MicroRNA-9-1慢病毒载体的构建及其对小鼠骨髓间质干细胞诱导分化为神经细胞的影响*景黎君，贾永林，鲁晶晶，韩瑞，王舒阳，李尽义，彭涛，贾延劼△（郑州大学第一附属医院神经内科，河南郑州450052）[摘要]目的：探讨microRNA-9-1在体外诱导小鼠骨髓间充质干细胞（MSCs）向神经细胞分化中的作用。方法:构建小鼠microRNA-9-1慢病毒载体（microRNA-9-1-LV）并感染小鼠MSCs，筛选最适感染复数（MOI）；实验分为未感染组、感染组（感染microRNA-9-1-LV组）、阴性对照组（感染FU-RNAi-NC-LV）；采用β-巯基乙醇诱导感染后小鼠MSCs分化为神经细胞。倒置荧光显微镜下观察MSCs感染后荧光表达情况；采用免疫细胞化学染色检测神经元烯醇化酶（NSE）、神经微管结合蛋白（MAP-2）和胶质纤维酸性蛋白（GFAP）的表达变化；PT-PCR检测MAP-2mRNA的表达变化；MTT方法检测细胞存活率。结果：(1)阳性克隆PCR证明小鼠microRNA-9-1慢病毒载体构建成功，孔稀释法测定病毒滴度为1×1012TU/L。(2)倒置显微镜下观察小鼠microRNA-9-1慢病毒载体感染成功，MOI值为20，感染4d时感染率最高，细胞存活率较高，感染率可达91.3%±4.2%。(3)β-巯基乙醇可以诱导MSCs向神经细胞分化，其中以感染组诱导效果最佳，NSE和MAP-2的表达率显著高于其它各组（P<0.05）。结论：(1)microRNA-9-1可高效感染小鼠MSCs。(2)感染miRNA-9-1-LV后MSCs经β-巯基乙醇诱导向神经细胞分化比率增加。[关键词]MicroRNA-9-1；骨髓间充质干细胞；神经元；慢病毒[]R329.21[文献标识码]A*[基金项目]国家自然科学基金资助项目（No.30770758）；河南省教育厅自然科学研究计划项目（No.2008A320032）---本文来源于网络，仅供参考，勿照抄，如有侵权请联系删除---△通讯作者Tel:0371-66295112;E-mail:激ayan激e1971@yahooEffectoflentiviralvectorofmicroRNA-9-1ondifferentiationofmousebonemarrowmesenchymalstemcellsintoneurons激NGLi-jun,激AYong-lin,LU激ng-激ng,HANRui,WANGShu-yang,LI激n-yi,PENGTao,激AYan-激e(DepartmentofNeurology,TheFirstAffiliatedHospital,ZhengzhouUniversity,Zhengzhou450052,China.E-mail:激ayan激e1971@yahoo)[ABSTRACT]AIM:ToinvestigatetheroleofmicroRNA-9ininducingbonemarrowmesenchymalstemcells(MSCs)todifferentiateintoneurons.METHODS:ThelentiviralvectorofmicroRNA-9-1(microRNA-9-1-LV)wasconstructedandtransfectedintomouseMSCs.Thecellsweredividedintonon-transfectedgroup,transfectedgroup(transfectedwithmicroRNA-9-1-LV)andnegativecontrolgroup(transfectedwithFU-RNAi-NC-LV).MSCsweretreatedwithβ-mercaptoethanol(β-ME)asaninducerfortriggeringthecellstodifferentiateintoneurons.ThefluorescenceexpressedbytransfectedMSCswereobservedunderinvertedfluorescencemicroscope.ThemRNAexpressionofmicrotubulin-associatedprotein2(MAP-2)wasdetectedbyRT-PCR.Theexpressionofneuralcellspecificmarkers,neuron-specificenolase(NSE),MAP-2andglialfibrillaryacidicprotein(GFAP),weremeasuredbyimmunocytochemicalmethod.TheviabilityofMSCswasdeterminedbyMTTmethod.RESULTS:TheresultsofPCRconfirmedsuccessfulconstructionofmousemicroRNA-9-1-LV.Thevirustiterwas1×1012TU/L(TU,transductionunit).Thebesttransfectionefficiency(upto91.3%±4.2%)andsurvivalrateappearedwhenmultiplyofinfection(MOI)was20andonthe4thday.β-MEinducedMSCstodifferentiateintoneuronsandthebestefficiencyoftheinductionwasobservedintransfectedgroup.TheexpressionlevelsofNSEandMAP-2intransfectedcellswerehigherthanthoseinthecellsofothergroups(P<0.05).CONCLUSION:MicroRNA-9-1-LVhashightransfectionefficiencyinmouseMSCs.HigherdifferentiationratefrommouseMSCstoneuronsisinducedbyβ-MEafterthecellsaretransfectedwithmicroRNA-9-1-LV.[KEYWORDS]MicroRNA-9-1;Marrowmesenchymalstemcells;Neurons;Lentivirus---本文来源于网络，仅供参考，勿照抄，...