运动训练对脑缺血大鼠神经干细胞移植治疗的影响
发布时间：2018-08-25 07:09
【摘要】： 第一部分神经干细胞的培养和鉴定 目的:掌握神经干细胞培养技术,为进一步实验提供可供移植的神经干细胞。方法:孕12-14天SD大鼠胚胎数只,分离获得大脑皮层和海马区脑组织,机械吹打制成单细胞悬液;离心后用神经干细胞培养液重悬细胞,按照1×10~6个细胞/ml密度接种于培养瓶中;定期观察,每2-3天半定量换液一次,7-10天传代一次。取培养的细胞行神经干细胞特异性抗原Nestin鉴定、增殖活性鉴定及分化能力鉴定。 结果:培养的神经干细胞聚集成细胞球以悬浮的方式生长,培养7-10天后细胞球形态致密,大小较均一,传代后仍然可得到大量的次代细胞球。培养细胞的Nestin及BrdU检测呈阳性,贴壁分化后可分化为β-Tublin或GFAP阳性细胞。 结论:此部分实验培养的细胞具有自我复制更新的能力和多向分化的潜能,是符合移植实验要求的神经干细胞。 第二部分神经干细胞体外分化抗原表达时程的研究 目的:研究体外神经干细胞向神经元分化过程中标志性抗原的表达情况,为进一步研究神经干细胞体外增殖分化影响因素以及进行体内移植的研究奠定基础。 方法:取12～14天胎鼠大脑皮层,体外培养神经干细胞;于第1次传代后第3天将培养的神经干细胞置于含有分化培养液的培养皿中进行分化;于不同的分化时间点(1、4、7、10和14天)采用免疫细胞化学方法检测Nestin、SOX2、DCX、TuJ1、NeuN各抗原的表达情况。 结果:Nestin和SOX2在开始时表达量非常近似,几乎在所有的细胞两者都同时表达,随时间的推移,两者的表达量都逐渐减少,但SOX2的表达时间相对Nestin而言持续更长;DCX表达量在分化1周内较高且变化不大,随后开始下降,至分化14天时仅有少数细胞呈DCX阳性;TuJ1在分化7天时就已经有少部分表达,随后其表达量逐渐增多;分化14天时有38.27%的细胞表达TuJ1;在贴壁分化的一周内未见NeuN阳性细胞出现,但至分化14天时,已有NeuN阳性细胞占21.11%;在贴壁分化第1天时就有大量的GFAP阳性细胞,其表达量随分化时间延长而逐渐减少,但下降趋势较Nestin缓和,至分化14天时仍有大量的GFAP阳性细胞。 结论:神经干细胞体外分化过程中抗原的表达是一个相互交错的过程,未分化状态标志性抗原表达的减少与分化状态标志性抗原表达的增加互相对应。 第三部分运动训练在缺血性脑梗死大鼠神经干细胞移植治疗中的作用 目的:探讨运动训练对缺血性脑梗死大鼠神经干细胞移植后神经功能和缺血脑区超微结构改变的影响。 方法:建立Sprague-Dawley大鼠大脑中动脉缺血/再灌注模型为实验对象,随机分为脑缺血对照组(对照组)、电动跑台训练组(运动组)、神经干细胞移植组(移植组)、神经干细胞移植联合运动训练组(联合组),每组6或10只。术后第5天时将超顺磁氧化铁(super paramagnetic iron oxide,SPIO)标记的神经干细胞移植到缺血侧纹状体区。术后第6天开始,运动组和联合组大鼠给予定量的电动跑台运动训练,4周运动训练期间所有4组大鼠均进行定期的神经功能评估。运动4周后处死大鼠,透射电镜观察SPIO标记的神经干细胞在宿主脑内存活迁徙以及宿主脑区超微结构变化情况。 结果:与对照组比较,运动组和联合组的神经功能评分在运动训练2周后,差异有统计学意义(P＜0.05,P＜0.01);移植组差异无统计学意义(P＞0.05)。联合组可见SPIO标记的神经干细胞密度较大,迁徙也相对广泛,宿主缺血脑区超微结构优于其他各组。 结论:运动训练可以促进神经干细胞移植对脑梗死大鼠的治疗作用,但其具体的机制还有待进一步研究。
[Abstract]:Part 1 cultivation and identification of neural stem cells
AIM: To master the culture technology of neural stem cells and provide transplantable neural stem cells for further experiments. Methods: The cerebral cortex and hippocampus were isolated from several embryos of SD rats from 12 to 14 days gestation, and the single cell suspension was made by mechanical blowing. The cultured cells were identified by Nestin, proliferation activity and differentiation ability.
Results: The cultured neural stem cells grew in suspension. After 7-10 days of culture, the cells were compact and uniform in size. After passage, a large number of secondary cell spheres could be obtained. Nestin and BrdU of cultured cells were positive, and they could differentiate into beta-Tublin or GFAP positive cells after adherent differentiation.
CONCLUSION: The cells cultured in this part of the experiment have the ability of self-replication and self-renewal and the potential of multi-differentiation. They are neural stem cells that meet the experimental requirements of transplantation.
The second part is about the time course of antigen expression in neural stem cells in vitro.
AIM: To study the expression of marker antigens during the differentiation of neural stem cells into neurons in vitro, so as to lay a foundation for further study on the factors influencing the proliferation and differentiation of neural stem cells in vitro and the study of transplantation in vivo.
Methods: Nestin, SOX2, DCX, TuJ1 and NeuN antigens were detected by immunocytochemistry at different time points (1, 4, 7, 10 and 14 days). Situation.
Results: The expression levels of Nestin and SOX2 were very similar at the beginning, and almost all cells expressed both at the same time. Over time, the expression of SOX2 decreased gradually, but the expression time of SOX2 lasted longer than that of Nestin. The expression of DCX was higher and unchanged during the first week of differentiation, then began to decline, and only at the 14th day of differentiation. A few cells were DCX-positive; TuJ1 was expressed in a small part at 7 days of differentiation, and then gradually increased; 38.27% of the cells expressed TuJ1 at 14 days of differentiation; no NeuN-positive cells were found within one week of adherent differentiation, but 21.11% of the cells were NeuN-positive at 14 days of differentiation; and a large number of GFA-positive cells were found on the first day of adherent differentiation. The expression of P-positive cells decreased gradually with the prolongation of differentiation time, but the downward trend was milder than that of Nestin. There were still a large number of GFAP-positive cells at the 14th day of differentiation.
CONCLUSION: The expression of antigens in neural stem cells during differentiation in vitro is an intertwined process, and the decrease of expression of undifferentiated marker antigens corresponds to the increase of expression of differentiated marker antigens.
The effect of third part exercise training on neural stem cell transplantation in rats with ischemic cerebral infarction
Objective: To investigate the effects of exercise training on nerve function and ultrastructure of ischemic brain after neural stem cell transplantation in rats with ischemic cerebral infarction.
METHODS: Sprague-Dawley rats were randomly divided into cerebral ischemia control group (control group), treadmill training group (exercise group), neural stem cell transplantation group (transplantation group), neural stem cell transplantation combined with exercise training group (combined group), 6 or 10 rats in each group. The super paramagnetic iron oxide (SPIO) labeled neural stem cells were transplanted into the ischemic striatum. The rats in the exercise group and the combined group were given quantitative treadmill exercise training on the 6th day after operation. All the rats in the four groups were assessed periodically during the exercise training period. The rats were sacrificed after 4 weeks of exercise, and the transmission electron microscopy was used. The migration of SPIO labeled neural stem cells in the host brain and the ultrastructural changes of the host brain were observed.
Results: Compared with the control group, the neurological function scores of the exercise group and the combined group were significantly different after 2 weeks of exercise training (P < 0.05, P < 0.01); there was no significant difference between the transplantation group and the control group (P > 0.05). Each group.
Conclusion: Exercise training can promote the therapeutic effect of neural stem cell transplantation on rats with cerebral infarction, but its specific mechanism needs further study.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
本文编号：2202119
[Abstract]:Part 1 cultivation and identification of neural stem cells
AIM: To master the culture technology of neural stem cells and provide transplantable neural stem cells for further experiments. Methods: The cerebral cortex and hippocampus were isolated from several embryos of SD rats from 12 to 14 days gestation, and the single cell suspension was made by mechanical blowing. The cultured cells were identified by Nestin, proliferation activity and differentiation ability.
Results: The cultured neural stem cells grew in suspension. After 7-10 days of culture, the cells were compact and uniform in size. After passage, a large number of secondary cell spheres could be obtained. Nestin and BrdU of cultured cells were positive, and they could differentiate into beta-Tublin or GFAP positive cells after adherent differentiation.
CONCLUSION: The cells cultured in this part of the experiment have the ability of self-replication and self-renewal and the potential of multi-differentiation. They are neural stem cells that meet the experimental requirements of transplantation.
The second part is about the time course of antigen expression in neural stem cells in vitro.
AIM: To study the expression of marker antigens during the differentiation of neural stem cells into neurons in vitro, so as to lay a foundation for further study on the factors influencing the proliferation and differentiation of neural stem cells in vitro and the study of transplantation in vivo.
Methods: Nestin, SOX2, DCX, TuJ1 and NeuN antigens were detected by immunocytochemistry at different time points (1, 4, 7, 10 and 14 days). Situation.
Results: The expression levels of Nestin and SOX2 were very similar at the beginning, and almost all cells expressed both at the same time. Over time, the expression of SOX2 decreased gradually, but the expression time of SOX2 lasted longer than that of Nestin. The expression of DCX was higher and unchanged during the first week of differentiation, then began to decline, and only at the 14th day of differentiation. A few cells were DCX-positive; TuJ1 was expressed in a small part at 7 days of differentiation, and then gradually increased; 38.27% of the cells expressed TuJ1 at 14 days of differentiation; no NeuN-positive cells were found within one week of adherent differentiation, but 21.11% of the cells were NeuN-positive at 14 days of differentiation; and a large number of GFA-positive cells were found on the first day of adherent differentiation. The expression of P-positive cells decreased gradually with the prolongation of differentiation time, but the downward trend was milder than that of Nestin. There were still a large number of GFAP-positive cells at the 14th day of differentiation.
CONCLUSION: The expression of antigens in neural stem cells during differentiation in vitro is an intertwined process, and the decrease of expression of undifferentiated marker antigens corresponds to the increase of expression of differentiated marker antigens.
The effect of third part exercise training on neural stem cell transplantation in rats with ischemic cerebral infarction
Objective: To investigate the effects of exercise training on nerve function and ultrastructure of ischemic brain after neural stem cell transplantation in rats with ischemic cerebral infarction.
METHODS: Sprague-Dawley rats were randomly divided into cerebral ischemia control group (control group), treadmill training group (exercise group), neural stem cell transplantation group (transplantation group), neural stem cell transplantation combined with exercise training group (combined group), 6 or 10 rats in each group. The super paramagnetic iron oxide (SPIO) labeled neural stem cells were transplanted into the ischemic striatum. The rats in the exercise group and the combined group were given quantitative treadmill exercise training on the 6th day after operation. All the rats in the four groups were assessed periodically during the exercise training period. The rats were sacrificed after 4 weeks of exercise, and the transmission electron microscopy was used. The migration of SPIO labeled neural stem cells in the host brain and the ultrastructural changes of the host brain were observed.
Results: Compared with the control group, the neurological function scores of the exercise group and the combined group were significantly different after 2 weeks of exercise training (P < 0.05, P < 0.01); there was no significant difference between the transplantation group and the control group (P > 0.05). Each group.
Conclusion: Exercise training can promote the therapeutic effect of neural stem cell transplantation on rats with cerebral infarction, but its specific mechanism needs further study.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
【参考文献】
相关期刊论文 前2条
1 张怡,赵连三,汪成孝,雷秉钧;小鼠胚胎成纤维细胞的分离与培养[J];四川大学学报(医学版);2003年02期
2 曹博,郑俊波,郭筠秋;大鼠睾丸支持细胞的分离纯化与鉴定[J];解剖科学进展;2004年01期
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