Lentivirus介导表达多基因的人胚基因工程神经干细胞的实验研究

The experimental study of genetic engineering human neural stem cell expressing multigene mediated by Lentivirus

目的:探索以Lentivirus为载体,构建同时携带并表达多基因的基因工程人胚神经干细胞(hum an neu鄄ral stem cell,hNSC)的可行性,为脊髓损伤治疗的研究提供材料。方法:培养和鉴定hNSC;用携带绿色荧光蛋白(green fluorescence protein,GFP)和神经营养因子-3(neurotrophic factor-3,NT-3)的Lentivirus转染hNSC;用荧光显微镜观察、鼠胚背根神经结培养(dorsal root ganglion,DRG)和Slot blot等方法检测基因工程hNSC的多基因表达情况。结果:培养获得了大量的hNSC;荧光显微镜观察到几乎100%的hNSC表达GFP;基因工程hNSC的培养液能促使大鼠DRG旺盛生长;Slot blot检测到基因工程hNSC能高效分泌NT-3蛋白。结论:以Lentivirus为载体能构建同时携带并稳定表达多基因的基因工程hNSC,为脊髓损伤治疗的基础研究及进一步临床应用提供了有价值的细胞资源。

Objective:To explore the feasibility to construct the genetic engineering human neural stem cell(hNSC) which were mediated by Lentivirus to express multigene,and afford the material for the further studies of spinal cord injury.Method:After the hNSC were isolated and identified,the hNSC were geneticly modified by Lentivirus expressing green fluorescence protein(GFP) and neurotrophic factor-3(NT-3).The transgenic complex expression were examined by the methods of fluorescence microscope,culturing with dorsal root ganglion of fetal rats and slot blot.Result:The genetic engineering hNSC was constructed successfully.Expression of bright green fluorscence were detected in all genetic engineering hNSC by the fluorescence microscope.The conditioned medium with transgenic hNSC could induce neurite flourishing outgrowth of dorsal root ganglion(DRG).The genetic engineering hNSC expressed high level of NT-3 which could be detected by the methods of slot blot.Conclusion:The genetic engineering hNSC mediated by Lentivirus expressing multigene can be applied widely in the basic research and the further clinic application as neural grafts for spinal cord injury will be great.