摘要
背景：外源性神经干细胞具有神经修复作用,可能对脑出血后的神经功能恢复起到一定的作用。目的：观察胎鼠神经干细胞的体外生长、分化及移植到脑出血大鼠后的存活、迁徙、分化情况,探讨神经干细胞对脑出血模型大鼠受损神经功能的修复作用。设计：完全随机分组设计,对照动物实验。单位：复旦大学附属华山医院神经外科材料：选用健康雄性成年SD大鼠18只为受体,体质量280～320g,由中国科学院上海实验动物中心提供。实验用鼠抗BrdU为Neomarkers产品,鼠抗胶质纤维酸性蛋白和兔抗微管相关蛋白2为Chemicon产品。方法：实验于2006-02/12在复旦大学附属上海医学院解剖组胚实验室完成。从胎龄14d的胎鼠海马中分离、培养、鉴定神经干细胞。16只受体SD大鼠被随机分为3组：对照组,PBS组和移植神经干细胞组。均通过尾状核内注射自体动脉血制作大鼠脑出血模型。移植NSC组在造模后30min在血肿腔周围四点分别移植浓度为2×10^11L^-1神经干细胞悬液5μL;PBS组于相同时间点在脑内相同部位注射PBS;PBS和神经干细胞的移植方法同自体血的移植方法。对照组大鼠在造模后30min只造成四点损伤,不注射任何物质。主要观察指标：在造模后立即,1,3,5,14,21,28d采用前肢评分和转身评分对大鼠神经功能进行评估。大鼠于造模后28d麻醉后取脑,并通过双标胶质纤维酸性蛋白、微管相关蛋白2、BrdU免疫组化来检测移植入脑的神经干细胞在体内的分化情况。结果：①神经功能评分：造模后5d,各组差异无显著性意义（P〉0.05）。造模后14～28d,干细胞移植组较其他3组明显改善（P〈0.05）。②脑组织切片双免疫组织学双标染色结果：干细胞移植组血肿周围凋亡细胞少于PBS组。受体大鼠脑组织切片显示有BrdU,微管相关蛋白2,胶质纤维酸性蛋白阳性细胞,说明神经干细胞可以在宿主脑内存活、迁徙和分化,可以分化为神经元样细胞和神经胶质样细胞。结论：神经干细胞移植可能通过分化为神经元样细胞和神经胶质细胞促进大鼠脑出血的神经功能恢复。
BACKGROUND： Exogenous neural stem cells （NSCs） can repair nerve and promote recovery of neurofunction following cerebral hemorrhage. OBJECTIVE： To observe the growth and development of NSCs in vitro, to evaluate the survival, migration and differentiation of transplanted NSCs surrounding hematoma and the possible recovery function of NSCs, and to investigate the repairing effect of NSCs on damaged neurofunction in cerebral hemorrhage model rats. DESIGN： Completely randomized grouping design and controlled animal study. SETTING： Department of Neurusurgery, Huashan Hospital, Fudan University. MATERIALS： Eighteen adult healthy male SD rats weighing 280-320 g were provided by Shanghai Animal Center of Chinese Science Academy. BrdU was provided by Neomarkers Company; rat-anti-glial fibrillary acidic protein （GFAP） and rabbit-anti-microtubule-associated protein-2 （MAP-2） by Chemicon Company. METHODS： This study was performed at the Laboratory of Anatomy and Histology ＆ Embryology, Shanghai Medical College, Fudan University from February to December 2006. The NSCs was isolated, cultured, and evaluated from hippocampus of day E14 fetal SD rats. Eighteen rats were randomly divided into control group, PBS group and NSC transplantation group. Cerebral hemorrhage rat models were established via injection of autologous arterial blood in caudate nucleus. Thirty minutes after model establishment, 5 μ L NSC suspension with the concentration of 2×10^11 L^-1 was transplanted at four points surrounding hematoma cavity in the NSC transplantation group. Transplantation of PBS and NSCs was the same as autoblood transplantation. Thirty minutes after model establishment, injuries at the four points were performed, and nothing was injected in the control group. MAIN OUTCOME MEASURES： Neurofunction was evaluated with forward limb scale and turning scale just soon after transplantation and at 1, 3, 5, 14, and 28 days after transplantation. Brain was colleted by anesthesia 28 days after model establishment. Differentiation of transplanted NSCs was detected through testing GFAP, MAP-2, and BrdU by using immunohistochemistry. RESULTS： （1） Neurofunction scores： There was no significant difference 5 days after model establishment （P 〉 0.05）. However, the scores were significantly improved in the NSC transplantation group 14-28 days after model establishment （P 〈 0.05）. （2） Immunofluorescent double labeling： Apoptosis cells around hemotoma in the NSC transplantation group were less than those in the PBS group. BrdU and MAP-2 or GFAP-positive cells were observed in cerebral tissue sections, and this suggested that NSCs could survive, migrate and differentiate in host brain and differentiate into neurons or astrocytes. CONCLUSION： NSC Transplantation contributes to the recovery of neurofunction in cerebral hemorrhage rats through differentiation into neurons or astrocytes.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第12期2364-2368,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
