HNF4α真核表达载体的构建及其在人脐带间充质干细胞中的表达  

Construction of HNF4α eukaryotic expression vector and its expression in human umbilical cord mesenchymal stem cells

作　　者：禹亚彬[1] 杭化莲[1] 卞建民[1] 

机构地区：[1]南京医科大学附属南京医院普外科,江苏南京210006

出　　处：《南京医科大学学报（自然科学版）》2012年第10期1356-1360,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金资助(81100306)

摘　　要：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 plasmid pcDNA3.1/HNF4a,transfect it into human umbilical cord mesenchymal stem cells （HUMSCs） ,and detect the expression in HUMSCs. Methods：pcDNA3.1/HNF4a vector was constructed with recombinant DNA technology. After detected by enzyme digestion and DNA sequencing,the plasmid was transfected into HUMSCs. The expression of HNF4a was detected simultaneously by using RT-PCR and Western blot. The hepatic specific protein ALB and AFP was detected by immunofluorescence after one week＇s transfeetion. Results：The plasmid peDNA3.1/HNF4a was constructed successfully. RT-PCR and Western blot results confirmed that HUMSCs expressed HNF4a after transfection. The green fluorescent protein could be identified by immunofluorescence after one week＇s transfection. Conclusion：Recombinant eukaryote plasmid pcDNA3.1/HNF4a was successfully constructed, and the HNF4a protien can be expressed in HUMSCs.

关 键 词：质粒构建 HNF4α 人脐带间充质干细胞 肝细胞分化 

分 类 号：R329.26[医药卫生—人体解剖和组织胚胎学]