腺病毒载体Ad-BDNF-EGFP的构建和在神经干细胞中的表达

目的研究腺病毒载体Ad-BDNF-EGFP的构建及其在神经干细胞(NSCs)中的表达。方法通过RT-PCR从大鼠海马中获得BDNF基因,通过基因克隆、HEK293包装,获得含增强绿色荧光蛋白基因的重组腺病毒表达载体pAd-BDNF-EGFP,将其感染原代培养的神经干细胞,观察EGFP及BDNF2种基因的表达,镜下测定转染率,并检测RT-PCR产物,证实BDNF的存在。转染后的神经干细胞经G418筛选,抗性细胞传代扩增后获得成功转染BDNF基因的NSCs克隆。结果荧光显微镜下可见感染后的NSCs表达EGFP而发出绿色荧光;通过RT-PCR证明感染后的NSCs具有表达BDNF的能力;用ELISA鉴定细胞上清中分泌的BDNF,72h的含量达到最高值,为12.78ng/mL;证明通过构建病毒的感染可以使神经干细胞获得分泌BDNF的能力,且EGFP基因可作为神经干细胞移植研究中良好的示踪剂。结论腺病毒病毒介导EGFP基因及BDNF基因在大鼠胚胎神经干细胞中成功表达,为应用以神经干细胞直接作为基因靶细胞,介导基因治疗中枢神经系统疾病奠定了基础。

Construction of Ad-EGFP-BDNF vector and its expressing in NSCs

Objective] To transfect neural stem cells with adenovirus vector of BDNF and EGFP,and also to investigate the expression of BDNF and EGFP in transfected neural stem cells. Methods] The BDNF cDNA was gained by RT-PCR from rat hippocampus. After packaged by HEK293. BDNF and EGFP gene was transfected into cultured neural stem cells through Ad-EGFP-BDNF vector. The neural stem cells with BDNF cDNA gene were examined by RT-PCR and immunoreactivity methods. The result of RT-PCR was examined. NSCs clones with BDNF gene were successfully selected by G418. Results] BDNF and EGFP expressed in transfected neural stem cells for a long time. The neural stem cells infected by adenovirus were incubated. G418 resistant clones of NSCs gave off strikingly bright green fluorescence, and showed stable EGFP expression detected by immunohistochemistry staining. The quality of BDNF was analyzed by RT-PCR and the quantity of it was detected by ELISA The result of RT-PCR was the same as Genbank. It was confirmed that infected NSCs could express BDNF. The BDNF cumulant could arrive to 12.68 ng/mL at 72 h. Conclusion] The BDNF and EGFP gene can be transfected into neural stem cells, and also can be expressed for a long time. So, EGFP is one of favorable tracers in the study of NSCs transplantation. Adenovirus mediated BDNF gene and EGFP gene transferring the NSCs and expressing of EGFP and BDNF gene successfully indicate that with the development of gene technology and molecular biology, NSCs can be used as target cells of gene transfer and therapy to SCI by transplantation.