双基因重组腺病毒载体的构建及转染大鼠骨髓间充质干细胞治疗脊髓损伤  

Treatment of spinal cord injury by transfection of double gene recombinant adenovirus vector into rat bone marrow mesecnhymal stem cells

作　　者：徐艳梅[1] 孙飞[2] 惠春影 王维[4] 

机构地区：[1]辽宁医学院附属第一医院重症医学科,辽宁省锦州市121001 [2]辽宁医学院附属第一医院急诊科,辽宁省锦州市121001 [3]辽宁医学院附属第一医院心脑外科,辽宁省锦州市121001 [4]辽宁医学院附属第一医院康复医学科,辽宁省锦州市121001

出　　处：《中国组织工程研究》2014年第23期3645-3652,共8页Chinese Journal of Tissue Engineering Research

基　　金：辽宁省科技厅社会发展处项目资助(2012408002)~~

摘　　要：背景:脑源性神经生长因子对多巴胺能神经元、胆碱能神经元等多种神经元都有广泛作用,能促进干细胞更多的向神经元样细胞分化;低氧诱导因子1α可提高组织和细胞在缺血环境下的生存能力,维持局部成血管微环境方面比任何基因均有更广泛的生理作用。目的:通过构建脑源性神经生长因子联合三点突变型低氧诱导因子1α双基因重组腺病毒载体,探索其转染大鼠骨髓间充质干细胞后2种基因在体外常氧条件下对脊髓损伤促神经再生及血管新生的作用。方法:①利用PCR方法定点突变人低氧诱导因子1α编码区的第402、564和803位氨基酸,将突变后低氧诱导因子1α基因联合脑源性神经生长因子重组入腺病毒pAdEasy-1系统,包装病毒并测定滴度。②以4种病毒液连同空白组共分5组进行后续实验;将病毒液转染入骨髓间充质干细胞内观察转染效率,检测各组转染细胞中脑源性神经生长因子基因及低氧诱导因子1αmRNA和蛋白表达情况。③进一步通过Western blot方法检测各组细胞中低氧诱导因子1α的下游成血管基因血管内皮生长因子蛋白表达情况。结果与结论:①编码区第402、564和803位氨基酸均定点突变为丙氨酸;4种腺病毒重组体构建成功并包装鉴定完毕。②实验组、阳性对照组1脑源性神经生长因子mRNA及蛋白表达量明显高于其他组(P<0.05);实验组、阳性对照组2细胞内低氧诱导因子1αmRNA及蛋白表达量、血管内皮生长因子蛋白表达均明显高于其他3组(P<0.05)。结果说明单载体双基因腺病毒系统转染骨髓间充质干细胞后不仅能够在常氧条件下大量且高效表达脑源性神经生长因子及低氧诱导因子1α蛋白,还同时能够促进其下游血管内皮生长因子的高效表达,为基因联合细胞移植治疗脊髓损伤提供了一种新的方向。BACKGROUND：Brain-derived neurotrophic factor （BDNF） has widespread effects on dopaminergic neurons, cholinergic neurons and other neurons, which can promote more differentiation of stem cells into neuron-like cells. Hypoxia inducible factor-1α（HIF1α） can improve tissue and cellviability in the ischemic environment and maintain the local microenvironment of hemangioblasts, which has a more far-ranging physiological role than genes. OBJECTIVE：By constructing double gene recombinant adenovirus vector with BDNF and three points mutant HIF1αto complete the transfection of adenovirus vector into rats mesenchymal stem cells and then study the promoting nerve regeneration and angiogenesis effect of these two genes to spinal cord injury in vitro in constant oxygen conditions. METHODS：（1）We finished the site-directed mutagenesis of 402, 564 and 803 amino acids in human HIF1αgene CDS region by PCR method and we finished recombination of mutation posterior HIF1αgene and BDNF into adenovirus pAdEasy-1 system. Packaging viral and titration determination were also finished. （2）Four kinds of virus fluids and blank group were selected for subsequent experiments. We observed transfection efficiency after transfection of virus into bone marrow mesenchymal stem cells and detected the expressions of BDNF and HIF1αmRNA and protein in transfection cells. （3） The protein expression of vascular endothelial growth factor, downstream formation vascular gene of HIF1α, in cells in al groups was detected by western blot assay. RESULTS AND CONCLUSION：（1）The site-directed mutagenesis of 402, 564 and 803 amino acids to alanine in coding sequence region was successful. The construction of four kinds of adenoviral recombinants was successful and identification of packaging was completed. （2）The level of BDNF gene mRNA and protein expression in experimental and positive control 1 groups was significantly higher than that in the other groups （P〈0.05）. The level of HIF1αmRNA and protein expression in

关 键 词：干细胞 干细胞移植 骨髓间充质干细胞 脊髓损伤 脑源性神经生长因子 低氧诱导因子1Α 血管内皮生长因子 

分 类 号：R394.2[医药卫生—医学遗传学]