TGF-β1诱导鼠肝细胞凋亡信号转导机制的研究

Study on the mechanism of apoptotic signal transduction of murine hepatocytes induced by TGF-β1

目的:本研究旨在明确转化生长因子β1(TGF-β1)诱导鼠肝细胞凋亡与caspase-3及caspase-8的关系。方法:通过50%CCl4腹腔注射,用8W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TGFβ1(5ng/ml)诱导的凋亡,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Hoechst33342对正常肝细胞进行了染色,并在荧光显微镜下观察比较caspase3和caspase8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TGFβ1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TGFβ1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58.76%和18.03%,两组具有明显的差别。但凋亡可以被caspase3和caspase8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-8和caspase-3的活化诱导鼠肝细胞产生凋亡。

Objective:The aim of present study was to determine the relationships between apoptosis induced by TGF-β1 and caspase-3 as well as caspase-8.Methods:Liver cirrhosis was induced in BALB/c mice by means of injection with 50% carbon tetrachloride for 8W. Hepatocytes were isolated from normal and cirrhotic mice by means of an in situ perfusion method with collagenase solution with minor modification. The induction of apoptosis by TGF-β1 was determined in these hepatocytes by electrophoresis on 1.5% agarose gel to observe DNA ladder. To detect apoptosis after treated with TGF-β1 (5ng/ml), normal hepatocytes were stained with fluorescent DNA-binding dyes Hoechst 33342 and viewed under fluorescent microscope to investigate the effects of caspase-3 and caspases-8 on TGF-β1-induced apoptosis. Results:The viability of hepatocytes isolated from mice by in situ two step collagenase digestion was 95.2%. Internucleosomal fragmented DNA ladder was observed using a 1.5% agarose gel in TGF-β1 treated normal hepatocytes. In comparison, no obvious DNA ladder from cirrhotic hepatocytes was observed. The percentage of apoptotic rate in TGF-β1 treated normal hepatocytes stained with Hoechst 33342 (58.76%) were much higher than hepatocytes untreated with TGF-β1 (18.03%) and could be blocked by Z-IETD-FMK and DEVD-CHO. Conclusion:It was revealed that cirrhotic hepatocytes showed poor response to apoptosis induced by TGF-β1, indicating that inhibitory regulation of cell growth has been impaired in hepatocytes of cirrhotic liver. TGF-β1 can induce apoptosis of murine hepatocytes via caspase-8 and caspase-3-transducted signal pathway.