肝细胞C3A的无血清培养研究  

A study of serum-free cultivation of human hepatocyte (C3A) cell line

作　　者：蔡理全[1] 赵逸超[2] 汪艳[1] 高毅[1] 

机构地区：[1]南方医科大学珠江医院肝胆二科,广东广州510280 [2]唐山工人医院肝胆外科,河北唐山063000

出　　处：《热带医学杂志》2013年第4期428-432,461,F0003,共7页Journal of Tropical Medicine

基　　金：国家"863"课题(2006AA02A141;2012AA020505);广东省教育部产学研结合项目(2007B090400094);广东省重大科技专项基金(2007A032100005);广东省全面战略合作引导项目(2011B090300015)

摘　　要：目的研制一种无血清培养基,并与其他三种常用无血清培养基进行比较,探讨自主研发无血清培养基在肝细胞培养上的可行性。方法以肝C3A细胞株为目标细胞,四组无血清培养基组均采取逐渐降低血清浓度方法(10%、5%、2.5%、0%),以达到完全无血清培养,与完全培养基组(阳性对照)及基础培养基组(阴性对照)在细胞活力、生长曲线、功能及相关功能基因上进行比较。结果细胞培养24 h后开始生长,并保持与阳性组相似的生长率。从第2天起,自主研发无血清培养基组尿素合成率就明显高于阳性及阴性对照组(P<0.001),在白蛋白合成上,实验组与阳性组比较差异无统计学意义。Real-time qPCR结果提示,在UGT、GST、AFP和ALB表达上,实验组与阳性组比较差异无统计学意义(P=0.640、0.075、0.504、1.000),但在CK18、CK19和HNF4α表达上,却明显低于阳性组(P<0.001)。结论新研发的无血清培养基在肝细胞培养中与完全培养基基本相同,能维持细胞形态、活力及主要功能,使肝细胞能更好地应用于生物人工肝治疗中。Objective To develop a serum-free culture condition for the cultivation of C3A human hepatocyte cell line. Methods C3A cells were cultivated under a stepwise condition of reducing the serum concentration from 10%, 5%, and 2.5% to 0%. The cells cultured in serum-supplemented medium （SSM） served as the positive control and the ceils cultured in basic medium served as the blank control. Cellular morphology was observed under an optical microscope, and the growth of C3A cell was tested by using CCK-8. Automatic biochemical analyzer was used to measure the level of ALT and urea in the culture supernatant. In addition, radioimmunoassay and real-time qPCR were adopted to analyze albumin secretion and gene expression. Results Cells began to growth after culturing for 24 h and the growth rates between the experimental group and the positive control groups were similar. The concentrations of urea in the SFM groups were significantly higher than the positive and blank control groups （P〈0.001） at day-2 after the cultivation. There was also a significant difference between the SFM groups and the control group in the albumin secretion. There was no significant difference in the gene expression of UGT,GST,AFP and ALB between the SFM groups and the positive control group （P=0.640, 0.075, 0.504, and 1.000, respectively）, but the expressions of CK18, CK19 and HNF4ct were significantly lower than the positive control group （P〈 0.001 ）. Conclusion A serum-free culture condition was developed for the cultivation of human C3A cells.

关 键 词：急性肝衰竭 细胞培养 无血清培养 

分 类 号：R575.3[医药卫生—消化系统]