人胚神经干细胞植入脑损伤大鼠的存活和分化状态(英文)

Survival and differentiation of human embryonic neural stem cells in rats with brain injury

背景:神经干细胞的发现为中枢神经系统损伤修复带来希望,但脑损伤后的内部环境对神经干细胞存活和分化的影响是一个复杂多变的过程。目的:观察大鼠脑液压冲击伤后植入的人胚神经干细胞存活情况和分化状态。设计:开放性实验。单位:广东省中医院神经外科,北京市神经外科研究所。材料:实验于2002-09/2003-03在北京市神经外科研究所神经干细胞室完成。选取SD雌性大鼠24只(购自中国医学科学院实验动物研究所,动物质量合格证号:SCXK(京)2002-2003),7周龄,体质量(250±10)g。8周龄流产胎儿大脑(产妇及其家属均同意提供),流产过程中B超监测胎儿的存活状况。BrdU单克隆抗体(Sigma公司),兔抗巢蛋白多克隆抗体(Chemicon公司),鼠抗微管相关蛋白2单克隆抗体(Neomarkers公司),兔抗胶质纤维酸性蛋白多克隆抗体(Biogenex公司)。方法:①取8周龄流产胎儿大脑皮层细胞,体外培养获得人胚神经干细胞。②大鼠制作液压冲击伤模型。大脑皮质运动感觉区骨窗位置:以前囟为零点,向后2.5mm,中线右3.0mm。液压冲击参数:冲击压力0.3MPa,冲击时间25ms,冲击次数为1次。③伤后24h在损伤区移植标有BrdU的人胚神经干细胞,1周和4周后处死大鼠,邻片行BrdU/微管相关蛋白2和BrdU/胶质纤维酸性蛋白免疫组织化学双染。主要观察指标:①人胚神经干细胞移植后的存活及迁移。②人胚神经干细胞移植后的分化。结果:①BrdU阳性细胞为椭圆形棕褐色,移植后1,4周均可见其存活并向周围迁移,且移植后4周迁移的范围更广。②移植后1周,皮质颗粒层和皮层下均见较多的BrdU阳性细胞,且BrdU/微管相关蛋白2双阳性细胞多于BrdU/胶质纤维酸性蛋白双阳性细胞;移植后4周,BrdU阳性细胞数明显减少,脉络丛和微血管中可见BrdU阳性细胞,且BrdU/胶质纤维酸性蛋白双阳性细胞多于BrdU/微管相关蛋白2双阳性细胞。结论:人胚神经干细胞能够存活于脑损伤区域,移植后逐渐分化为星形胶质细胞,且易被内皮吞噬细胞所消化。提示免疫排斥反应可能影响人胚神经干细胞的存活。

BACKGROUND： Finding of neural stem cells （NSCs） brings new hope for repairing central nervous system （CNS） injury. However, the influence of internal environment after brain injury on the survival and differentiation of NSCs is a complex and variable process. OBJECTIVE： To observe the survival and differentiation of human embryonic NSCs following implantation into rats with fluid percussion brain injury. DESIGN ： Open experiment. SETTING： Department of Neurosurgery, Guangdong Provincial Hospital of Traditional Chinese Medicine; Beijing Institute of Neurosurgery. MATERIALS： This experiment was carried out in the Laboratory of Neural Stem Cells, Beijing institute of Neurosurgery from September 2002 to March 2003. Twenty-four female SD rats, aged 7 weeks, with body mass of （250±10） g, were provided by the Experimental Animal Institute, Chinese Academy of Medical Sciences [License No. SCXK （Jing） 2002-2003]. Cerebrum of 8-week aborted fetus was obtained （Informed consents were obtained from parturients and their relatives）. Fetal survival was monitored by B ultrasonic wave during abortion. BrdU monoclonal antibody （Sigma Company）, rabbit anti-nidogen polyclonal antibody （Chemicon Company）, mouse anti-microtubule-associated protein 2 （MAP-2） monoclonal antibody （Neomarkers Company）, rabbit anti-cjlial fibrillary acidic protein （GFAP） polyclonal antibody （Biogenex Company）. METHODS： （1）Cerebral cortex cells of 8-week aborted human fetus was harvested and cultured in vitro for obtaining human embryonic NSCs. （2）Rat models of hydraulic impact injury were developed. Bone window of motor sensory area of cerebral cortex was set at 2.5mm posterior to bregma which was zero point and 3.0 mm lateral to midline. Hydraulic impact injury parameters were set as impact pressure 0.3 MPa, impact time 25 ms and impact time once. BrdU-labeled human embryonic NSCs were implanted into injured area at 24 hours after injury. After 1 and 4 weeks, rats were sacrificed. Adjacent sections were doubly stained by BrdU/MAP-2 and BrdU/GFAP. MAIN OUTCOME MEASURES： （1） Survival and immigration of implanted human embryonic NSCs. （2） Differentiation of implanted human embryonic NSCs. RESULTS： （1）BrdU-positive cells were oval and brown. At 1 and 4 weeks after implantation, BrdU-positive cells survived and migrated, and they migrated more widely at 4 weeks after implantation. （2）At 1 week after implantation, more BrdU-positive cells were found in the subcortical granular layer and subcortex, and BrdU/MAP-2 -positive cells were more than BrdU/GFAP-positive cells; At 4 weeks after implantation, BrdU-positive cells were significantly reduced, and found in choroid plexus and blood capillary, and BrdU/GFAP-positive cells were more than BrdU/MAP-2- positive cells. CONCLUSION： Implanted human embryonic NSCs can survive in the region of brain injury, gradually differentiate into astrocytes during rehabilitation and are easily digested by endothelial phagocytes. It indicates that immunological rejection possibly influences the survival of human embryonic NSCs.