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目的:探讨Bcl-2基因在人膀胱癌细胞中表达对细胞毒性T淋巴细胞(CTL)凋亡诱导作用的影响。方法:采用基因重组技术构建真核表达载体pcDNA3.1(+)/Bcl-2;应用脂质体介导的基因转染技术将pcDNA3.1(+)/Bcl-2导入膀胱癌BIU-87细胞,RT-PCR检测Bcl-2基因表达水平;不同浓度抗Fas单克隆抗体(Anti-Fasmab)模拟CTL的Fas-配体(Fas-L)处理转染与未转染Bcl-2基因的膀胱癌细胞,采用四甲基偶氮唑蓝(MTT)比色法、吖啶橙(AO)荧光染色法和流式细胞检测仪(FCM)分析细胞存活和凋亡情况。结果:酶切和核酸测序证明真核表达载体pcDNA3.1(+)/Bcl-2构建成功。将重组质粒转染膀胱癌细胞后,RT-PCR分析表明,转染Bcl-2基因的BIU-87/Bcl-2细胞的Bcl-2基因表达水平较BIU-87细胞和转染空载体的BIU-87/neo细胞显著增高,P<0.01。An-ti-Fasmab作用后,BIU87/Bcl-2细胞的存活率显著高于BIU-87和BIU-87/neo细胞,P<0.05或P<0.01。3种细胞虽均发生凋亡的形态学改变,但BIU87和BIU87/neo细胞改变更为明显,两者的凋亡率分别为(25.33±3.00)%和(27.05±1.75)%,而BIU-87/Bcl-2细胞的凋亡率为(18.06±1.41)%,显著低于前两者,P<0.01。结论:Bcl-2基因高表达通过阻断CTL杀伤膀胱癌细胞的Fas/Fas-L途径,使膀胱癌细胞能够抵御免疫系统中CTL的攻击。
Objective: To investigate the effect of Bcl-2 gene on the apoptosis of human bladder cancer cells induced by cytotoxic T lymphocytes (CTLs). Methods: The eukaryotic expression vector pcDNA3.1 (+) / Bcl-2 was constructed by gene recombination technology. The pcDNA3.1 (+) / Bcl-2 was transfected into bladder cancer BIU-87 The expression of Bcl-2 gene was detected by RT-PCR. Anti-Fasmab anti-Fasmab was used to mimic the CTL Fas-Ligand expression in the bladder of untransfected Bcl-2 The cell survival and apoptosis were analyzed by MTT colorimetric assay, acridine orange (AO) fluorescence staining and flow cytometry (FCM). Results: The eukaryotic expression vector pcDNA3.1 (+) / Bcl-2 was successfully constructed. RT-PCR analysis showed that the expression level of Bcl-2 gene in BIU-87 / Bcl-2 cells transfected with Bcl-2 gene was significantly higher than that in BIU-87 cells transfected with empty vector -87 / neo cells was significantly increased, P <0.01. The survival rate of BIU87 / Bcl-2 cells was significantly higher than that of BIU-87 and BIU-87 / neo cells after An-ti-Fasmab treatment (P <0.05 or P <0.01) (25.33 ± 3.00)% and (27.05 ± 1.75)% in BIU87 and BIU87 / neo cells, respectively, while the apoptotic rates of BIU87 and BIU87 / neo cells were (25.33 ± 3.00)% and (18.06 ± 1.41)%, significantly lower than the former two, P <0.01. CONCLUSION: High expression of Bcl-2 gene can kill bladder cancer cells by blocking CTL-induced Fas / Fas-L pathway in bladder cancer cells, which can prevent bladder cancer cells from attacking CTL in the immune system.