Bcl-2基因在人膀胱癌细胞中表达对CTL凋亡诱导作用的影响

Research of the effect and its mechanism of Bcl-2 on the immune escape of human bladder cancer cells

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 study the effect of Bcl-2 on the apoptosis of human bladder cancer cells induced by cytotoxic T lymphocyte （CTL）. METHODS： The technology of gene recombination was used to constructed the eucaryotic expression vector pcDNA3. 1（ ＋ ）/Bcl-2. Inciding with enzymes and nucleinic acid sequencing were used to verify the vectors. The human bladder cancer BIU-87 cells were transfected with Bcl-2 by liposome introduced transfection and the genetic transcription level was evaluated by RT-PCR. Bladder cancer cells transfected with Bcl-2 or not were exposed to Anti-Fas monoclonal antibody （Anti-Fas mAb） which mimicked the cytotoxic activity of CTL, then MTT method, fluorescent staining with acridine orange （AO）, and flow cytometer （FCM） were used to observe the differences of survival rates, morphology of apoptosis and apoptosis rates. RESULTS： The inciding with enzymes and nucleinic acid sequencing verified that the recombinant vector pcDNA3. 1 （＋）/Bcl-2 was constructed successfully. The result of RT-PCR indicated that the transcription level of Bcl-2 was higher in BIU-87/Bcl-2 than in other two kinds of cells,P〈0.01. Compared with BIU-87 and BIU-87/neo, BIU-87/Bcl-2 showed a higher survival rate （P〈 0.05 or P〈0.01） and appeared a more obvious apoptosis morphology changes under Anti-Fas mab. The apoptosis rates of BIU-87 and BIU-87/neo were （ 25.33 ± 3.00） % and （ 27.05 ± 1.75）%, while that of BIU-87/Bcl-2 was （18.06± 1.41）%, which was significantly lower than those of the other two kinds of ceils, P〈0.01. CONCLUSIONS： Over expression of Bcl-2 may inhibit the apoptosis of human bladder cancer cells by interrupting the Fas/FasL pathway of CTL, which results in the resistance of human bladder cancer cells to the attack of CTL.