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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 clone the oxalate-degrading genes frc and oxc of OXF into the retroviral vectors pLEGFP-N1 and pBaBE-puro respectively and co-transfect them into normal human bone marrow mesenchymal stem cells (hMSCs) Stable table in hMSCs. METHODS: The coding sequence of frc and oxc genes were amplified by PCR from OXF genome in our laboratory. The hCRCs and oxc were introduced into hMSCs by retroviral vector. The expression of frc and oxc was detected by Q-PCR and Western blot. Results: The recombinant vectors pLEGFP-N1-myc-frc and pBaBE-puro-flag-oxc were successfully constructed. The stable expression of frc and oxc gene in hMSCs was detected. CONCLUSION: The retroviral vector-mediated transfection of frc and oxc into hMSCs can lay the foundation for the subsequent induction of differentiation into hepatocytes and treatment of endogenous hyperoxaluria, providing the raw materials for further experiments.