过表达胶质细胞神经营养因子基因转染骨髓间充质干细胞移植治疗脊髓损伤  

Transplantation of bone marrow mesenchymal stem cells overexpressing glial cell line derived neurotrophic factor gene for spinal cord injury

作　　者：黄成[1] 刘元兵[1] 戴永平[1] 王亮亮[1] 崔益华[1] 杨建东 Huang Cheng;Liu Yuanbing;Dai Yongping;Wang Liangliang;Cui Yihua;Yang Jiandong(Department of Orthopedics,Rugao People’s Hospital,Rugao 226500,Jiangsu Province,China;Department of Spine Surgery,Northern Jiangsu People’s Hospital,Yangzhou 225000,Jiangsu Province,China)

机构地区：[1]如皋市人民医院骨科,江苏省如皋市226500 [2]苏北人民医院脊柱外科,江苏省扬州市225000

出　　处：《中国组织工程研究》2020年第7期1037-1045,共9页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81071466)，项目负责人：杨建东;扬州市十三五科教兴卫领军人才计划资助(LJRC20182)，项目负责人：杨建东;如皋市科研计划项目(SRG(15)3015)，项目负责人：黄成~~

摘　　要：背景:胶质细胞神经营养因子(glial cell line derived neurotrophic factor,GDNF)在诱导骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)体外定向分化及促进脊髓损伤大鼠神经功能恢复过程中起到重要作用。目的:观察过表达GDNF基因的BMSCs分化情况及其促进脊髓损伤大鼠神经功能恢复的潜在分子机制。方法:①以重组目的基因腺病毒转染BMSCs并分为Ad-GDNF-GFP转染组、Ad-GFP转染组、未转染组,免疫荧光鉴定各组细胞神经元特异性烯醇化酶及微管相关蛋白2的表达,Western blot检测各组细胞GDNF、Wnt3a、Wnt7a蛋白表达。②以改良Allen法制备大鼠脊髓损伤模型,将造模成功的45只SD大鼠随机分为3组,分别以过表达GDNF基因BMSCs(GDNF-BMSCs)、BMSCs、PBS移植至脊髓损伤局部。移植后4周采用BBB评分法评估大鼠运动功能恢复情况,苏木精-伊红染色观察脊髓形态变化,免疫组化检测损伤局部神经元特异性烯醇化酶、突触素Ⅰ及胶质纤维酸性蛋白表达,Western blot检测损伤局部Bcl-2、肿瘤坏死因子α蛋白表达。结果与结论:①Ad-GDNF-GFP转染组BMSCs可向神经元样细胞形态转变并表达神经元特异性烯醇化酶、微管相关蛋白2;Wnt3a、Wnt7a蛋白表达量显著高于Ad-GFP转染组、未转染组;②移植后4周,GDNF-BMSCs移植组大鼠BBB评分明显提高、脊髓空洞面积显著缩小。GDNF-BMSCs移植组脊髓损伤局部胶质纤维酸性蛋白、肿瘤坏死因子α表达量显著低于BMSCs移植组及PBS移植组,而神经元特异性烯醇化酶、突触素Ⅰ及Bcl-2表达量显著高于BMSCs移植组、PBS移植组;③结果表明,Wnt信号通路参与过表达GDNF基因BMSCs向成熟神经元分化过程,移植后通过降低脊髓损伤局部炎症反应、减少细胞凋亡及胶质瘢痕形成、促进轴突再生,提高BMSCs移植治疗脊髓损伤的疗效。BACKGROUND:Glial cell line derived neurotrophic factor(GDNF)plays an important role in inducing differentiation of bone marrow mesenchymal stem cells(BMSCs)in vitro and promoting neurological function recovery in rats with spinal cord injury.OBJECTIVE:To observe potential molecular mechanisms of differentiation of BMSCs overexpressing GDNF gene and promoting neurological function recovery after spinal cord injury in rats.METHODS:(1)BMSCs transfected with recombinant target gene adenovirus were divided into Ad-GDNF-GFP transfection group,Ad-GFP transfection group and non-transfection group.Microtubule-associated protein 2 and neuron-specific enolase expression levels were detected by immunofluorescence in each group.Western blot assay was used to detect the expression of GDNF,Wnt3a and Wnt7a protein in each group.(2)The rat spinal cord injury model was prepared by modified Allen method.The 45 Sprague-Dawley rat models were randomly divided into three groups.GDNF-BMSCs,BMSCs and PBS were transplanted into the site of spinal cord injury.The motor function recovery of rats was evaluated 4 weeks after operation.The morphological changes of spinal cord were observed by hematoxylin-eosin staining.The local neuron-specific enolase,Synapsin Ⅰ and glial fibrillary acidic protein were analyzed with immunohistochemistry.The expression levels of Bcl-2 and tumor necrosis factor-α protein were detected by western blot assay.RESULTS AND CONCLUSION:(1)BMSCs overexpressing GDNF gene could differentiate into neuron-like cells and express neuron-specific enolase and microtubule-associated protein 2 in vitro in the Ad-GDNF-GFP transfection group.The expression of Wnt3a and Wnt7a protein was significantly higher in the Ad-GDNF-GFP transfection group than in the Ad-GFP transfection group and non-transfection group.(2)The Basso,Beattie and Bresnahan score in GDNF-BMSCs group was significantly increased and the stenosis area was significantly reduced at 4 weeks after transplantation.The expression of glial fibrillary acidic protein an

关 键 词：胶质细胞源性神经营养因子 骨髓间充质干细胞 细胞分化 WNT信号通路 脊髓损伤 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]