CAJ | 학술논문

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.
배경:생물형인공간채용저간세포혹간암세포작위이식물래원존재동물원성질병화치류성적담심,이정상성인간세포야구유일정적국한성.목적:통과검측전염전、후정상성인간세포적활솔、생장곡선화세포주기적변화,료해전염SV40영생화기인대정상성인간세포생장특성적영향.방법:배양불동시간적정상인간세포화영생화정상성인간세포,채용태반람염색화AO-PI염색,우배양후1～8 d채용MTT염색법계수세포활솔;용MTT비색법측정세포적A치병회제생장곡선;채용류식세포술검측세포적생장주기.결과여결론:3충염색법검측현시량충세포적활솔위95%～99%,존활솔무명현차이.전염전、후적량충정상성인간세포적생장곡선무명현차이,균재배양3～5 d시정지수형생장,단전염후정상성인간세포교전염전증식략쾌.채용류식세포술측득적량충간세포적세포주기:전염후간세포S기세포점65.64%,G_0～G_1기세포점34.36%,G_2기세포점0%;전염전간세포S기점21.27%,G_0～G_1기세포점62.64%,G2기세포점12.09%,제시전염후간세포적S기세포명현증다,증식능력증강.전염SV40영생화기인적정상성인간세포교전염전세포적증식활력증고,존활솔무명현차이,제시전염후세포증식능력경강.
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.