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目的:观察凋亡素诱导不同膀胱癌细胞株凋亡的效果并结合文献评价其临床应用前景。方法:采用凋亡素原核表达载体pBV220/Apoptin、真核表达载体PCDNA3、人膀胱移行细胞癌细胞株BIU-87及EJ、RT-PCR试剂盒以及质粒提取试剂盒。将原核表达载体中的Apoptin基因克隆入真核表达载体PCDNA3,经双酶切及基因测序鉴定无误后,利用脂质体转染法将真核表达质粒PCDNA3/Apoptin转染膀胱癌细胞株BIU-87及EJ。然后于转染后24h利用RT-PCR观察基因表达情况,并分别于转染后24、48、72h利用透射电镜检测肿瘤细胞超微结构变化、MTT法检测细胞存活率、流式细胞仪TUNAL法检测各组细胞的凋亡率,并对结果行统计学分析。结果:成功构建真核表达载体PCDNA3/Apoptin并转染膀胱癌细胞株BIU-87及EJ。转染24h后,通过RT-PCR法可见凋亡素基因在细胞内表达,并于转染后不同时段观察到实验组细胞发生高效凋亡,并随时间的延长而逐步升高,转染后72h,两种细胞株的凋亡率约为65%,而MTT法所测细胞存活率约为25%,与对照组相比,差异有统计学意义(P<0.05)。同时通过透射电镜可观察到实验组多数细胞出现凋亡的超微结构变化,如核边集、核碎裂及凋亡小体等。结论:凋亡素可诱导不同的膀胱癌细胞株发生高效的凋亡。考虑到其具有与小分子化疗药物不同的作用机制,我们认为凋亡素有可能成为克服膀胱癌细胞多药耐药性的新途径之一。
Objective: To observe the apoptosis induced by apoptin in different bladder cancer cell lines and to evaluate the clinical application of apoptin. Methods: Prokaryotic expression vector pBV220 / Apoptin, eukaryotic expression vector PCDNA3, human bladder transitional cell carcinoma cell line BIU-87, EJ, RT-PCR kit and plasmid extraction kit were used. The prokaryotic expression vector Apoptin gene was cloned into the eukaryotic expression vector PCDNA3, after double enzyme digestion and gene sequencing identification is correct, using liposome transfection method the eukaryotic expression plasmid PCDNA3 / Apoptin transfected bladder cancer cell line BIU- 87 and EJ. Then the expression of the gene was observed by RT-PCR 24h after transfection. The ultrastructural changes of tumor cells were detected by transmission electron microscopy at 24, 48 and 72 hours after transfection. The cell viability was detected by MTT assay and TUNAL method by flow cytometry The apoptosis rate of each group was detected, and the results were statistically analyzed. Results: The eukaryotic expression vector pcDNA3 / Apoptin was successfully constructed and transfected into bladder cancer cell lines BIU-87 and EJ. Transfection 24 h later, the expression of apoptin gene was observed in the cells by RT-PCR, and the apoptosis of cells in the experimental group was observed to be highly efficient at different time points after transfection. After transfection, 72h, the apoptosis rate of both cell lines was about 65%, while the cell viability measured by MTT method was about 25%, compared with the control group, the difference was statistically significant (P <0.05). At the same time, ultrastructural changes of most cells in the experimental group were observed by transmission electron microscopy, such as nuclear edge set, nuclear fragmentation and apoptotic bodies. Conclusion: Apoptosis can induce efficient apoptosis in different bladder cancer cell lines. Considering that it has a different mechanism of action than small molecule chemotherapeutic agents, we believe that apoptin may be one of the new ways to overcome the multi-drug resistance of bladder cancer cells.