【摘要】 目的:探讨凋亡相关基因Bcl-2过表达对多柔比星(ADM)诱导的膀胱癌细胞凋亡和NF-κB活化的影响。方法:采用脂质体转染法将Bcl-2基因转入膀胱癌细胞,G418筛选获得抗性亚克隆细胞株,RT-PCR检测Bcl-2基因的表达。用6.25、12.5和25μg/mL的ADM分别作用于细胞24 h,流式细胞术检测细胞凋亡,蛋白质印迹法检测NF-κB活化情况。结果:建立分别稳定过表达Bcl-2基因和新霉素抗性基因(neo)的膀胱癌亚克隆株BIU87/Bcl-2和BIU87/neo,RT-PCR结果显示,BIU87/Bcl-2细胞的Bcl-2 mRNA水平明显高于BIU87/neo和BIU87细胞,F=63.107,P<0.01。经6.25、12.5和25μg/mL ADM作用24 h后,BIU87/Bcl-2细胞凋亡率较BIU87/neo细胞明显降低,t=11.216,P<0.01。与BIU87/neo细胞相比,ADM作用24 h后BIU87/Bcl-2细胞胞质IκB明显减少,t=-0.255,P=0.018;而胞核NFκ-B p65明显增多,t=2.088,P=0.049。结论:Bcl-2基因能够抑制ADM诱导的膀胱癌BIU87细胞凋亡,其抗凋亡作用涉及NF-κB活化。更多还原

【Abstract】 OBJECTIVE: To explore the effect of Bcl-2 overexpression on apoptosis and transcription factor NF-κB activation induced by adriamycin in bladder carcinoma cells.METHODS: Under the induction of liposome,the whole length of Bcl-2 cDNA was transfected into humen bladder carcinoma cell line BIU87.The subclone cell lines were obtained by persistent G418 selection.The Bcl-2 gene expression of cancer cells was detected by RT-PCR methods.After being treated with adriamycin at concentrations of 6.25 μg/mL,12.5 μg/mL and 25 μg/mL for 24 hours repectively,the apoptosis rates of BIU87/neo and BIU87/Bcl-2 cells were determined by using flow cytometric analysis,and the expression of NF-κB p65 in nucleus and IκBα in cytoplasm of them were detected by using Western blot analysis.RESULTS: The subclone bladder carcinoma cell lines,stably expressing Bcl-2 and neo gene respectively,were successfully selected and named as BIU87/Bcl-2 and BIU87/neo.RT-PCR results demonstrated Bcl-2 mRNA levels of BIU87/Bcl-2 cells were significantly higher than those of BIU87 and BIU87/neo cells(F=63.107,P<0.01).After being treated with adriamycin at concentrations of 6.25 μg/mL,12.5 μg/mL and 25 μg/mL for 24 hours,the percentage of apoptosis in BIU87/Bcl-2 cells was lower significantly(P<0.01).Compared with BIU87/neo cells,the expression of NF-κB p65 in nucleus was higher and IκBα in cytoplasm was lower significantly(t=2.088,P=0.049;t=0.255,P=0.018) in BIU87/Bcl-2 cells after being treated with adriamycin.CONCLUSION: Bcl-2 can block apoptosis induced by adriamycin in bladder carcinoma BIU87 cells and its effect involves in the activation of NF-κB.更多还原