BDNF修饰神经干细胞的建立及脑内移植研究

Establish the BDNF-modified neural stem cells and transplant into the brain of rats with cerebral ischemia

目的建立脑源性神经营养因子(BDNF)修饰的神经干细胞,观察其体外活性及移植大鼠脑缺血模型后在大鼠脑内的基因表达和存活情况。方法体外培养、纯化大鼠胚胎神经干细胞并鉴定。构建BDNF重组基因质粒后转染神经干细胞并鉴定其体外活性。建立20只大鼠暂时性大脑中动脉阻塞模型(tMCAO)后3d通过立体定向法将B rdU标记的BDNF修饰神经干细胞(BDNF-NSCs)注射到其右侧纹状体缺血半暗区。移植后1周随机抽取10只大鼠,杀鼠取脑,观察BDNF基因的表达情况。移植后12周观察剩余10只大鼠脑内所移植神经干细胞的增殖、迁徙情况。结果培养获得Nestin标记呈阳性,并具有增殖和分化潜能的神经干细胞。构建BDNF重组基因质粒后转染NSCs证实其具有体外BDNF表达能力,与背根神经节共培养能促进神经元突起伸出。立体定向移植后1周,BDNF-NSCs在大鼠脑内存活并部分表达BDNF基因产物。移植后12周,BDNF-NSCs在大鼠脑内增殖、迁徙并部分分化为成熟神经元。结论BDNF修饰神经干细胞移植大鼠tMCAO模型后早期即可表达BDNF基因产物,并具有增殖、迁徙、分化的能力,为进一步开展其对大鼠脑缺血后功能修复的研究奠定基础。

Objective To establish the BDNF-modified neural stem cells, observe their activity in vitro as well as the gene expression and survival in vivo after transplantation into the brain of rats with cerebral ischemia damage. Methods Cultured, purified and identified the rats＇ embryo NSCs in vitro. The plasmid carrying the BDNF gene was constructed, and the activities in vitro of the NSCs were evaluated after transfection. Twenty SD rats of temporary middle cerebral artery occlusion （tMCAO） models were established. Three days later, the BruU-labeled BDNF-NSCs were transplanted into the penumbra zones of right striatum. One week after the transplantation, ten rats in random were killed and BDNF expression in the transplanted cells was measured. Twelve weeks later, the proliferation and migration of transplanted NSCs were ob- served on the other 10 rats. Result The highly purified NSCs were cultured successfully and the gene expression of the plasmid carrying the BDNF gene in the NSCs were detected in vitro too. We found BDNF could stimulate the neurogenesis. 1 week after the transplantation,it showed that BDNF-transfected NSCs survived and BNDF was expressed partly;12 weeks later, the transplanted NSCs proliferated, migrated and some of them differentiated to mature neurons. Conclusion The BDNF-modified NSCs can express gene products in the early phase after transplantation and have capacity of proliferation, migration and differentiation. The study can offer the evidence for the research of functional recovery after transplantation in rats with cerebral ischemia.