永生化人肝细胞中TNK1介导细胞凋亡的机制研究

Mechanism research of TNK1 mediated apoptosis in immortalized human hepatocytes

目的探究酪氨酸激酶非受体1(TNK1)在永生化人肝细胞中介导细胞凋亡的机制。方法采用免疫组织化学法检验TNK1在人肝细胞癌和正常组织中的表达水平,流式细胞术检测被激活细胞凋亡的细胞比例,逆转录病毒载体包装感染法构建人永生化肝细胞系IHH rtTA-TNK1细胞模型,免疫印迹法检测TNK1和细胞凋亡相关蛋白的表达。结果TNK1在人肝细胞癌细胞组织中表达水平明显低于正常组织;TNK1激活c-含半胱氨酸的天冬氨酸蛋白水解酶(caspase)-3、c-caspase-9的表达;TNK1使线粒体中的细胞色素C部分转移至细胞质中;TNK1上调Bak并降低B淋巴细胞瘤(Bcl)-xL、Mcl-1的蛋白水平。结论在永生化人肝细胞中TNK1介导了Bcl-2家族蛋白表达水平使线粒体外膜通透性增加,导致细胞色素C释放从而激活caspase-9和caspase-3,最终诱导细胞凋亡。

Objective To explore the mechanism of TNK1-mediated apoptosis in immortalized human hepatocytes.Methods The immunohistochemical method was adopted to detect the expression level of TNK1 in human hepatocellular carcinoma(HCC)and normal tissues.The flow cytometry was used to examine the proportion of cells activated to apoptosis.The retrovirus vector packaging and infection method was used to construct the immortalized human hepatocytes line IHH rtTA-TNK1 cell model.Immunoblotting was used to examine the expressions of TNK1 and apoptosis-related proteins.Results The level of TNK1 in human hepatocellular carcinoma cellular tissue was significantly lower than that in normal tissues.TNK1 activated the expressions of c-caspase-3 and c-caspase-9.TNK1 caused the partial translocation of cytochrome C in mitochondria into cytoplasm.TNK1 up-regulated the expression of Bak,while down-regulated the expression of Bcl-xL and Mcl-1.Conclusion In immortalized human hepatocytes,TNK1 mediates the expression level of Bcl-2 family proteins,makes the increase of mitochondrial outer membrane permeability,causes the release of cytochrome C,thus activates caspase-9 and caspase-3,and eventually induces apoptosis.