NT-3真核过表达载体的构建及其在大鼠MSCs中的表达  

Construction of Eukaryotic Overexpression Vector of NT-3 and Its Expression in Rats Mesenchymal Stem Cells

作　　者：赵元淑 邓镇[1] 马猛[2] 罗亚楠[3] 朱晓琴[2] 雷水生[3] 

机构地区：[1]广州医科大学机能学实验中心,广东广州511436 [2]广州医科大学生理教研室,广东广州511436 [3]广州医科大学附属第五医院皮肤科,广东广州510700

出　　处：《临床医学工程》2016年第2期158-160,共3页Clinical Medicine & Engineering

基　　金：广东省省级科技计划项目(No.2013B021800191;No.2014A020212512);广州市属高校科研计划资助项目(No.2012c128;No.2012D001)

摘　　要：目的构建p CDNA3.1-NT-3真核过表达载体,鉴定其在大鼠骨髓间充质干细胞(MSCs)中的表达。方法从SD大鼠脑中提取并扩增NT-3基因,并将其克隆入p CDNA3.1中,构建p CDNA3.1-NT-3真核载体;原代培养大鼠间充质干细胞,将p CDNA3.1-NT-3通过Lip2000介导转染至大鼠MSCs;用RT-PCR、免疫荧光法、Western blot法检测NT-3在MSCs中的表达。结果扩增的NT-3基因序列正确,并成功连接到真核载体p CDNA3.1中;转染MSCs后,RT-PCR、免疫荧光、Western blot结果显示MSCs中过表达NT-3的m RNA和蛋白。结论 p CDNA3.1-NT-3真核载体构建成功,转染MSCs后能在细胞中正确过表达,为进一步NT-3基因修饰的MSCs治疗癫痫的研究奠定了实验基础。Objective To construct the eukaryotic overexpression vector of NT-3 and analyze its expression in rats mesenchymal stem cells（MSCs）. Methods The NT-3 gene was extracted from the brain of SD rats and amplified by PCR to clone into p CDNA3.1, thus constructing the eukaryotic vector of p CDNA3.1-NT-3. The rat MSCs was given primary culture, the recombinant plasmid p CDNA3.1-NT-3was transfected into MSCs via Lip2000. The expression of NT-3 in MSCs was detected by RT-PCR, Western blot method and immunofluorescence method. Results The amplified NT-3 gene sequence was correct, and had been correctly cloned into the eukaryotic expression vector p CDNA3.1. After the transfection of MSCs, the results of RT-PCR, immunofluorescence method and Western blot method showed m RNA and protein of NT-3 overexpression in MSCs. Conclusions p CDNA3.1-NT-3 eukaryotic expression vector is successfully constructed, and has correct overexpression in cells after the transfection of MSCs. The results establish the experimental foundation for further MSCs in treatment of epilepsy with NT-3 genetic modification.

关 键 词：NT-3 骨髓间充质干细胞 癫痫 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]