脑皮层创伤灶注射法移植神经干细胞实验研究  

Transplantation of neural stem cells by injection into traumatic lesions in the cerebral cortex

作　　者：李钢[1] 徐如祥[1] 柯以铨[1] 姜晓丹[1] 

机构地区：[1]南方医科大学珠江医院神经外科,广东神经外科研究所,广州510282

出　　处：《中华神经医学杂志》2008年第2期125-130,共6页Chinese Journal of Neuromedicine

基　　金：总后勤部卫生部“十五”重点攻关课题（012054）;广东省重大科技项目基金[粤财企（2001）367]

摘　　要：目的观察食蟹猴自体骨髓源神经干细胞移植到大脑皮层创伤灶内的存活、生长情况。方法取骨髓分离、纯化骨髓基质细胞，培养诱导成神经干细胞，nestin染色表达阳性后再加入细胞标记物BrdU进行共培养7d待移植用。移植前将BrdU标记的神经干细胞收集、离心，制成干细胞悬液，要求活细胞〉80％。食蟹猴在采用Allen’s打击法制作皮层创伤灶后，分即时移植（术后即在创伤灶注入，n=3）和延迟移植组（术后30d在创伤灶注入，n=3），用微量注射针将干细胞悬液注入到猴脑皮质损伤灶。同时设置对照组（n=1），在动物的左侧皮层制作创伤灶并按上述方法注射含同样浓度BrdU的神经干细胞培养液0．5mL，右侧皮层注入干细胞悬液。移植后1、3、6个月即时移植组、延迟移植组各灌杀1只食蟹猴，移植后6月灌杀对照组食蟹猴，做移植区组织切片染色检查。结果即时移植组和延迟移植组都可观察到脑皮层创伤灶内有棕褐色的B耐U标记阳性细胞，而对照组组织切片中则未见BrdU阳性表达细胞。移植后BrdU阳性表达细胞早期呈簇状分布，随后散在分布，6个月后细胞形态多样。移植后6个月在移植区邻近的脑白质内也可观察到有BrdU阳性表达的细胞。结论移植的骨髓源性神经干细胞在脑皮层创伤灶内有存活并可向邻近区域迁移。Objective To determine whether autologous bone marrow strom cells-derived neural stem cells （BMSCs-d-NSCs） can survive and grow after transplantated into traumatic lesions in monkey cerebral cortex. Methods Stromal cells from bone marrow were separated, adjusted to 1×10^6 cells/mL and seeded onto multi-well plates. We added medium, fetal calf serum, leukemia inhibitory factor and basic fibroblast growth factor before incubating at 37 ℃ and 5% CO2 in a humidified atmosphere. Adherent cells were cultured in neural stem cell medium with FCS and retinoic acid and then characterized by nestin staining. BMSCs-d-NSCs were cultured with BrdU for 7 d and BrdU-stained cells were collected and resuspended. Traumatic lesions were made by Allen＇ s method in the cerebral cortex of monkeys （Macaca fascicularis）. Animals were assigned to immediate transplantation （IT） group （NSCs marked by BrdU staining were injected into the traumatic lesion immediately, n=3） or delayed transplantation （DT） group （BrdU marked NSCs were injected into the traumatic lesion at day 30 aRer injury, n=3）. One animal was enrolled in the control group： traumatic lesions were made on each side of the monkey brain and the leR side was injected with 0.5 mL NSCs culture medium without NSCs but containing the same concentration of BrdU, the right side was injected with NSCs marked by BrdU staining. Two animals in both IT and DT groups were sacrificed at each of the following post-transplantation times （1, 3 or 6 months） and tissue sections from the transplantation area were stained; two animals in control group were sacrificed 6 month aRer transplantation. Results In both IT and DT groups, brown, BrdU-positive cells were observed in the lesions. There were no BrdU＋ cells in the control group. Over the 6 months, transplanted cells first distributed as clusters and later as scattered. Cell morphology varied and cells had protuberances. After 6 months, BrdU positive cells were observed in white matter nearby the transplantat

关 键 词：骨髓基质细胞 神经干细胞 颅脑损伤 细胞移植 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]