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探讨可溶性TRAIL蛋白对体外培养的正常人肝细胞增殖和凋亡的影响机制。应用体外培养的张氏肝细胞株,分别加入不同浓度的TRAIL蛋白,在不同时间点,用MTT法检测细胞增殖,Annexin V-FITC检测细胞凋亡,荧光显微镜及流式细胞仪检测线粒体损伤情况。结果表明,低浓度TRAIL对肝细胞生长抑制作用不明显,而中高浓度TRAIL作用3 d后对肝细胞有显著的抑制作用。再更换为培养液后,肝细胞生长抑制率均有明显降低。培养24 h后,100 ng/ml TRAIL诱导肝细胞早期凋亡率比对照组显著增加(P<0.05);培养48 h后,20、100 ng/ml TRAIL诱导肝细胞晚期凋亡率分别为(9.1%±2.6%)及(10.7%±2.5%),与对照组相比,具有统计学意义(P<0.05)。TRAIL作用12 h后荧光强度明显降低,随着作用时间延长,荧光减弱更明显,表明线粒体损伤严重。结果提示,高浓度TRAIL蛋白通过线粒体损伤途径,诱导张氏肝细胞凋亡。
To investigate the mechanism of soluble TRAIL on the proliferation and apoptosis of normal human hepatocytes cultured in vitro. Applying different concentrations of TRAIL protein in vitro, the cell proliferation was detected by MTT assay at different time points, apoptosis was detected by Annexin V-FITC, and mitochondrial damage was detected by fluorescence microscopy and flow cytometry . The results showed that low concentrations of TRAIL on hepatocyte growth inhibition was not obvious, and high concentrations of TRAIL 3 days after the liver cells had a significant inhibitory effect. After changing to culture solution, the inhibition rate of hepatocyte growth decreased obviously. After cultured for 24 h, the apoptosis rate of hepatocytes induced by 100 ng / ml TRAIL was significantly higher than that of the control group (P <0.05), and the apoptosis rate induced by TRAIL at 20,100 ng / ml after 48 h culture was ( 9.1% ± 2.6%) and (10.7% ± 2.5%) respectively, which was statistically significant compared with the control group (P <0.05). Fluorescence intensity of TRAIL after 12 h was significantly reduced, with the extension of time, the fluorescence decreased more significantly, indicating that mitochondrial damage is serious. The results suggest that high concentrations of TRAIL protein through the mitochondrial damage pathway, induce apoptosis of Zhang’s hepatocytes.