人热休克蛋白72转染人脂肪间充质干细胞的研究  

Transfection and expression of human heat shock protein 72 gene mediated by adenoviral vector into human adipose derived stem cells

作　　者：冯浩[1] 张腾[1] 伍博[1] 易成刚[2] 赵建辉[2] 吴东辉[3] 贾晓敏[4] 

机构地区：[1]湖南师范大学第一附属医院皮肤科,湖南长沙410005 [2]第四军医大学西京医院全军整形外科研究所,陕西西安710032 [3]湖南师范大学第一附属医院整形美容科,湖南长沙410005 [4]湖南师范大学第一附属医院病理科,湖南长沙410005

出　　处：《中国现代医学杂志》2014年第33期19-22,共4页China Journal of Modern Medicine

基　　金：长沙市科技局基金(No:K1303034-31);湖南省发展改革委员会一般项目(No:2013-1199-3);湖南省研究生创新项目(No:CX2013B235)

摘　　要：目的观察人热休克蛋白72(HSP72)基因体外转染人脂肪间充质干细胞(ASCs)及其HSP72表达情况。方法通过腹部取皮术获得的皮下脂肪利用胶原酶消化法进行体外分离培养,体外扩增至第3代,用腺病毒载体系统将人源性HSP72转染至第3代ASCs,在倒置荧光显微镜下观察转染后的细胞形态和生长变化,应用实时荧光定量PCR、Western blot法鉴定HSP72在ASCs中的表达。结果皮下脂肪经消化,体外培养可获得ASCs P3代;ASCs转染24 h后,绿色荧光蛋白表达明显增强。经实时荧光定量PCR证实转基因ASCs表达HSP72m RNA在24 h后逐渐增强。Western blot检测证实转基因ASCs在24 h后出现特征条带,而空白组未出现条带。结论采用腺病毒介导的基因转染技术可将HSP72基因转染至ASCs中,并可以在HSP72m RNA和蛋白中有效表达,ASCs可以作为HSP72基因治疗的载体。[Objective ] To observe the expression of human heat shock protein 72 gene （HSP72） after being trans- fected into human adipose derived stem ceils （ASCs） in vitro. [Methods] ASCs were isolated by collagenase from intact fat of abdomen harvested from skin grafts and expand to the 3rd passage in vitro. The HSP72 gene was trans- fected into lt.SCs by adenovirus vector. The mRNA and protein expression of HSP72 gene in the transfected cells was analyzed by Real-Time Reverse Transcription PCR and Western blotting respectively. [ Results ] ASCs could be separated from human fat tissue and amplified in vitro. After transfection for 24 h, the expression of green fluores- cent protein was significantly increased. The Real-Time Reverse Transcription PCR and Western blotting revealed that there was HSP72 mRNA and protein expression in the transfected cells, and a significant positive zone of hy- bridization was visible at 70 kD. No positive band was found in control groups. [Conclusions] Gene transfer technology mediated by adenoviral vector can transfect human HSP72 gene into ASCs, which can stably express exogenous HSP72 mRNA and protein. ASCs can be used as the vector for genetic therapy of HSP72.

关 键 词：热休克蛋白72 脂肪干细胞 转染 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]