中国组织工程研究

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (23): 3645-3652.doi: 10.3969/j.issn.2095-4344.2014.23.007

• 干细胞移植 stem cell transplantation • 上一篇    下一篇

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

徐艳梅1，孙 飞2，惠春影3，王 维4   

  1. 辽宁医学院附属第一医院，1重症医学科，2急诊科，3心脑外科，4康复医学科，辽宁省锦州市 121001
  • 修回日期:2014-03-08 出版日期:2014-06-04 发布日期:2014-06-04
  • 通讯作者: 王维，硕士，副教授，辽宁医学院附属第一医院康复医学科，辽宁省锦州市 121001
  • 作者简介:徐艳梅，女，1983年生，吉林省松原市人，汉族，2007年吉林大学护理学院毕业，护师，主要从事脊髓损伤的基因治疗。
  • 基金资助:

    辽宁省科技厅社会发展处项目资助(2012408002)

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

Xu Yan-mei1, Sun Fei2, Hui Chun-ying3, Wang Wei4   

  1. 1Department of Critical Care Medicine, 2Department of Emergency, 3Department of Cardiac and Brain Surgery, 4Department of Rehabilitation Medicine, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001, Liaoning Province, China
  • Revised:2014-03-08 Online:2014-06-04 Published:2014-06-04
  • Contact: Wang Wei, Master, Associate professor, Department of Rehabilitation Medicine, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001, Liaoning Province, China
  • About author:Xu Yan-mei, Senior nurse, Department of Critical Care Medicine, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001, Liaoning Province, China
  • Supported by:

     the grant from the Social Development Department of Liaoning Science and Technology Bureau, No. 2012408002

PDF

490

被引次数

3

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1α mRNA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1α mRNA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。


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程


全文链接：

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

Abstract:

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 cell viability 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 HIF1α to 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 HIF1α gene CDS region by PCR method and we finished recombination of mutation posterior HIF1α gene 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 HIF1α mRNA and protein in transfection cells. (3) The protein expression of vascular endothelial growth factor, downstream formation vascular gene of HIF1α, in cells in all 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 HIF1α mRNA and protein expression in experimental and positive control 2 groups was significantly higher than that of the other groups (P < 0.05). The level of vascular endothelial growth factor protein was also increased in the experimental and positive control 2 groups, which was significantly different from other groups (P < 0.05). These findings indicate that the BDNF and HIF1α proteins are largely and efficiently expressed in constant oxygen conditions after single vector double gene adenovirus system transfection into mesenchymal stem cells and high-efficiency expression of vascular endothelial growth factor protein is promoted so that this is a new direction for treatment of spinal cord injury by gene therapy combined with cell transplantation.


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程


全文链接：

Key words: stem cells, bone marrow, spinal cord injuries, nerve growth factor, vascular endothelial growth factors, adenoviruses

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