人胚神经干细胞植入脑出血大鼠的存活和分化状态(英文)

Survival and differentiation of human embryonic neural stem cells following implantation into rats with cerebral hemorrhage

背景:研究证实外源性神经干细胞能修复神经,促进脑出血后神经功能障碍的恢复。然而,脑出血后局部内环境对移植神经干细胞存活分化的影响是一个复杂多变的过程。目的:观察人胚神经干细胞植入脑出血大鼠脑内的存活和分化状态。设计、时间及地点:免疫组织化学水平的开放性实验,于2007-05/2008-04在泸州医学院附属医院分子生物实验室完成。材料:纳入40只SD雌性大鼠,由中国医学科学院实验动物研究所提供;8周龄流产胚胎大脑由德阳市人民医院医院妇产科提供。方法:取8周龄流产人胚胎大脑皮质细胞,体外培养获得人胚神经干细胞。通过注射自体动脉血到尾状核制作大鼠脑出血模型,出血后2d将标有5'-溴脱氧尿嘧啶的人胚神经干细胞悬液移植到血肿腔周围的4点,1,2周后处死大鼠,相邻脑组织切片行5'-溴脱氧尿嘧啶/微管相关蛋白2和5'-溴脱氧尿嘧啶/胶质纤维酸性蛋白免疫组织化学双染。主要观察指标:应用免疫组织化学及免疫荧光染色方法观察移植入脑出血大鼠脑内的人胚神经干细胞存活、分化状态和迁徙情况。结果:5'-溴脱氧尿嘧啶阳性细胞为椭圆形棕褐色,移植后1周及2周均可见其存活并向周围迁移,且移植后2周迁移的范围广。移植后1周,脑组织切片见5'-溴脱氧尿嘧啶/微管相关蛋白2和5'-溴脱氧尿嘧啶/胶质纤维酸性蛋白双阳性细胞,且5'-溴脱氧尿嘧啶/微管相关蛋白2双阳性细胞多于5'-溴脱氧尿嘧啶/胶质纤维酸性蛋白双阳性细胞。移植后2周,5'-溴脱氧尿嘧啶阳性细胞数明显减少,在脉络丛和微血管中可见,且5'-溴脱氧尿嘧啶/胶质纤维酸性蛋白双阳性细胞多于5'-溴脱氧尿嘧啶/微管相关蛋白2双阳性细胞。结论:人胚神经干细胞移植入脑出血大鼠脑内能够存活,移植后逐渐分化为神经细胞和星形胶质细胞。

BACKGROUND： Studies have demonstrated that exogenous neural stem cells （NSCs） could repair nerve and promote recovery of neurofunction following cerebral hemorrhage. However, the influence of internal environment after cerebral hemorrhage on the survival and differentiation of NSCs is a complex and variable process. OBJECTIVE： To observe the survival and differentiation of human embryonic NSCs implanted in rats with cerebra hemorrhage. DESIGN, TIME AND SETTING： Open, immunohistochemistry, experiment was performed at the Laboratory of Molecular Biology, Affiliated Hospital of Luzhou Medical College from May 2007 to April 2008. MATERIALS： A total of 40 female SD rats were provided by the Experimental Animal Institute, Chinese Academy of Medical Sciences. Brain of 8-week aborted fetus was obtained from Department of Gynaecology and Obstetrics, the People＇s Hospital of Deyang City. METHODS： Cerebral cortex cells of 8-week aborted human fetus were harvested and cultured in vitro to obtain human embryonic NSCs. Cerebral hemorrhage rat models were established via injection of autologous arterial blood in caudate nucleus. Two days after modeling, 5 μL BrdU-labeled human embryonic NSCs suspension was transplanted at four points surrounding hematoma cavity in the rats. After 1 and 2 weeks, rats were sacrificed. Adjacent sections were doubly stained by BrdU/microtubule-associated protein 2 （MAP-2） and BrdU/glial fibrillary acidic protein （GFAP）. MAIN OUTCOME MEASURES： The survival, immigration and differentiation of human embryonic NSCs implanted in rats were observed by immunohistochemistry and immunofluorescence staining. RESULTS： BrdU-positive cells were oval and brown. At 1 and 2 weeks after implantation, BrdU-positive cells survived and migrated, and they migrated more widely at 2 weeks after implantation. At 1 week after implantation, BrdU/MAP-2-positive cells and BrdU/GFAP-positive cells were observed in cerebral tissue sections, and the number of BrdU/MAP-2-positive cells was more than BrdU/GFAP-positive cells; At 2 weeks after implantation, BrdU-positive cells found in choroid plexus and blood capillary were significantly reduced, and BrdU/GFAP-positive cells were more than BrdU/MAP-2- positive cells. CONCLUSION： Implanted human embryonic NSCs can survive and migrate in the hemorrhage region, gradually differentiate into neurons or astrocytes.