重组人硫氧化还原蛋白对原代培养肝细胞的影响

目的探讨重组人硫氧化还原蛋白(rhTrx)对原代培养肝细胞生存生长的影响。方法逆转录聚合酶链反应法扩增出hTrx cDNA,并在原核表达系统中表达。IgM还原实验和胰岛素还原实验测定rhTrx的生物学活性。3H胸腺嘧啶核苷(TdR)掺入试验和细胞乳酸脱氢酶(LDH)释放试验检测rhTrx对SD大鼠原代培养肝细胞生存生长的影响。结果克隆出hTrx开放阅读框cDNA并获得高效表达,可溶性目的蛋白回收率为23．20％,蛋白纯度为96．30％。IgM还原和胰岛素还原实验均显示制备的rhTrx具有较好的生物学活性(t=7．5860,P<0．01)。加入rhTrx培养的肝细胞生长旺盛,并维持了良好的细胞形态。rhTrx可促进原代培养肝细胞的DNA合成,并以剂量依赖方式明显抑制乙醇造成的肝细胞损害。结论制备出具有生物学活性的rhTrx,hTrx对原代培养肝细胞的生存具有促进作用。

Effects of recombinant human thioredox on primary cultured hepatocytes

Objective To investigate the effects of recombinant human thioredox ( rhTrx) on primary cultured hepatocytes. Methods The cDNA coding hTrx was amplified by using RT-PCR. The objective protein was expressed in E. Coli. The activity of purified rhTrx protein was determined by IgM and insulin disulfide reduction assay. DNA synthesis was determined by 3H-TdR mixing assay to realize the proliferating roles of rhTrx in murine primary cultured hepatocytes. Cell viability was assessed by lactate dehydrogenase ( LDH) releasing assay. Results The cloning hTrx open reading frames cDNA was confirmed by sequence analysis. The soluble recombinant hTrx are expressed efficiently in the prokaryotic expression system. The recovery ratio was 23. 20% of total bacteria protein with a purity of the expressive protein of 96. 30%. The rhTrx had stronger IgM and insulin reduced activity compared with the controls (P<0.01). Hepatocytes exposed to rhTrx was growing luxuriantly. DNA synthesis was accelerative with times as determined by H-TdR mixing assay. rhTrx suppressed the cytotoxicity induced by ethanol in a dose-dependent manner as determined by LDH releasing assay. Conclusion The soluble rhTrx was expressed efficiently in E. Coli with biological activity which enhances the survival and growth of primary cultured hepatocyte.