低氧诱导因子1α基因三点突变体体外转染骨髓间充质干细胞  

In vitro transfection of triple-point mutants of hypoxia-inducible factor 1 alpha into bone marrow mesenchymal stem cells

作　　者：惠春影 肖洪艳[2] 何欣玲 王维[3] 

机构地区：[1]辽宁医学院附属第一医院心脑外科,辽宁省锦州市121001 [2]辽宁医学院附属第一医院介入科,辽宁省锦州市121001 [3]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19期2953-2960,共8页Chinese Journal of Tissue Engineering Research

基　　金：辽宁省科学技术厅社会发展处资助项目(2012408002)~~

摘　　要：背景:脊髓损伤后只有尽早地在损伤局部构建有效的血管网络,才能为各种细胞的分化提供营养支持和代谢保证,加速损伤局部的愈合。目的:构建三点突变的低氧诱导因子1α重组腺病毒表达载体,将其转染大鼠骨髓间充质干细胞后检测常氧条件下对脊髓损伤促血管新生的作用。方法: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基因重组入腺病毒pAdEasy-1系统,包装病毒并测定滴度,同理包装未突变组和空病毒组;以3种病毒液连同空白组共分4组进行后续实验。将病毒液转染入大鼠骨髓间充质干细胞内,通过示踪因子增强型绿色荧光蛋白观察病毒转染效率,通过RT-PCR方法检测4组细胞中低氧诱导因子1α基因mRNA表达情况;进一步通过Western blot方法检测4组细胞中低氧诱导因子1α基因及其下游成血管基因血管内皮细胞生长因子的蛋白表达情况。结果与结论:编码区第402,564和803位氨基酸均定点突变为丙氨酸;3种腺病毒重组体构建成功并包装鉴定完毕。含突变基因病毒液组、含未突变基因病毒液组低氧诱导因子1αmRNA表达量明显高于空病毒液组、空白组(P<0.05)。含突变基因病毒液组细胞低氧诱导因子1α蛋白及血管内皮细胞生长因子蛋白表达量显著高于其他3组(P<0.05)。提示三点突变后低氧诱导因子1α基因不仅能够在常氧条件下大量且高效表达,还同时能够促进其下游血管内皮细胞生长因子基因的高效表达,为脊髓损伤疾病的血管新生治疗提供了一种新的治疗方向。BACKGROUND：The incidence of spinal cord injury is increasing year by year in China so that the construction of effective vascular network in local injury as soon as possible is the guarantee of metabolism and nutritional support to differentiation of al kinds of cels and healing of injury is also promoted by vascular network. OBJECTIVE：To study the effect of triple-point mutants of hypoxia-inducible factor1α （HIF1α） to promote angiogenesis after spinal cord injury in normoxic condition. METHODS：Site-directed mutagenesis of 402, 564 and 803 amino acids in human HIF1α coding sequence area was completed by PCR, and the adenovirus pAdEasy-1 system was recombined with post-mutation HIF1α gene. Packaging viral and titration determination of experimental group was completed and the same was done to non mutation group and control virus group. The future experiment was continued with three virus groups and blank group （A group： including mutation HIF1α gene virus liquid; B group： including non mutation HIF1α gene virus liquid; C group： including control virus liquid; D group： blank group）. Then, virus liquid was transferred into nbsp;bone marrow mesenchymal stem cels. We observed transfection efficiency of virus by enhanced green fluorescent protein and to detect mRNA and protein expression of HIF1α gene in al transfection cels. We also detected protein expression of vascular endothelial growth factor acting as downstream angiogenesis gene of HIF1α in four groups by Western blot. RESULTS AND CONCLUSION：Three adenoviral recombinants were successfuly constructed and the packaging and identification were accomplished. The site-directed mutations of 402, 564 and 803 amino acids in coding sequence area were successful and al of them were changed to alanine. The level ofHIF1α mRNA expression in both A group andB group were significantly higher than that in the C group and D group （P〈0.05）. The expression levels of HIF1α and vascular endothelial growth factor proteins in A group was

关 键 词：干细胞 骨髓干细胞 骨髓间充质干细胞 脊髓损伤 低氧诱导因子1Α 基因突变 血管内皮细胞生长因子 

分 类 号：R394.2[医药卫生—医学遗传学]