腺病毒介导神经营养因子基因在骨髓间充质干细胞中表达及意义  

Adenovirus -mediated neurotrophic factor gene expression and significance in bone marrow mesenchymal stem cells

作　　者：黄成[1] 吴朗 朱银圣[3] 卢宏栩 吴颖[4] 王静成[5] 冯新民[5] 杨建东[5] 

机构地区：[1]如皋市人民医院骨科,江苏226500 [2]扬州大学临床医学院,225001 [3]如皋市人民医院神经内科,江苏226500 [4]如皋市人民医院普外科,江苏226500 [5]苏北人民医院骨科,扬州225001

出　　处：《中华实验外科杂志》2016年第1期175-178,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金面上项目（NSFC81071466）;江苏省六大人才高峰（2014-WSN-076）;南通市青年医学人才科研基金（WQZ2015010）;扬州市“科教兴卫工程-学术技术带头人”

摘　　要：目的采用基因转染方法，以增强骨髓间充质干细胞（BMSCs）内源性胶质细胞源性神经营养因子（GDNF）基因表达，观察BMSCs在内源性GDNF作用下分化。方法分离、纯化大鼠BMSCs，流式细胞术鉴定细胞表型分子。实验组以载鼠源性GDNF基因、绿色荧光蛋白（GFP）基因重组腺病毒（Ad-rGDNF—GFP）转染第3代BMSCs，即BMSCsrGDNF—GFP转染组，对照组分别为BMSCsGFP转染组、未转染组（BMSCsNull组）。转染72h后察各组中BMSCs对GFP的表达。荧光定量聚合酶链反应（qPCR）比较各组BMSCs对GDNF基因表达水平。噻唑蓝（MTT）法测定各组细胞活力。分别于转染后3、7d对比观察各组BMSCs形态变化，免疫荧光检测各组BMSCs对神经核蛋白（NeuN）及低分子神经丝蛋白（NF-L）的表达。结果全骨髓贴壁法可获取梭形贴壁细胞，低表达表型分子CD11b（1．4％）、CD34（1．5％），高表达表型分子CD29（99．8％）、CD90（95．6％）；转染72h后，BMSCsrGDNF．GFP转染组、BMSCsGFP转染组均表达GFP，未转染组无GFP表达；统计学分析qPCR结果表明BMSCsrGDNF—GFP转染组细胞GDNF基因表达水平高于BMSCsGFP转染组与BMSCsNull组，且差异有统计学意义；统计学分析MTT检测结果表明BMSCsrGDNF-GFP转染组细胞活力于转染后3d显著高于对照组。转染后3、7d，免疫荧光鉴定结果证实BMSCsrGDNF—GFP转染组中细胞表达NeuN、NF—L，并呈现出较为典型的神经样细胞形态，对照组细胞无NeuN、NF—L表达，且细胞基本维持原梭形形态。结论Ad—rGDNF—GFP转染BMSCs增强细胞内源性GDNF表达后，BMSCs可向神经样细胞定向分化，并保持较高的细胞活力。Objective It has been demonstrated that bone marrow stem cells have multi -lineage differentiation potential. Previous studies have found that it could differentiate into neural - like cells induced by neurotrophic factor. The aims of this article are to enhance the expression of endogenous glial cell linederived neurotrophic factor （GDNF） of bone marrow mesenchymal stem cells （BMSCs） with the method of gene transfection and observe the neural - like differentiation of BMSCs under the effect of endogenous GDNF. Methods BMSCs were isolated from the SD rats with the method of direct adherence. Surface phenotype assays for ceils were identified by flow cytometry. The BMSCs at passage 3 were transfected by recombinant adenovirus. In experiment group, the cells were transfected by recombinant adenovirus containing the gene of rGDNF and green fluorescent protein （ BMSCsrGDNF - GFP group）. In control groupl, the cells were transfected with GFP （BMSCsGFP group）. In control group2, the cells were not transfected （ BMSCsNull group）. Expression of GFP was observed after transfection for 72 h. Expression of GDNF from BMSCs was detected by Realtime fluorescence quantitative PCR（ Q - PCR） at the different time - point after transfection. Immunofluorescence was used to detect theexpression of the neuronal marker NeuN and NF - L at 3 and 7d after the transfection. The viability of cells was measured and analyzed by statistical method after the transfection. Results The BMSCs could he acquired by the method of whole marrow direct adherence. Phenotypic cell surface marker analysis of P3 BMSCs using flow cytometry found that BMSCs highly expressed CD29 （99.8%）, CD90 （95.6%）, and expressed low levels of CD11b （ 1.4% ） and CD34（ 1.5% ）. Adenovirus - mediated gene transfer could improve expression of endogenous GDNF and the viability of BMSCs. The resuh of immunofiuorescence indicated that the BMSCs could express the maker of neurons and showed significant morphology of neural - like cells with

关 键 词：骨髓间充质干细胞 胶质细胞源性神经营养因子 分化 神经元 转染 

分 类 号：R651.2[医药卫生—外科学]