BDNF重组腺病毒的构建及在大鼠骨髓间质干细胞的表达  

Construction of recombinant adenovirus expressing BDNF and its expression in expanded rat mesenchymal stem cells in vitro

作　　者：李红乐[1] 李浩威[1] 邢飞跃[2] 孙学刚[2] 邓宇斌[1] 张秀明[1] 姜勇[2] 李树浓[1] 

机构地区：[1]中山大学中山医学院病理生理学教研室,广东广州510080 [2]第一军医大学病理生理学教研室和全军休克微循环重点实验室,广东广州510515

出　　处：《中国病理生理杂志》2003年第4期438-442,T001,共6页Chinese Journal of Pathophysiology

基　　金：国家"973"计划项目 (No.2 0 0 1CB5 0 990 4) ;广东省"十五"重大专项资助项目 (A30 2 0 10 1)

摘　　要：目的 :利用大肠杆菌细菌内同源重组构建带有增强型绿色荧光蛋白 (EGFP)报告基因的脑源性神经营养因子前体 (proBDNF)和脑源性神经营养因子 (BDNF)重组腺病毒并在大鼠骨髓间质干细胞 (rMSC)高效表达。方法 :采用两步亚克隆的方法将proBDNF和BDNF构建入带有EGFP表达盒的腺病毒穿梭质粒pAdTrack -CMV中 ,形成转移载体pAdTrack -proBDNF和pAdTrack -BDNF ,采用化学转化法在大肠杆菌BJ5 183内与腺病毒骨架质粒pAdEasy- 1同源重组 ,得到重组腺病毒载体pAd -proBDNF和pAd -BDNF ;转染 2 93细胞 ,包装成重组病毒颗粒 ;将重组病毒上清感染rMSC ,用荧光显微镜下观察和Western -blotting鉴定重组病毒在rMSC表达 ;用Ad -proBDNF和Ad -BDNF感染的rMSC在体外诱导向神经样细胞分化 ;用Ad -proBDNF和Ad -BDNF的感染的rMSC接种于裸鼠肌肉内 ,两周后荧光显微镜下直接观察。结果 :成功地构建了proBDNF和BDNF重组腺病毒载体并制备出高滴度重组病毒 ,重组病毒能在体外培养的rMSC高效表达并不影响其分化潜能 ,体内移植实验表明Ad -proBDNF和Ad -BDNF感染的rMSC能在体内表达。结论 :重组腺病毒具有较高的介导proBDNF和BDNF基因表达于rMSC的效率 ,带有报告基因EGFP的Ad -proBDNF和Ad -BDNF感染的rMSC可以用于体内移植实验。AIM: To construct recombinant adenovirus vector containing brain derived neurotrophic factor, (BDNF) gene using bacterial homogenous recombination, and investigate the expression in expanded rat mesenchymal stem cells (rMSC) in vitro. METHODS: BDNF gene and proBDNF gene were subcloned into adenovirus shuttle plasmid pAdTrack-CMV containing enhanced green fluorescent protein gene (EGFP) expression cassette, forming shuttle vector of pAdTrack-BDNF, and pAdTrack-proBDNF, and co-transformed into BJ5183 bacterial cells with adenovirus backbone vector pAdEasy-1 using chemical transformation. After the recombinant adenovirus vector was obtained, the identified recombinant adenovirus plasmid DNA was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. rMSC were infected by recombinant adenovirus and EGFP expression was detected using fluorescent microscope. Infection efficiency was assessed by flow cytometrics. Western blotting identified expression of Ad -proBDNF and Ad-BDNF in rMSC. rMSC infected with Ad -proBDNF and Ad-BDNF were induced to differentiate into neuron-like cells. rMSC infected with Ad -proBDNF and Ad-BDNF were injected into nude mice and assessd in vivo. RESULTS: We successfully constructed the recombinant adenovirus Ad -proBDNF and Ad-BDNF that expressed in expanded rMSC in vitro.CONCLUSION: Recombinant adenovirus high-effectively mediates Ad -proBDNF and Ad-BDNF expression in expanded rMSC in vitro and in vivo.

关 键 词：BDNF 重组腺病毒 构建 大鼠 骨髓间质干细胞 表达 增强型绿色荧光蛋白 基因融合 脑源性神经营养因子 

分 类 号：Q78[生物学—分子生物学] R338.8[医药卫生—人体生理学]