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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。
Objective: To investigate the effect of apoptosis-related gene Bcl-2 overexpression on apoptosis and NF-κB activation induced by doxorubicin (ADM) in bladder cancer. Methods: The Bcl-2 gene was transfected into bladder cancer cells by lipofection method. The resistant subclone cell line was screened by G418. The expression of Bcl-2 gene was detected by RT-PCR. The cells were treated with 6.25,12.5 and 25μg / mL ADM for 24 h respectively. The apoptosis was detected by flow cytometry. The activation of NF-κB was detected by Western blotting. Results: The subclones BIU87 / Bcl-2 and BIU87 / neo of bladder cancer stably overexpressing Bcl-2 and neo were established respectively. The results of RT-PCR showed that the expression of BIU87 / Bcl- Bcl-2 mRNA level was significantly higher than BIU87 / neo and BIU87 cells, F = 63.107, P <0.01. The apoptotic rate of BIU87 / Bcl-2 cells was significantly lower than that of BIU87 / neo cells at 6.25, 12.5 and 25 μg / mL ADM for 24 h (t = 11.216, P <0.01). Compared with BIU87 / neo cells, the cytoplasmic IκB of BIU87 / Bcl-2 cells decreased significantly at 24 h after ADM treatment (t = -0.255, P = 0.018) 0.049. CONCLUSION: Bcl-2 gene can inhibit ADM-induced apoptosis of bladder cancer BIU87 cells and its anti-apoptotic effect involves activation of NF-κB.