Superfect高效介导PDX-1基因在骨髓间质干细胞表达  

High expression of PDX-1 in bone marrow mesenchymal stem cells mediated by superfect

作　　者：孙吉平[1] 贾延劼[2] 王晓莉[1] 杨于嘉[1] 

机构地区：[1]中南大学湘雅医院儿科,湖南长沙410008 [2]郑州大学第一附属医院神经内科,河南郑州450000

出　　处：《中国病理生理杂志》2007年第1期12-17,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30200128)

摘　　要：目的:构建含有胰腺十二指肠同源框1(PDX-1)基因的真核表达载体,并提高重组载体在骨髓间质干细胞(MSCs)的表达效率,为骨髓MSCs作为糖尿病基因治疗的靶细胞奠定基础。方法:RT-PCR体外扩增PDX-1基因,双酶切后整合入相同双酶切的真核表达载体构建重组载体,对重组载体进行酶切分析和序列测定进行鉴定;利用Percoll细胞分离液体外培养大鼠骨髓MSCs,流式细胞仪检测细胞周期后,Superfect介导正确重组的载体转染骨髓MSCs,检测转染效率和细胞存活率从而对转染条件进行优化;G418筛选阳性细胞后,RT-PCR和Western blotting检测PDX-1基因在骨髓MSCs中的表达。结果:重组载体双酶切分析和序列测定证实重组载体构建成功。流式细胞仪显示85.9%的MSCs处于G0/G1期,提示骨髓MSCs具有强大的分化潜能;荧光显微镜下成功转染的细胞呈现全细胞绿色荧光,Superfect介导的转染效率和细胞存活率与其绝对用量和孵育细胞的时间有关;RT-PCR显示转染后PDX-1基因在骨髓MSCs表达,随转染时间延长,表达逐渐增强,与Western blotting结果一致。结论:成功构建了含有PDX-1基因的真核表达载体;Superfect能高效介导外源性基因在骨髓MSCs中表达;转染外源性基因的MSCs可望成为一种理想的基因治疗的载体细胞。AIM： To construct a eukaryotic expression vector containing pancreatic duodenal homebox - 1 （ PDX - 1 ） and to elevate the expression efficiency of exogenous gene in rat bone marrow mesenchymal stem cells （MSCs）. METHODS： Recombinant vector containing PDX - 1 was constructed. Flow cytometry was used to identify the cell cycle of bone marrow mesenchymal stem cells （MSCs） cultured in vitro. Recombinant vector containing PDX - 1 was transfected into bone marrow MSCs using superfect in medium. After being selected by G418, RT - PCR and Western blotting were used to investigate the expression of PDX - 1 in MSCs. RESULTS： Restricted enzyme analysis and sequencing showed that PDX - 1 gene segment was consistent with that in GenBank. Flow cytometry showed that there were about 85.9% cells at the cell cycle of G0/G1. The whole cells transfected emitted green fluorescence under flow cytometry. The efficiency of tranafection was above 40%. RT - PCR and Western blotting demonstrated that there was expression of PDX - 1 in transfected bone marrow MSCs. CONCLUSION： Recombinant vector containing PDX - 1 was constructed successfully. Superfect mediated expression of exogenous gene in bone marrow MSCs in a high efficiency, and bone marrow MSCs containing exogenous gene are an ideal cells for gene therapy.

关 键 词：骨髓间质干细胞 基因 胰腺十二指肠同源框1 糖尿病 

分 类 号：R363[医药卫生—病理学]