人微小抗肌萎缩蛋白基因转染大鼠骨髓间充质干细胞及其基因表达  

Transfection and in vitro expression of human microdystrophin gene in rat mesenchymal stem cells

作　　者：王淑辉[1] 张成[1] 陈松林[1] 于美娟[2] 张雅妮[1] 李美山[1] 熊符[2] 尚延昌[1] 冯善伟[1] 申本昌[1] 

机构地区：[1]中山大学附属第一医院神经内科,广东广州510080 [2]中山大学干细胞与组织工程研究中心,广东广州510080

出　　处：《南方医科大学学报》2006年第3期261-265,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(30370510;30170337);广东省自然科学基金(31693);卫生部临床重点项目基金(2001321);高等学校博士学科点专项科研基金(20030558058);教育部骨干教师基金(082004)~~

摘　　要：目的构建人微小抗肌萎缩蛋白基因(microdystrophin)的真核表达载体,转染大鼠骨髓间充质干细胞(rMSC),研究其体外表达情况。方法限制性酶切PBSK-MICRO质粒的微小抗肌萎缩蛋白基因片段,插入到真核表达质粒pcD-NA3.1(+)的NotI位点内,克隆出真核表达载体pcDNA3.1(+)/micro dystrophin。通过酶切和测序对质粒进行鉴定。用脂质体将重组质粒转染入rMSCs中,经过G418筛选后,用RT-PCR及间接免疫荧光技术检测表达产物。结果pcDNA3.1(+)/micro dystrophin经过NotI、Hind III酶切鉴定及测序证实插入片断正确。提取转染重组质粒rMSCs的总RNA,进行RT-PCR可见mRNA的转录;对G418筛选后rMSCs进行免疫荧光检测可见细胞内亮丽的红色荧光,说明转染后的rMSCs有微小抗肌萎缩蛋白的表达。结论成功构建了人microdystrophin基因真核表达载体,将其转染入rMSCs内有微小抗肌萎缩蛋白的表达,为进一步研究体外修饰自体干细胞移植治疗DMD奠定了基础。Objective To consla＇uct the eukaryotic expression vector of human microdystrophin gene and observe its expression in rat mesenchymal stem cells （rMSCs） in vitro. Methods The plasmid PBSK-MICRO containing human microdystrophin cDNA was digested by restriction endonuclease, and the resultant microdystrophin fragment was inserted into the NotI site of pcDNA3.1 （＋） to prepare the eukaryotic expression vector-pcDNA3.1 （＋）/microdystrophin, which was identified by endonuclease digestion and sequencing. The recombinant plasmid was transfected into rMSCs via lipofectamine, and after G418 selection, the expression ofmicrodystrophin was detected by RT-PCR and indirect immunofluorescence assay. Results Microdystrophin gene fragment was correctly inserted into the plasmid pcDNA3.1 （＋）, as conformed by sequencing and digestion with Not I and Hind HI. The total mRNA of the transfected rMSCs was extracted and microdystrophin mRNA expression was found in the cells by RT-PCR. Indirect immtmofluorescence assay for the protein expression of microdystrophin showed bright red fluorescence in the transfected rMSCs. Conclusion Eukaryotic expression plasmid pcDNA3.1 （＋）/microdystrophin has been constructed successfully and microdystrophin can be expressed in transfected rMSCs in vitro, which may facilitate further research of Duchenne muscular dystrophy treatment by genetically modified allogeneic stem cell transplantation.

关 键 词：微小抗肌萎缩蛋白基因 真核表达 转染 骨髓间充质干细胞 

分 类 号：R346[医药卫生—基础医学]