肝细胞凋亡率改变对肝再生过程的调控作用研究

目的 探讨肝部分切除(partial hepatectomy,PH)后TGF-β1诱导的肝细胞凋亡率的改变在肝再生不同阶段中的作用.方法 建立70%肝切除模型,原位灌注分离正常和PH后1,3,5,7 d大鼠的肝细胞,流式细胞检测各组肝细胞的凋亡率;用DNA荧光染料Hoechst33258对分离培养的原代肝细胞进行染色,荧光显微镜观察TGF-β1(5 μg/L)诱导的凋亡;免疫细胞化学检测TGF-β1对原代肝细胞Bcl-2表达的影响.结果 分离肝细胞的活率达92%;正常肝细胞的凋亡率为(18.89±3.14)%,PH后1 d肝细胞凋亡率为(11.85±2.51)%,1 d后开始下降,第3天降至最低,而后逐渐升高,第7天为(28.82±5.53)%;正常大鼠肝细胞经TGF-β1作用后凋亡率为(58.13±6.63)%,高于对照组(17.81±3.19)%.有显著差异(P＜0.05);而PH后1,3,5 d分离的肝细胞经过和未经过TGF-β1诱导的凋亡率相比无差别;Bcl-2的表达在PH后早期(1～3 d)分离的肝细胞中逐渐升高;经TGF-β1作用后Bcl-2的表达明显下降.结论 TGF-β1诱导的肝细胞凋亡率在肝再生不同阶段有明显差异,对于促进和终止肝再生可能具有重要的调控作用.

Regulating effect of apoptotic rate of hepatocyte on course of hepatic regeneration

Objective To investigate the role of different apoptotic rate induced by TGF-β1 in different stages of hepatic regeneration after partial hepatectomy(PH).Methods Rat model of 70% PH was established.Primary hepatocytes were isolated by in situ perfusion with collagenase solution from normal rats and those 1 d,3 d,5 d,7 d after PH.Flow cytometry was used to detect apoptotic rate in each group.The apoptosis of primary hepatocytes induced by TGF-β1(5 μg/L)was determined by staining with fluorescent dyes of DNA-binding Hoechst33258 and observed under fluorescent microscope.The effect of TGF-β1 on Bcl-2 expression in hepatoeytes was detected with immunocytochemistry.Results The viability of primary hepatocytes was 92%.The apoptotic rate of normal hepatocyte was 18.89%±3.14%,the apoptotic rate in 1 d was 11.85%±2.51%.After that it began to decrease,it was the lowest on the 3rd d.Then it began to increase gradually,it was 28.82%±5.53% on day 7 after PH.The apoptotic rate of normal hepatocytes induced by TGF-β1 was 58.13%±6.63%,which was higher than that in control groups(17.81%±3.19%,P＜0.05).However,the apoptotic rate of isolated primary hepatocytes had no difference between the groups induced and not induced by TGF-β1 1,3,5 d after PH.Bcl-2 expression in hepatocytes isolated in the early stage after PH was gradually increased and higher than that of normal group.The Bcl-2 expression was decreased significantly after being treated with TGF-β1.Conclusion TGF-β1 induced apoptotic rate has a marked difference in different stages of hepatic regeneration and it possesses an important regulating role for initiation and termination of hepatic regeneration.