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目的 本研究旨在明确TGF β1诱导鼠肝细胞凋亡与Caspase 3及Caspase 8的关系。方法 通过 5 0 %CCl4腹腔注射 ,用 8周时间诱发BALB/c小鼠形成肝硬化模型。应用改良的胶原酶原位灌注法分离小鼠的肝细胞。为检测TGF β1(5ng/ml)诱导凋亡 ,利用 1.5 %的琼脂糖凝胶电泳观察DNA梯形条带 ,应用DNA荧光染料Hoechst 33342对正常肝细胞进行染色 ,并在荧光显微镜下观察比较Caspase 3和Caspase 8在TGF β1诱导的凋亡中的作用。 结果 胶原酶原位二步灌流法分离肝细胞活率为 95 .2 %。正常肝细胞予以TGF β1处理后 ,应用 1.5 %的琼脂糖凝胶电泳可以发现具有凋亡特征的梯形条带。硬化肝细胞则很少出现梯形条带。经TGF β1处理的鼠正常肝细胞应用Hoechst染色发现 ,其凋亡率明显高于未处理组 ,分别为 5 8.76 %和 18.0 3% ,两组具有明显的差别。但凋亡可以被Caspase 3和Caspase 8抑制剂阻止。 结论 硬化肝细胞不能象正常肝细胞那样发生TGF β1诱导的凋亡 ,提示硬化肝细胞的抑制性生长调控机制已受损。TGF β1通过Caspase 3和Cas pase 8的活化诱导鼠肝细细胞产生凋亡。
Purpose This study aimed to clarify the relationship between TGFβ1-induced hepatocyte apoptosis and Caspase 3 and Caspase 8. Methods BALB / c mice were induced by intraperitoneal injection of 50% CCl4 for 8 weeks to induce cirrhosis. Mouse hepatocytes were isolated by modified collagenase in situ perfusion. To detect the apoptosis induced by TGFβ1 (5ng / ml), DNA ladder was observed by 1.5% agarose gel electrophoresis. The normal liver cells were stained with DNA fluorescent dye Hoechst 33342 and observed under a fluorescence microscope. Caspase 3 And Caspase 8 in TGFβ1-induced apoptosis. Results Collagenase in situ two-step perfusion method to isolate liver cell viability was 95.2%. Normal liver cells treated with TGFβ1, 1.5% agarose gel electrophoresis can be found with traits of apoptosis trapezoidal band. Hardened hepatocytes are rarely trapezoidal bands. Hoechst staining showed that the apoptotic rate of TGFβ1-treated murine normal hepatocytes was significantly higher than that of untreated hepatocytes (5 8.76% and 18.0 3% respectively). There was a significant difference between the two groups. But apoptosis can be blocked by Caspase 3 and Caspase 8 inhibitors. Conclusion The sclerotic hepatocytes can not induce TGFβ1-induced apoptosis as normal hepatocytes, suggesting that the inhibitory growth regulatory mechanism of sclerotic hepatocytes has been impaired. TGFβ1 induces apoptosis in rat hepatic cells through the activation of Caspase 3 and Caspase 8.