转染SV40永生化基因对正常成人肝细胞生长特性的影响

背景:生物型人工肝采用猪肝细胞或肝癌细胞作为移植物来源存在动物源性疾病和致瘤性的担心,而正常成人肝细胞也具有一定的局限性.目的:通过检测转染前、后正常成人肝细胞的活率、生长曲线和细胞周期的变化,了解转染SV40永生化基因对正常成人肝细胞生长特性的影响.方法:培养不同时间的正常人肝细胞和永生化正常成人肝细胞,采用胎盘蓝染色和AO-PI染色,于培养后1～8 d采用MTT染色法计数细胞活率;用MTT比色法测定细胞的A值并绘制生长曲线;采用流式细胞术检测细胞的生长周期.结果与结论:3种染色法检测显示两种细胞的活率为95%～99%,存活率无明显差异.转染前、后的两种正常成人肝细胞的生长曲线无明显差异,均在培养3～5 d时呈指数型生长,但转染后正常成人肝细胞较转染前增殖略快.采用流式细胞术测得的两种肝细胞的细胞周期:转染后肝细胞S期细胞占65.64%,G_0～G_1期细胞占34.36%,G_2期细胞占0%;转染前肝细胞S期占21.27%,G_0～G_1期细胞占62.64%,G2期细胞占12.09%,提示转染后肝细胞的S期细胞明显增多,增殖能力增强.转染SV40永生化基因的正常成人肝细胞较转染前细胞的增殖活力增高,存活率无明显差异,提示转染后细胞增殖能力更强.

Effects of SV40 immortalized gene transfection on growth characteristics of normal adult hepatocytes

BACKGROUND:Bioartificial liver using porcine hepatocytes or hepatoma cells as sources of transplanted material encounter the danger of zoonotic disease or oncogenicity. The normal adult hepatocytes also have some limitations. OBJECTIVE: To detect the survival rate, growth curve and cell cycle of normal adult hepatocytes before and after transfection,and to observe the effects of SV40 immortalized gene transfection on growth characteristics of normal adult hepatocyte. METHODS: Cultured normal adult hepatocytes and immortalized normal adult hepatocytes were performed placenta blue staining and AO-PI staining. MTT staining was used to count the cell survival rate at days 1-8 after culture. MTT assay was used to measure absorbance value of cells, and to draw cell growth curves. The cell growth cycle was detected by flow cytometry. RESULTS AND CONCLUSION: The three kinds of staining assay showed that the survival rates of the two kinds of cells were 95%-99%, which had no significant difference. The two normal adult hepatocytes had no obviously difference in growth curves, both of which presented as exponential growth at days 3-5 after culture, but the cells grew faster after transfection. Flow cytometry results demonstrated that: after transfection, 65.64% cells were in the S phase, 34.36% cells in the G_0-G_1 phase, and no cells in G_0 phase; however, S phase cells accounted for 21.27%, G_0-G_1 phase cells accounted for 62.64%, G_2 phase cells accounted for 12.09% before transfection. This suggested that the S-phase cells were markedly increased in hepatocytes after transfection, and the proliferation capacity was enhanced. Compared with normal adult hepatocytes, the SV40 immortalized gene trasfected cells exhibit stronger proliferative activity.