人胎肝细胞分离与培养技术的研究

The researches on isolation and culture technique of human fetal hepatocytes

目的 探讨分离及培养人胎肝细胞的最佳方法。方法 比较两种灌流消化法对分离人胎肝细胞的效果 ,观察不同条件对体外培养人胎细胞生长及生存时间的影响。结果 经腹主动脉及脐静脉双灌流消化法分离人胎肝细胞效果明显优于单纯经脐静脉灌流消化法 ,前者肝细胞获得率为 (5.97± 1 .81 )× 1 0 8/kg体重 ,后者肝细胞获得率为 (4 .1 2± 2 .3 1 )× 1 0 8/kg体重 (P <0 .0 5)。促肝细胞生长素对体外培养的人胎肝细胞生长和生存时间无明显影响 ,均在培养第 1 0～ 1 4天开始变性、坏死。鼠尾胶可延长体外培养人胎肝细胞的生存时间达 2 0～ 2 8d。与球体细胞培养方法相结合 ,人胎肝细胞在体外生存时间延长至 3 0～ 3 8d。结论 人胎肝细胞的分离应首选经腹主动脉及脐静脉双灌流消化法。人胎肝细胞在体外生存时间受培养条件的影响。

Objective To explore the optimal method for isolate and in vitro culturing hepatocytes from human fetus.Methods Two kinds of irrigation digestive methods for isolating human fetal hepatocytes (HFH) were compared to evaluate their isolation efficiency and observe the influence of some circumstances on growth and survival time of HFH in vitro culture.Results The irrigation digestive method by abdominal aorta and umbilical vein was marked superior to that by umbilical vein alone on isolating HFH.The production rate of hepatocytes in the former was (5.97±1.81)×10 8/kg body weight, that in the latter was (4.12±2.31)×10 8/kg body weight(P<0.05).Promoting hepatocyte growth factor (PHGF) didn't present significant influence on growth and survival time of HFH in vitro culture.Whether PHGF was added into the culture medium or not, HFH would present degeneration and /or necrosis on the 10?th 14?th day after the culture.Rat tail gel technique could prolong survival time of HFH in vitro culture to 20 28 days.The combined technique of rat tail gel and cellular sphere culture could prolong the survival time of HFH in vitro culture to 30 38 days.Conclusion Double irrigation digestive method via abdominal aorta and umbilical vein should first be selected in isolating HFH.Survival time of HFH in vitro culture was influenced by cultured conditions.