体外培养人胎肝细胞与永生化L-02肝细胞的生物学性状比较

Comparison of the biological features between human fetal hepatocyte and immortalized L-02 hepatocyte in vitro

目的 对比体外培养的人胎肝细胞与L 0 2肝细胞形态学及增殖分化特征的异同 ,初步评价其用于移植的可行性 ,探讨介入性肝细胞移植术的理想供体来源。方法 分离培养 14～ 2 4周的引产胎儿肝细胞 ,放射免疫法测定上清液中甲胎蛋白 (AFP)与白蛋白 (ALB)含量 ,免疫细胞化学法检测细胞角蛋白 19(CK 19)的表达。同法检测传代培养L 0 2肝细胞的蛋白表达。结果 人胎肝细胞分离活率达 95 % ,在体外存活最长达 3周 ,可同时检测到AFP、ALB及CK 19表达 ,其中ALB分泌峰值 4 2 0 6 μg/ml;L 0 2肝细胞增殖迅速 ,AFP与CK 19表达阴性 ,ALB表达在 10 μg/ml水平 ,部分细胞多次传代后发生形态变异 ,ALB表达缺失。结论 人胎肝细胞具有潜在的双向分化能力 ,是肝细胞移植较为合适的供体来源 ;L 0 2肝细胞不适用于移植。

Objective To evaluate the feasibilities of the potential donors in liver cell transplantation using the human fetal hepatocytes and immortalized L-02 hepatocytes by comparing their biological features. Methods Human fetal hepatocytes were isolated from aborted fetal livers (gestational ages from 14 w to 24 w) by an improved two-stage perfusion method and cultured in a conditioned medium without any growth factors.α-fetal protein (AFP) and albumin (ALB) were detected by radioimmunoassay (RIA) and cytokeratin-19 (CK-19) was identified by cellular immunochemistry study.Immortalized L-02 hepatocytes were cultured in the same condition and the characteristic proteins were detected by the same methods.Results The viability of human fetal hepatocytes was approximately 95% using the perfusion method, and the maximum survival time of the cultured hepatocytes was 3 weeks.The expression of AFP, ALB, and CK19 was detected at the same time, especially during Day 3 to Day 7 in the culture.By comparison, the proliferation ability of L-02 hepatocyte was greater, although with a lower level of ALB secretion.The expression of AFP and CK19 was not detected.Furthermore, during the long culture, L-02 hepatocytes may undergo a morphologic change and fail to express ALB.Conclusion Human fetal hepatocyte may be a practical donor for hepatocyte transplantation with its high-level protein expression and potential bi-differentiation ability.In view of the absent expression of ALB and the morphologic change in culture, although with better proliferation, L-02 hepatocyte seems not useful for hepatocyte transplantation.