Construction of recombinant adenovirus expressing BDNF and its expression in expanded rat mesenchymal stem cells in vitro
LI Hong-le1, LI Hao-wei1, XING Fei-yue2, SUN Xue-gang2, DENG Yu-bin1, ZHANG Xiu-ming1,2, JANG Yong1
1. Department of Pathophysiology, Sun Yat-sen University of Medical Sciences, Guangzhou 510080, China;
2. Department of Pathophysiology, Key Lab for Shock and Microcirculation of PLA, The First Military Medical University, Guangzhou 510515, China
Abstract：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.
李红乐, 李浩威, 邢飞跃, 孙学刚, 邓宇斌, 张秀明, 姜勇, 李树浓. BDNF重组腺病毒的构建及在大鼠骨髓间质干细胞的表达[J]. 中国病理生理杂志, 2003, 19(4): 438-442.
LI Hong-le, LI Hao-wei, XING Fei-yue, SUN Xue-gang, DENG Yu-bin, ZHANG Xiu-ming, JANG Yong, LI Shu-nong. Construction of recombinant adenovirus expressing BDNF and its expression in expanded rat mesenchymal stem cells in vitro . Chin J Pathophysiol, 2003, 19(4): 438-442.
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