转基因神经干细胞移植治疗暂时性脑缺血的实验研究  

Transplantation of gene-transfected neural stem cells for transient cerebral ischemia in rats

作　　者：朱巍[1] 周良辅[1] 汪洋[2] 朱剑虹[1] 毛颖[1] 

机构地区：[1]复旦大学附属华山医院神经外科,上海200040 [2]复旦大学医学院解剖组胚系

出　　处：《中华医学杂志》2004年第12期1029-1034,共6页National Medical Journal of China

基　　金：2 0 0 2年上海市科技发展基金重点资助项目 (0 2DJ14 0 2 5 )

摘　　要：目的　探讨转染血管内皮细胞生长因子 (VEGF)基因的神经干细胞在宿主缺血区的基因表达情况 ,以及对神经功能的保护作用。方法　用脂质体介导法将VEGF12 1基因转染到大鼠神经干细胞中。经RT PCR及免疫荧光染色检测转基因神经干细胞及其分化细胞的基因表达情况。建立大鼠大脑中动脉梗塞模型 (tMCAO) ,并将其随机分成 :(1)对照组 ,(2 )细胞悬液PBS移植组 ,(3)神经干细胞移植组 ,(4)转基因神经干细胞移植组 ,前 3组每组 10只大鼠 ,第 4组 2 0只大鼠。立体定向法将BrdU标记的转基因神经干细胞移植到tMCAO大鼠的纹状体缺血半暗区。移植后 2～ 12周进行神经损害严重程度评分 (NSS)并与其他 3组比较。用免疫荧光染色方法观察移植后 1周转基因神经干细胞在脑内的基因表达情况和移植后 12周转基因神经干细胞在脑内的分化、迁徙情况。结果　转基因神经干细胞及其分化的子代细胞均有VEGF12 1的表达并持续 2周左右。移植后 2、4、6、8、10、12周(4)组大鼠的NSS评分分别为 5 8± 1 5、5 0± 1 0、4 6± 1 0、4 0± 0 7、4 0± 1 0、3 8± 0 4 ,均低于其他3组。其中第 8周显著低于 (1)、(2 )组 (均P =0 0 0 8) ,第 12周显著低于 (1)、(2 )、(3)组 (均P =0 0 0 0 )。转基因神经干细胞移植后 1周?ObjectiveTo detect the expression and the role of vascular endothelial growth factor (VEGF)-transfected neural stem cells (NSCs) in rat brain subjected to ischemia. MethodsFetal NSCs were cultured from E14 days SD rats and transfected with VEGF121 gene by using lipofectAMINE technique. The gene expression of transfected cells was detected by RT-PCR and immunofluorescent staining in vitro. Temporary middle cerebral artery occlusion (tMCAO) model was established in 40 SD rats and then rate were randomly divided into (1) control group (n=10),(2) PBS transplantation group (n=10),(3) neural stem cells transplantation group (n=10),and (4) VEGF-secreting neural stem cells transplantation group (n=10). BrdU-labelled NSCs and VEGF-secreting NSCs were transplanted into the penumbra zones respectively 3 days after the tMCAO model was established. Neurological Severity Score (NSS) was checked in all groups 2,4,6,8,10,12 weeks respectively after transplantation. One and 12 weeks after the transplant-ation,10 rats in the group (4) were killed and then brains taken out respectively. By using immunofluorescent staining,the VEGF expression of transplanted cells 1 week after transplantation,differentiation and migration of transplanted neural stem cells 12 week after transplantation were detected respectively. ResultsVEGF-transfected neural stem cells could continuously express gene products during the first 2 weeks. Both transfected NSCs and their progeny expressed VEGF gene products,which was demonstrated by fluorescence study. There were no significant differences in NSS in groups 4 when tMCAO models were just established. However,the values of NSS in (4) group were 5.8±1.5,5.0±1.0,4.6±1.0,4.0±0.7,4.0±1.0,3.8±0.4 from 2～12 weeks after transplantation,significantly lower than those in groups (1) and (2) 8 weeks (P=0.008) and those in groups (1),(2) and (3) 12 weeks (P=0.000) after transplantation. NSS in group (3) was also lower than that in groups (1) and (2) 8 and 12 weeks after transplantation. One week after transpla

关 键 词：转基因神经干细胞移植 暂时性脑缺血 VEGF 内皮生长因子 神经功能 

分 类 号：R743.3[医药卫生—神经病学与精神病学]