转基因神经干细胞移植治疗暂时性脑缺血的实验研究

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VEGF121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20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VEGF121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=0.008),第12周显著低于(1)、(2)、(3)组(均P=0.000).转基因神经干细胞移植后1周在宿主脑内迁徙并表达VEGF基因产物,移植后12周在宿主脑内存活、迁徙,部分分化成神经元.结论转染VEGF基因的神经干细胞移植后在缺血早期表达基因产物,并对宿主局部血管和神经结构具有保护作用.转基因神经干细胞移植是治疗脑缺血性疾病的可行途径.

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

OBJECTIVE: To detect the expression and the role of vascular endothelial growth factor (VEGF)-transfected neural stem cells (NSCs) in rat brain subjected to ischemia. METHODS: Fetal 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 transplantation, 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. RESULTS: VEGF-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 approximately 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 transplantation, immunofluorescent staining showed that VEGF-transfected NSCs migrated and expressed VEGF into hosts' brains. Twelve weeks after transplantation, transplanted NSCs survived and migrated, some of them differentiated to neurons and integrated well with hosts' cytoarchitectural components. CONCLUSION: VEGF-transfected NSCs express gene products during the early time after transplantation, which reduce brain injury through protecting the vascular system against ischemia and reperfusion injury. Transplantation of VEGF-transfected NSCs might be a novel method for treatment of cerebral ischemia.