Shh基因转染大鼠骨髓间充质干细胞的可行性  

Experimental study of rat mesenchymal stem cells transfected with Sonic Hedgehog gene

作　　者：杨进福[1] 吴晓明[1] 唐滔[1] 谭志平[1] 张伟[1] 

机构地区：[1]中南大学湘雅二医院心胸外科,长沙410011

出　　处：《中华医学杂志》2012年第10期705-708,共4页National Medical Journal of China

基　　金：国家自然科学基金（30871424）;教育部新世纪优秀人才支持计划（NCET-07-0871）;湖南省自然科学基金（11JJ7005）

摘　　要：目的探讨SonicHedgehog（Shh）基因有效转染大鼠骨髓间充质干细胞（BMSC）的可行性。方法构建pcDNA3．1-Shh真核表达质粒，酶切和测序鉴定。密度梯度离心．贴壁培养法获取Wistar大鼠BMSC，电击穿孔法将pcDNA3．1-Shh、pcDNA3．1（-）空质粒和pmaxGFP报告质粒转染进BMSC，48h后Western印迹法检测Shh基因的表达情况。结果酶切和测序结果证实pcDNA3．1．Shh正确性。转染48h后，荧光显微镜可观察到绿色荧光蛋白（GFP）的表达，有40％以上的细胞发出绿色荧光。Western印迹法检测证实有Shh基因表达，而转染pcDNA3．1（-）空质粒的BBMSC未检测到表达。结论重组真核表达质粒pcDNA3．1-Shh转染大鼠BMSC后能有效表达Shh基因，为进一步Shh基因与细胞联合治疗大鼠心肌缺血模型提供了实验依据。Objective To evaluate the feasibility of genetic modification of mesenchymal stem cells （MSC） with Sonic Hedgehog（Shh）gene. Methods The pcDNA3, l-Shh eukaryotic expression plasmid was constructed and its correctness evaluated by the restriction enzyme analysis and sequencing. MSC were isolated from Wistar rats by density gradient centrifugation and purified, transfected with pcDNA3.1-Shh, blank plasmid pcDNA3.1 （ - ） or pmaxGFP respectively by NucleofectorTM. The protein expression of Shh in MSC was detected by Western blot after 48 hours. Results Correct construction of pcDNA3.1-Shh was identified by the methods of restriction enzyme analysis and nucleotide sequence determination. The expression of green fluorescent protein （GFP） could be observed by fluorescence microscopy after 48 hours. The expression of Shh gene was detected by Western blot. But the MSC transfected with empty plasmid expression was not detected. Conclusions Recombinant Eukaryotic expression plasmid pcDNA3.1-Shh is successfully detected in rat MSC. It may provide experimental rationales for the future gene therapy.

关 键 词：基因 间质干细胞 基因疗法 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]