group was significantly higher than that in other groups. Conclusions FHIT gene expression and DOC can cooperate to promote apoptosis of gastric cancer cells, which may be related to the upregulation of Caspase-3 protein.