摘要
目的观察无血清微载体培养对永生化人肝细胞(IHH)凝血因子mRNA和蛋白表达的影响,以进一步提高生物人工肝的安全性。方法实验分为有血清微载体培养IHH组和无血清微载体培养IHH组,分别培养0、5、10、15、20 d后观察两组细胞的增殖情况;应用RT-PCR和Western-blot方法检测两种培养方式对IHH凝血因子I、II、V、VII、VIII、IX、X、XI的mRNA和蛋白表达的影响。结果经0、5、10、15、20 d培养后,镜下两组细胞同时间点细胞密度相似,细胞活力差异无统计学意义(P>0.05)。RT-PCR结果显示两组细胞均能表达FI、FII、FV、FVII、FVIII、FIX、FX、FXI的mRNA,无血清培养组mRNA表达量相对较高(P<0.05)。Western-blot结果显示两组细胞均可表达以上8种凝血因子蛋白,两组细胞的FI、FII、FVIII、FX、FXI蛋白表达量差异无统计学意义(P>0.05),无血清培养组FV、FVII、FIX的蛋白表达量相对增高(P<0.05)。结论成功建立无血清微载体培养IHH体系,且无血清微载体培养对IHH凝血因子的产生影响不大。
Objective To observe the impact on the proliferation and production of blood coagulation factors in immortalizedhuman hepatocyte（IHH）,and further improve the safety of bio-artificial liver. Methods This experiment was divided into2 groups ：the group of IHH cultured with serum micro-carriers and the group of IHH cultured with serum-free micro-carriers. After culturing for 0,5,10,15 and 20 d,proliferation of cells were measured. The expression levels of mRNAand protein for factor I（FI）,factor II（FII）,factor V（FV）,factor VII（FVII）,factor VIII（FVIII）,factor IX（FIX）,factor X（FX）and factor XI（FXI）were determined by RT-PCR and Western blot. Results After culturing for 0,5,10,15 and 20 d,the cell density of the hepatocytes in the 2 groups were almost similar at the same time point. No significantdifference was found in the cell activities of the 2 groups（P〉0.05）. RT-PCR results showed that two groups cells could ex-press mRNA of FI,FII,FV,FVII,FVIII,FIX,FX and FXI,NS the expression levels of these mRNA were significantlyhigher in serum-free culture group（P〈0.05）. Western blot results showed that cells of the 2 groups could produced thesecoagulation factors proteins,and there was no significant differences in FI,FII,FVIII,FX and FXI between the two groups（P〉0.05）,while the levels of FV,FVII and FIX were significantly higher in serum-free group（P〈0.05）. Conclusion This study successfully established a serum-free micro-carrier culture IHH system and serum-free micro-carrier culture has lit-tle effect on the generation of coagulation factors.
出处
《热带医学杂志》
CAS
2017年第4期430-434,F0004,共6页
Journal of Tropical Medicine
基金
国家高技术发展研究计划(863计划)(2006AA02Z482)
关键词
无血清微载体培养
永生化人肝细胞
凝血因子
Serum-free micro-carrier culture
Immortalized human hepatocytes
Blood coagulation factors
