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目的通过将肝实质细胞分离、纯化、体外培养,全面分析原代肝细胞的生长状态,研究乙醛诱发大鼠肝细胞毒性和胞内游离Ca2+变化及Ca2+螯合剂的干预效应,探讨乙醛致肝细胞损伤机制及Ca2+螯合剂的保护作用。方法分离培养大鼠原代肝细胞,以含300mmol/L乙醛的培养基培养肝细胞24、48h,相差倒置显微镜下观察肝细胞的形态变化。结果相差倒置显微镜下,新分离的大鼠肝细胞折光性强,有立体感,乙醛处理肝细胞24h后肝细胞体积肿胀,颜色变深,颗粒粗糙,部分细胞脱壁,少量细胞出现细胞壁缺失,呈放星状;乙醛处理48h后,可见到肝细胞质变稀疏,细胞核固缩,表面出现胞膜疱,细胞膜破碎;相当一部分细胞破碎脱落可见到一些核已消失的肝细胞浆或残屑。结论①乙醇可导致严重的肝细胞极性紊乱,导致细胞损伤,形成不同程度的病理状态,从而在细胞及分子水平对导致肝病形成的机理进行了探讨;②乙醛诱发肝细胞毒性和损伤可能与肝细胞[Ca2+]i升高有关。
OBJECTIVE: To study the growth of primary hepatocytes by isolating, purifying and culturing hepatic parenchymal cells in vitro. To study the hepatotoxicity and intracellular free Ca2 + in rat induced by acetaldehyde and the intervention effect of Ca2 + chelator, Mechanism of hepatocyte injury and protective effect of Ca2 + chelator. Methods Primary rat hepatocytes were isolated and cultured, and hepatocytes were cultured for 24,48 h in medium containing 300 mmol / L acetaldehyde. The morphological changes of hepatocytes were observed under inverted phase contrast microscope. Results Under inverted phase contrast microscope, the newly isolated rat hepatocytes showed strong refraction and stereopsis. After treated with acetaldehyde for 24 hours, the hepatocytes became swollen and darker in color, with rough particles and part of the cells with detached cells and few cells with cell wall defects , Showing star; acetaldehyde treated 48h, we can see the liver cytoplasm thinning, nuclear pyknosis, the surface of the membrane blisters, the cell membrane broken; a considerable number of cells broken off to see some of the nucleus has disappeared hepatocyte plasma or debris . Conclusions ①Ethanol can cause severe hepatocyte polarity disorder, leading to cell injury, forming different degrees of pathological conditions, and thus at the cellular and molecular levels on the mechanism leading to liver disease were discussed; ② acetaldehyde induced hepatotoxicity and damage may And hepatocytes [Ca2 +] i increased.