Bcl-2基因对丝裂霉素C诱导的膀胱癌细胞凋亡抑制及其机制的研究

Research on effect and its mechanism of over expression of Bcl-2 in inhibition of apoptosis of bladder cancer cells induced by mitomycin C

目的:探讨Bcl-2基因对丝裂霉素C(MMC)诱导的膀胱癌细胞凋亡的抑制作用及其分子机制。方法:采用DNA重组技术构建真核表达载体pcDNA3.1(+)/Bcl-2;利用脂质体将重组载体pcDNA3.1(+)/Bcl-2和空载体pcDNA3.1(+)转入人膀胱癌BIU-87细胞,RT-PCR检测基因转录水平;MMC作用于转染前、后的细胞,应用四甲基偶氮唑蓝(MTT)比色法、吖啶橙(AO)荧光染色法、Westernblot技术对细胞生长、凋亡、Caspase-3活性及细胞色素C在细胞内分布的变化进行检测。结果:1)成功构建真核表达载体pcDNA3.1(+)/Bcl-2,建立Bcl-2基因高表达的膀胱癌细胞株BIU-87/Bcl-2。2)在MMC作用下,与未转染Bcl-2基因的膀胱癌细胞株BIU-87和BIU-87/neo相比,BIU-87/Bcl-2细胞株存活率上升,P<0.01,其IC50是后者的15倍,q=6.41,P<0.01;BIU-87/Bcl-2的凋亡率显著降低,q值分别为22.62和20.45,P值均<0.01,Caspase-3激活和线粒体细胞色素C的释放受到抑制,BIU-87/Bcl-2的A405显著低于BIU-87和BIU-87/neo,q值分别为28.20和26.70,P值均<0.01,表明BIU-87/Bcl-2的Caspase-3激活受限。结论:Bcl-2基因高表达通过抑制线粒体细胞色素C的释放和Caspase-3的激活,使膀胱癌细胞对MMC诱导的凋亡产生抗性,导致耐药性的产生。

OBJECTIVE： To study the inhibitory effect and molecular mechanism of Bcl-2 on the apoptosis of bladder cancer cells induced by mitomycin C. METHODS： Eucaryotic expression vector pcDNA3.1 （＋）/Bcl-2 was constructed based on DNA recombination technology. Two vectors including pcDNA3.1 （＋）/Bcl-2 and pcDNA3.1（＋ ） were both transfered into BIU-87 cells. RT-PCR was used to detect the transcriptional level of Bcl-2. Before and after incubated with mitomycin C （MMC）, MTT method, acridine orange fluorescence staining and Westernblot were used to detect the cell growth, apoptosis, Caspase-3 activity and the intracellular distribution of cytochrome C to all the cells. RESULTS： 1 ） Bcl-2 was highly expressed in BIU-87/Bcl-2. 2） Compared with the cells that did not highly express Bcl-2, BIU-87/Bcl-2 showed a high survival rate under MMC. The survival rate was significantly higher than BIU-87 and BIU-87/neo, P〈0.01, and IC50 of BIU-87/Bcl-2 was 15-fold higher than that of BIU-87 and that of BIU-87/neo, q = 6.41, P〈0.01. BIU-87/Bcl-2 showed a lower apoptosis rate under MMC （q was 22.62 and 20.45 respectively, both P〈0.01）, and the activation of caspase-3 and the release of cytochrome C from mitochondria were inhibited. A405 of BIU-87/Bcl-2 was significantly lower than that of BIU-87 and that of BIU-87/neo （q was 28. 28 and 26.70 repectively, both P〈0. 01）, which indicated that the activation of caspase-3 was inhibited under MMC. CONCLUSION： Over expres- sion of Bcl-2 may inhibit the release of cytochrome C from mitochon- dria and the activation of caspase-3, which could render bladder cancer cells resistant to apoptosis induced by MMC.