产甲酸草酸杆菌基因frc和OXC在人骨髓间充质干细胞中的表达

Expressions of frc and oxc genes of oxalobacter formigenes in human bone marrow mesenchymal stem cells

目的:将产甲酸草酸杆菌(OXF)草酸分解基因frc和oxc分别克隆到逆转录病毒载体pLEGFP-N1和pBaBE-puro,共转染正常成人骨髓间充质干细胞(hMSCs),使frc和oxc基因在hMSCs中稳定表。方法:以本实验室保存的OXF基因组为模板,PCR法扩增出frc和oxc基因的编码序列,采用逆转录病毒载体将frc和oxc导入hMSCs中,Q-PCR和Western blot检测其表达。结果:重组载体pLEGFP-N1-myc-frc、pBaBE-puro-flag-oxc构建成功;并检测出frc和oxc基因在hMSCs中的稳定表达。结论:逆转录病毒载体介导的frc和oxc能成功转染hMSCs,能为以后体外诱导分化为肝细胞,治疗内源性高草酸尿奠定基础,为下一步的实验提供原材料。

Objective To investigate the expressions of oxalate-degredation genes frc and oxc of oxalobaeter formigenes （OXF） in human bone marrow mesenchymal stem cells （hMSCs）. Methods Genome of OXF saved in our laboratory was as the mould, the frc and oxc genes were amplified by PCR, and were cloned into retrovirus vectors pLEGFP-N1 and pBaBE-puro. The expressions of frc and oxc genes in hMSCs were detected by Q-PCR and Western blot. Results The recombinant retrovirus vectors pLEGFP-Nl-myc-frc and pBaBE-puro-flag-oxe were successfully constructed. The frc and oxc genes could be stably expressed at mRNA and protein level in hMSCs. Conclusion Frc and oxc genes could be transfected into normal hMSCs by retrovirus vectors successfully.