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目的:构建pcDNA3.1/HNF4α重组质粒,转染人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,HUMSCs),并检测其在HUMSCs中的表达。方法:采用基因重组技术构建载体pcDNA3.1/HNF4α,经酶切和DNA测序鉴定,通过脂质体法转染HUMSCs后,进行RT-PCR和Western blot分析,免疫荧光检测转染后1周肝特异性生化指标。结果:成功构建真核表达质粒pcDNA3.1/HNF4α,转染人脐带间充质干细胞后,RT-PCR示转染后HNF4αmRNA表达,Western blot分析见HNF4α蛋白表达,免疫荧光示转染1周后HNF4α促进了HUMSCs向肝细胞方向分化。结论:成功构建真核表达载体,并在HUMSCs中正确表达,为进一步研究HNF4α在干细胞向肝细胞分化中作用提供了实验基础。
OBJECTIVE: To construct pcDNA3.1 / HNF4α recombinant plasmids and transfect them into human umbilical cord mesenchymal stem cells (HUMSCs), and to detect their expression in HUMSCs. Methods: The recombinant plasmid pcDNA3.1 / HNF4α was constructed by gene recombination. The recombinant plasmid was identified by restriction enzyme digestion and DNA sequencing. The transfected HUMSCs were analyzed by RT-PCR and Western blot. Specific biochemical indicators. Results: The eukaryotic expression plasmid pcDNA3.1 / HNF4α was successfully constructed and transfected into human umbilical cord mesenchymal stem cells. The expression of HNF4α mRNA was detected by RT-PCR. The expression of HNF4α protein was detected by Western blot analysis. After 1 week of transfection, HNF4α promotes the differentiation of HUMSCs towards hepatocytes. CONCLUSION: The eukaryotic expression vector was successfully constructed and correctly expressed in HUMSCs, which provided the experimental basis for further studying the role of HNF4α in the differentiation of stem cells into hepatocytes.