胶质细胞源性营养因子及内皮素B受体基因共转染小鼠神经干细胞(英文)

Cotransfection of glial cell-derived neurotrophic factor and endothelin receptor type B gene into mouse neural stem cells

背景:胶质细胞源性神经营养因子与内皮素B受体基因的缺失将导致肠神经系统发育异常。神经干细胞移植不仅可以从解剖和功能上修复神经系统,还可以作为基因转染的载体。目的:拟将携带胶质细胞源性神经营养因子和内皮素B受体基因的重组腺病毒转染至小鼠神经干细胞,并观察目的基因的表达。设计:细胞-基因学观察。材料:新生昆明小鼠由华中科技大学同济医学院动物实验中心提供。jetPEI转染试剂为PolyPlus公司产品;携带绿色荧光蛋白的胶质细胞源性神经营养因子、内皮素B受体基因共表达腺病毒由本室孙念峰博士和张景辉博士构建并惠赠。方法:无菌条件下取新生小鼠脑组织,制备单细胞悬液。取携带绿色荧光蛋白的胶质细胞源性神经营养因子和内皮素B受体基因共表达腺病毒,溶解于NaCl中制备JetPEI/DNA复合体。用DMEM/F12完全培养基调整次代神经干细胞密度为5×108L-1,向24孔板中每孔加入400μL细胞悬液、100μLJetPEI/DNA复合体,置37℃、体积分数为5%的CO2培养箱中,分别于转染24,48,72h后收集神经干细胞。主要观察指标:采用荧光显微镜、流式细胞仪检测转染效率,RT-PCR检测目的基因在神经干细胞内的表达。结果:转染24h后即可在荧光显微镜下观察到绿色荧光蛋白的表达,转染24,48,72h后绿色荧光蛋白阳性率分别为15.36%,24.67%,25.73%。各转染时间点神经干细胞均表达胶质细胞源性神经营养因子及内皮素B受体基因,转染24h条带亮度较低,72h时外源基因表达水平最高。结论:运用jetPEI试剂成功将目的基因转染至小鼠神经干细胞内,且胶质细胞源性神经营养因子和内皮素B受体基因在靶细胞中得到有效转录和表达。

BACKGROUND：Deletion of glial cell-derived neurotrophic factor and endothelin receptor type B gene will induce abnormal development of enteric nervous system.Neural stem cell transplantation can repair nervous system from anatomy and function,and be considered as a vector of gene transfection.OBJECTIVE：To transfect recombinant adenovirus carrying glial cell-derived neurotrophic factor and endothelin receptor type B gene into mouse neural stem cells,and to observe expression of target gene.DESIGN：A cell-gene study.MATERIALS：New-born Kunming mice were provided by the Animal Center of Tongji Medical College,Huazhong University of Science and Technology,China.jetPEI reagent was purchased from PolyPlus Co,France.The pAdTrack-CMV-GE with green fluorescent protein （GFP） was gifted by Doctor Sun Nianfeng and Zhang Jinghui in our laboratory.METHODS：Neonatal mouse brain tissues were sterilely obtained to prepare monoplast suspension.Adenovirus expressing glial cell-derived neurotrophic factor and endothelin receptor type B gene with GFP was dissolved in NaCl to prepare JetPEI/DNA complex.Subcultured neural stem cells in DMEM/F12 were regulated to 5×10^8/L,and 400 μL cell suspension and 100 μL JetPEI/DNA complex were seeded on a 24-well plate at 37 ℃ in 5% CO2 incubator.Neural stem cells were harvested at 24,48 and 72 hours following transfection.MAIN OUTCOME MEASURES：The efficiency of transfection was detected using fluorescence microscope and flow cytometry.Target gene expression in neural stem cells was determined using RT-PCR.RESULTS：Bright green fluorescence of the transfected cells could be observed under fluorescence microscope after 24 hours of transfection.The positive rate of GFP was 15.36%,24.67%,25.73% at 24,48 and 72 hours following transfection respectively.Neural stem cells expressed glial cell-derived neurotrophic factor and endothelin receptor type B gene at various time points.Strap brightness was low at 24 hours,and exogenous gene expression was great at 72 hours.CONCLUSION：The target genes were successfully transfected into neural stem cells by using jetPEI reagent.Moreover,glial cell-derived neurotrophic factor and endothelin receptor type B gene effectively transcribed and expressed in target cells.