生长因子对大鼠肝细胞增殖和酶活性表达的影响

Effect of Growth Factors on Proliferation and Expression of Enzyme Activity of Rat Hepatocytes in Primary Culture

目的研究表皮生长因子 (EGF)、肝细胞生长因子 (HGF)、EGF联合HGF对大鼠原代培养肝细胞增殖和功能的影响 ,为肝细胞移植建立细胞库、提高逆转录病毒载体感染肝细胞的效率寻找适当的培养条件。 方法分离、培养肝细胞 ,采用3 H -TdR掺入试验测定肝细胞内DNA合成 ,应用细胞化学方法观察培养的肝细胞内葡萄糖 -6 -磷酸酶活性的表达。 结果培养肝细胞受EGF(1 0ng/ml)、HGF(5ng/ml)作用后 ,肝细胞内DNA合成分别增加了 5 9倍 (P <0 .0 1 )和 1 6倍 (P <0 .0 1 )。EGF和HGF联合应用不表现出明显的协同作用。培养的肝细胞可合成葡萄糖 - 6 -磷酸酶 ,具有肝细胞特异性功能。 结论提示EGF和HGF在体外可刺激肝细胞增殖并维持肝细胞功能 ,为肝细胞移植、肝病基因治疗提供了一个完善的肝细胞培养系统。

objective The study was undertaken to evaluate the effect of growth factors on DNA synthesis and function of adult rat hepatocytes maintained in primary culture, and to provide a useful, well defined culture system for establishment of a cell bank and for efficient, stable transduction of hepatocytes by replication-defective retroviruses. Methods The in situ two-step collagenase perfusion technique was used for the isolation of hepatocytes from the adult liver. Cells were maintained in culture on exposure to EGF, HGF, and EGF combined with HGF. 3 H-TdR incorporation was used to determine DNA synthesis. Results The expression of glucose-6-phosphatase activity was detected by the cytochemical procedure. EGF(10ng/ml) and HGF(5ng/ml) could stimulate the DNA synthesis of cultured hepatocytes significantly. The average increase in the control group was 59 times with EGF(P<0.01) and 16 times with HGF(P<0.01) respectively. No synergic effect was observed in hepatocytes treated with EGF plus HGF. Most of the cultured cells exhibited glucose-6-phosphatase activities. The presence of EGF and HGF in rat hepatocyte cultures can increase DNA synthesis and maintain hepatocyte function. Conclusion It suggests that the hepatocyte cultures described in the present study may be a useful, optimal culture system for the hepatocyte transplantation and liver gene therapy.