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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 best method to isolate and culture human fetal liver cells. Methods The effects of two kinds of perfusion digestion on human fetal hepatocytes were compared. The effects of different conditions on the growth and survival time of human fetal cells in vitro were observed. Results The isolation of human fetal liver cells by double perfusion of abdominal aorta and umbilical vein was superior to that of simple transvaginal umbilical vein digestion. The former achieved a rate of (5.97 ± 1.81) × 108 / kg body weight, The latter liver cell yield was (4.12 ± 2.31) × 108 / kg body weight (P <0.05). Hepatocyte growth-promoting factor had no significant effect on the growth and survival time of human fetal liver cells cultured in vitro. All of them were denatured and necrotic on day 10-14. Rat tail gel can extend the survival of human fetal liver cells in vitro survival time of 20 ~ 28 days. Combined with the method of spheroid cell culture, the survival time of human fetal liver cells in vitro extended to 30 ~ 38 days. Conclusion The separation of human fetal hepatocytes should be the first choice of abdominal aorta and umbilical vein by double perfusion digestion. Human fetal liver cells in vitro survival time by the culture conditions.