Bcl-2过表达抑制乙醇诱导的HCC-9204肝癌细胞凋亡

Overexpression of bcl-2 protects HCC-9204 hepatoma cells from ethanol-induced apoptosis

目的 探讨凋亡相关基因bcl 2过表达对乙醇诱导的肝细胞癌 (HCC)细胞凋亡的影响。方法 将含有人bcl 2cDNA的逆转录病毒表达载体pDOR SB质粒转染到不表达bcl 2蛋白的人肝癌细胞系HCC 92 0 4细胞中 ,用免疫组织化学ABC法检测bcl 2蛋白的表达情况。连续克隆化直至获得10 0 %表达bcl 2蛋白的单克隆细胞株 ,并进行流式细胞术检测。用 6 %乙醇分别作用于上述细胞 6h后 ,用MTT法和TUNEL法和流式细胞仪检测细胞凋亡发生程度。结果 转染pDOR SB的细胞大部分为bcl 2蛋白表达阳性 ,而转染pDOR空载体或未转染的HCC 92 0 4细胞均为bcl 2蛋白阴性。连续 3次克隆化后 ,流式细胞检测表明所获得的单克隆细胞株 10 0 %稳定表达bcl 2蛋白 ,命名为HCC bcl 2。6 %乙醇作用后 ,HCC bcl 2细胞和未转染的HCC 92 0 4细胞经MTT法检测的吸光度值分别为 0 5 19±0 0 5 3和 0 36 6± 0 0 46 ,前者明显高于后者 (P <0 0 1) ;TUNEL指数分别为 0 .387和 0 .6 13,亚二倍体凋亡峰比例分别为 3.8%和 10 .7% ,前者均明显低于后者 (P <0 .0 5 )。结论 bcl 2蛋白过表达能够抑制乙醇诱导的HCC 92 0 4肝癌细胞凋亡。

Objective To explore the effect of apoptosis-associated gene bcl-2 overexpression on ethanol-induced apoptosis in hepatocellular carcinoma (HCC) cells. Methods The plasmid of retrovirus expression vector pDOR-SB, which contains human bcl-2 cDNA, was transfected into a HCC cell line HCC-9204 cells whose bcl-2 protein expression is below detectable level. The expression of bcl-2 protein was detected by immunohistochemical ABC method. The cells were cloned continually until a monoclonal cell strain which expressed bcl-2 protein at a 100% positive rate was obtained, and the cells were detected with flow cytometry. After the above cells were treated with 6% ethanol for 6 h, the happenning of apoptosis were detected by MTT assay, TUNEL assay and flow cytometry. Results bcl-2 protein expression was detected in most of the cells which had been transfected with pDOR-SB, while it was bcl-2 protein negative in cells transfected with pDOR empty vector or non-transfected HCC-9204 cells. The obtained monoclonal cell strain expressed bcl-2 protein at a 100% positive rate under flow cytometer after being cloned 3 times in succession, and was named HCC-bcl-2. After being treated with 6% ethanol, the optical absorbance data of HCC-bcl-2 cells and non-transfected HCC-9204 cells detected by MTT assay were 0.519±0.053 and 0.366±0.046, respectively, the former was significantly higher than the latter (P<0.01). Their TUNEL index was 0.387 and 0.613 respectively, and their sub-G1 apoptotic peak scale was 3.8% and 10.7% respectively, the former were all lower than the latter (P<0.05). Conclusion Overexpression of bcl-2 protein can suppress ethanol-induced apoptosis in HCC-9204 hepatoma cells.