同种异体胚胎神经干细胞移植修复脊髓损伤的时效性

背景:研究表明神经干细胞在脊髓损伤中具有潜在的治疗作用,但何时移植效果较好?目的:观察同种异体胚胎神经干细胞移植对脊髓损伤大鼠双后肢运动功能的修复作用,并分析其移植时效性.设计:观察对比实验.单位:大连医科大学分子生物学实验室和大连理工大学环境与生命学院生物医学实验室.方法:实验于2003-07/08在大连医科大学分子生物学实验室和大连理工大学环境与生命学院生物医学实验室完成.1] 取孕,14～16d的大白鼠1只,引颈处死,剖腹,取出胎鼠,钳取大脑皮质及皮质下脑室旁的脑组织,体外培养大鼠胚胎神经干细胞.2]将30只成年SD大鼠按随机数字表法分为3组,即损伤对照组、早期移植组和延期移植组,每组10只.3组大鼠均制作脊髓横断损伤模型,造成大鼠下肢瘫痪,早期移植组、延期移植组大鼠分别在伤后3d及3周移植胎鼠脑组织神经干细胞.观察移植后大鼠双后肢的运动功能恢复情况.神经干细胞移植4周后,取出移植区脊髓,作切片进行免疫组织化学染色,观察比较各组大鼠的脊髓组织形态学变化.实验过程中对动物处置符合动物伦理学要求.主要观察指标:1]各组大鼠神经干细胞移植后双后肢的功能恢复情况.2]神经干细胞移植4周后各组大鼠脊髓移植区的组织形态学变化.结果:脊髓损伤建模实验纳入大鼠30只,全部进入结果分析,无脱失.1]各组大鼠神经干细胞移植后双后肢的功能恢复情况:早期移植组和延期移植组大鼠双后肢的运动功能均有明显改善,但早期移植组表现尤为显著.早期移植组和延期移植组细胞移植后五六天即可见瘫痪大鼠的后肢肌力开始恢复,两三周后可出现爬行,4周后后肢活动活跃;损伤对照组大鼠的瘫痪肢体无任何恢复.2]移植4周后早期移植组大鼠移植区脊髓组织的形态学变化;脊髓移植区肉眼可见有增生的组织充填,镜下有大量新生的细胞,表现为神经元和神经胶质细胞阳性染色.损伤对照组肉眼见两断端间呈空腔状.镜下脊髓残端变性坏死明显,空泡变性等.延期移植组脊髓移植区的组织形态学变化介于早期移植组和损伤对照组之间,无典型的组织形态学变化特征,镜下有一定量的新生细胞,但数量较早期移植组明显少.结论:同种异体胚胎神经干细胞移植对脊髓横断大鼠运动功能恢复有促进作用,早期移植疗效更好.

Time effectiveness of allotransplantation of rat embryonic neural stem cells for repairing spinal cord injury

BACKGROUND: Previous studies have demonstrated that neural stem cells play potential therapeutic effects on the repair of spinal cord injury. However, the time for acquiring the best allotransplantation effects remains unclear.OBJECTIVE: This study was designed to observe the repairing effects of allotransplantation of embryonic neural stem cells on the motor function of rat two posterior limbs after spinal cord injury and investigate the time effectiveness of the allotransplantation.DESIGN: A controlled observational experiment.SETTING: Laboratory of Molecular Biology, Dalian Medical University; Laboratory of Biomedicine, School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian, Liaoning Province, China.METHODS: This study was performed at the Laboratory of Molecular Biology, Dalian Medical University & Laboratory of Biomedicine, School of Environmental and Biological Science and Technology, Dalian University of Technology between July and August 2003. One albino rat of gestational 14-16 days was sacrificed for harvesting embryonic rat brain cells. Embryonic rat cerebral cortex and subcortical periventricular brain tissue were taken for in vitro culture of rat embryonic neural stem cells. An additional 30 adult Sprague Dawley rats were randomly divided into 3 groups with 10 rats in each group: control, early allotransplantation and delayed allotransplantation groups. All 30 rats were subjected to spinal cord transection injury, leading to rat paralysis of both lower extremities. Embryonic rat neural stem cells were transplanted into the rats in the early and delayed transplantation groups at 3 days and 3 weeks after injury, respectively. Following allotransplantation, motor function of rat two lower extremities was followed. At 4 weeks after allotransplantation of neural stem cells, rat spinal cord was harvested from transplanted region for immunohistochemistry in order to observe and compare the morphological change of rat spinal cord tissue among the 3 groups. The following protocol was performed in accordance with ethical guidelines stated in Guide for the use and care of laboratory animals, approved by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources Commission on Life Scineces, National Research Council, China (1985).MAIN OUTCOME MEASURES: Motor functional recovery of rat two lower extremities after neural stem cell transplantation. Histomorphological change of rat spinal cord at 4 weeks after neural stem cell transplantation.RESULTS: Thirty rats were included in the final analysis. In the early and delayed transplantation groups, the motor function of rat two lower extremities was noticeably improved, in particular in the early transplantation group. In the two experimental groups, muscular strength of paralyzed rat two lower extremities began to recover 5 or 6 days after transplantation of neural stem cells. Two or three weeks later, all rats in the two experimental groups could crawl and four weeks later, two extremities could move actively (approximately approaching to score 3 prescribed as follows). In the control group, no recovery of paralyzed extremities was found. At 4 weeks after transplantation, in the early transplantation group, proliferative tissue could be visible in the spinal cord transplantation region. Through the use of microscope, a considerable number of new cells were found that presented with neuronal and glial cell-positive staining. In the control group, a cavity between two broken ends could be visible. Meanwhile, necrosis and vacuolar degeneration, and other symptoms in the stump of spinal cord were observed with a microscope. In the delayed transplantation group, the histomorphological change of spinal cord region was between the other two groups. No typical histomorphological change was found. A number of new cells were apparent with a microscope, but the number was less compared with the early transplantation group.CONCLUSION: Allotransplantation of embryonic neural stem cells promotes the recovery of rat motor function after spinal cord transection. Early transplantation acquires better therapeutic effects.