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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: The absence of glial cell line-derived neurotrophic factor and endothelin B receptor genes leads to abnormalities in the development of the intestinal nervous system. Neural stem cell transplantation can not only repair the nervous system anatomically and functionally, but also serve as a vector for gene transfection. OBJECTIVE: To transfect the recombinant adenovirus carrying glial cell line-derived neurotrophic factor and endothelin B receptor gene into mouse neural stem cells and observe the expression of the target gene. Design: Cell-genetics observation. Materials: Newborn Kunming mice were provided by Animal Experiment Center, Tongji Medical College, Huazhong University of Science and Technology. The jetPEI transfection reagent is a product of PolyPlus company. The glial cell line-derived neurotrophic factor carrying green fluorescent protein and the adenovirus expressing endothelin B receptor gene are constructed and donated by Dr. Sun Nianfeng and Dr. Zhang Jinghui. Methods: The brain tissue of newborn mice was taken under aseptic conditions to prepare single cell suspension. GFP-gDNF and ET-1 gene were co-expressed with adenovirus and dissolved in NaCl to prepare JetPEI / DNA complex. The density of the secondary neural stem cells was adjusted to 5 × 10 8 L -1 with DMEM / F12 complete medium, and 400 μL of cell suspension and 100 μL of JEEPI / DNA complex were added to each well of the 24-well plate and incubated at 37 ° C. with 5% CO 2 In the box, neural stem cells were harvested 24, 48 and 72 hours after transfection respectively. MAIN OUTCOME MEASURES: Transfection efficiency was detected by fluorescence microscopy and flow cytometry, and the expression of the target gene in neural stem cells was detected by RT-PCR. Results: The expression of GFP was observed under fluorescence microscope 24h after transfection. The positive rates of GFP were 15.36%, 24.67% and 25.73% respectively at 24, 48 and 72 hours after transfection. The neural stem cells expressed glial cell line-derived neurotrophic factor and endothelin B receptor gene at each time point of transfection. The brightness of the transfected cells was lower at 24h and reached the peak at 72h. Conclusion: The target gene was successfully transfected into mouse neural stem cells by using jetPEI reagent, and the glial cell line-derived neurotrophic factor and endothelin B receptor gene were efficiently transcribed and expressed in target cells.