慢病毒介导骨形态发生蛋白7基因转染诱导大鼠骨髓间充质干细胞向神经元样细胞的分化  

Neuronal differentiation of rat bone marrow mesenchymal stem cells via lentivirus-mediated bone morphogenetic protein 7 transfection

作　　者：张文 雷堃 高磊[1] 李宽新[1] Zhang Wen;Lei Kun;Gao Lei;Li Kuanxin(Department of Joint and Spine Surgery,the Second Affiliated Hospital of the Medical College of Shihezi University(Xinjiang Production and Construction Corps Hospital),Urumqi 830000,Xinjiang Uygur Autonomous Region,China)

机构地区：[1]石河子大学第二附属医院(新疆生产建设兵团医院)关节脊柱外科

出　　处：《中国组织工程研究》2020年第7期985-990,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81560216)，项目负责人：李宽新~~

摘　　要：背景:骨髓间充质干细胞具备向神经元样细胞分化的潜能,已经被列为治疗脊髓损伤的首选干细胞,但其分化效率低,寻找一种具有高效诱导能力的因子尤为重要。基于文献查阅及课题组研究基础,推测骨形态发生蛋白7(bone morphogenetic protein 7,BMP-7)基因可能在促进骨髓间充质干细胞向神经元样细胞分化上发挥着重要作用。目的:探讨骨形态发生蛋白7慢病毒载体转染诱导大鼠骨髓间充质干细胞向神经元样细胞分化的作用。方法:采用全骨髓贴壁法培养SD大鼠骨髓间充质干细胞,以感染复数为50,25,10,1进行LV-GFP转染,转染后3 d在荧光倒置显微镜下观察各组GFP表达情况,确定最佳感染复数。取第3代骨髓间充质干细胞,分为空白对照组(常规培养)、LV-GFP组、LV-BMP-7-GFP组,以最佳感染复数转染24,48,72,96,120 h后,采用MTT法检测细胞存活率;转染3 d后,采用免疫细胞化学实验检测神经细胞标记物神经丝蛋白200、突触素1表达。结果与结论:①LV-GFP转染后3 d,感染复数为1组未见GFP阳性细胞,感染复数为10,25,50组可见GFP阳性细胞,其中感染复数为10组平均荧光强度高于其余组(P<0.05),为最适感染复数;②空白对照组、LV-GFP组神经丝蛋白200、突触素1表达呈阴性;LV-BMP-7-GFP组神经丝蛋白200、突触素1表达呈阳性,胞体和轴突被染成亮棕黄色;③结果表明,LV-BMP-7转染可促进骨髓间充质干细胞向神经元样细胞分化。BACKGROUND:Bone marrow mesenchymal stem cells have the potential to differentiate into neuron-like cells,which have been listed as the preferred stem cells for the treatment of spinal cord injury.However,due to their low differentiation efficiency,it is particularly important to find a factor with high induction ability.Based on literature review and our previous studies,it is speculated that bone morphogenetic protein 7(BMP-7)gene may play a vital role in promoting the differentiation of bone marrow mesenchymal stem cells into neuron-like cells.OBJECTIVE:To investigate the differentiation of rat bone marrow mesenchymal stem cells into neurons induced by BMP-7 lentivirus vector transfection.METHODS:Bone marrow mesenchymal stem cells of Sprague-Dawley rats were cultured by whole bone marrow adherence method,and then were transfected with LV-GFP when multiplicities of infection were 50,25,10,and 1.Green fluorescent protein expression was observed using fluorescence inversion microscope in each group at 3 days after transfection,to confirm the best multiplicity of infection.Passage 3 bone marrow mesenchymal stem cells were divided into blank control group(routine culture),LV-GFP group,and LV-BMP-7-GFP group,followed by transfection at the best multiplicity of infection.After 24,48,72,96,and 120 hours of transfection,MTT assay was used to detect cell survival rate in each group.Immunocytochemical assay was used to detect the expression of nerve cell markers(neurofilament protein 200,synaptophysin-1)after 3 days of transfection.RESULTS AND CONCLUSION:(1)After 3 days of LV-GFP transfection,GFP-positive cells were observed under fluorescence microscopy when multiplicities of infection were 10,25,and 50,whereas no GFP-positive cells were found when the multiplicity of infection was 1.The average fluorescence intensity was the highest when the multiplicity of infection was 10(P<0.05),indicating that multiplicity of infection=10 had the best infection effect.(2)Immunocytochemical results showed that the expression of neuro

关 键 词：骨形态发生蛋白7 骨髓间充质干细胞 神经细胞 细胞分化 神经丝蛋白200 突触素1 

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