比较新生大鼠脑部不同部位的神经干细胞被诱导分化为少突胶质细胞的差异

发布时间：2017-12-31 15:10

本文关键词：比较新生大鼠脑部不同部位的神经干细胞被诱导分化为少突胶质细胞的差异　出处：《福建医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 【目的】体外培养获得大脑皮质、纹状体、海马三个不同部位来源的神经干细胞(neural stem cells, NSCs)并对其进行诱导分化,比较三个不同部位来源的神经干细胞增殖传代能力的差别以及定向诱导分化为少突胶质细胞的分化率的差别。为体外培养获得更多的少突胶质细胞的研究提供实验依据。【方法】 1.分别取新生24h内SD大鼠的大脑皮层、海马、纹状体部位来源的神经干细胞在碱性成纤维生长因子和B27联合作用下使其稳定增殖,单细胞克隆实验检测其自我更新能力;免疫荧光染色法对NSCs标志物nestin以及分化为神经元和胶质细胞的多向分化能力进行鉴定。 2.对三个不同部位来源的神经干细胞进行传代培养,CCK8法检测不同部位来源的NSCs的增殖能力,绘制细胞生长曲线;比较不同部位来源的NSCs的增殖及传代能力差别。 3.分别以胰岛素样生长因子-1、甲状腺素、神经胶质母细胞瘤培养上清液诱导NSCs分化,并通过激光共聚焦显微镜及Western blot法对成功诱导为OLC的细胞分别进行阳性细胞计数与Galc的定量检测,比较各组神经干细胞定向分化为少突胶质细胞的诱导分化率。【结果】 1.采用无血清培养基培养,可获取高纯度的大鼠原代NSCs;nestin标志物检测阳性,自然分化后可分化为少突胶质细胞、神经元及星形胶质细胞,免疫荧光染色阳性;单细胞克隆实验可以获得单细胞克隆球。 2.大脑皮质来源的NSCs可以传代9-12次,海马来源的NSCs可以传代8-11次,纹状体来源的NSCs可以传代6-9次;CCK8法绘制细胞生长曲线,统计分析得:海马与纹状体来源的NSCs的增殖活性无差异(P㧐0.05);皮质与海马来源的NSCs增殖活性有差异(P㩳0.05),结果有统计学意义;皮质与纹状体来源的NSCs增殖活性有差异(P㩳0.05),结果有统计学意义。且随着传代次数的增加,该差异性依然维持。 3.胰岛素样生长因子-1、甲状腺素诱导能力无差异(P㧐0.05);神经胶质母细胞瘤培养上清液与胰岛素样生长因子-1诱导能力有差异(P㩳0.05),结果有统计学意义;神经胶质母细胞瘤培养上清液与甲状腺素诱导能力有差异(P㩳0.05),结果有统计学意义;皮质来源的神经干细胞与海马及纹状体来源的神经干细胞向少突胶质细胞分化率有差异(P㩳0.05),结果有统计学意义; 【结论】 1.采用无血清培养基悬浮培养,可获取高纯度的具有自我更新及多向分化能力的大鼠原代NSCs。 2.皮质来源的神经干细胞增殖及传代能力强于海马、纹状体部位来源的神经干细胞。并且体外培养的神经干细胞一直维持该特性。 3.神经胶质母细胞瘤培养上清液的诱导神经干细胞向少突胶质细胞分化的能力强于胰岛素样生长因子-1、甲状腺素;皮质来源的神经干细胞向少突胶质细胞的分化能力强于海马、纹状体部位来源的神经干细胞。
[Abstract]:[Objective]
Get the cerebral cortex in vitro, striatum, hippocampus three neural stem cells of different origins (neural stem, cells, NSCs) and of its differentiation, three from different parts of neural stem cell proliferation ability difference and differentiation to the differences in the rate of differentiation of oligodendrocytes provide experimental. According to the research get more oligodendrocytes in vitro.
[method]
Hippocampus were obtained from 1. newborn SD rats 24h in the cerebral cortex, striatum derived neural stem cells in basic fibroblast growth factor and B27 under the combined effect of the proliferation of experimental single cell clone detection their self-renewal ability; immunofluorescence staining for NSCs and nestin markers were identified to differentiate into multilineage differentiation the ability of neurons and glial cells.
2., three sources of neural stem cells from different locations were cultured. The proliferation ability of NSCs from different parts was detected by CCK8, and cell growth curve was drawn. The proliferation and passage ability of NSCs from different locations were compared.
3. respectively by insulin-like growth factor -1, thyroxine, NSCs induced differentiation of human glioblastoma supernatant and cultured by confocal microscopy and Western blot method for quantitative OLC induced cells were Galc positive cell counting and detection, comparison of neural stem cells to differentiate into oligodendrocyte differentiation rate cells.
[results]
1. using serum-free culture medium can obtain high-purity primary rat NSCs; nestin markers positive natural differentiation can differentiate into oligodendrocytes, neurons and astrocytes, immunofluorescence staining; single cell clone experiments can get cell clone ball.
2. from the cerebral cortex of NSCs can be passaged 9-12 times, hippocampus derived NSCs can be passaged 8-11 times, from the striatum of NSCs can be passaged 6-9 times; the cell growth curve CCK8 method, statistical analysis: no difference between hippocampus and striatum derived the proliferation activity of NSCs (P? 0.05); NSCs proliferation activity of cortex and hippocampus sources there are differences (P? 0.05), the results were statistically significant; the proliferation of NSCs cortex and striatum derived differences (P? 0.05), the results were statistically significant. With the increase in the number of passages, the differences are still maintained.
3. insulin like growth factor -1, no difference in ability induced by thyroxine (P? 0.05); nerve glioblastoma supernatant and insulin-like growth factor -1 induced ability are different (P? 0.05), the results were statistically significant; and the supernatant induced by thyroxine ability is different in glioblastoma culture (P? 0.05) and the results have statistical significance; cortex derived neural stem cells and hippocampus and striatum derived neural stem cells to oligodendrocyte differentiation rate difference (P? 0.05), the results were statistically significant;
[Conclusion]
1. by suspension culture of serum-free medium, the primary NSCs. of rats with high purity and self renewal and multidirectional differentiation can be obtained.
2., the proliferation and passage ability of cortical derived neural stem cells are stronger than those of hippocampus and striatum. Neural stem cells cultured in vitro maintain this characteristic.
3. human glioblastoma culture to oligodendrocyte differentiation in insulin-like growth factor -1, neural stem cells induced by supernatant of thyroxine; cortical source of neural stem cells to oligodendrocyte differentiation ability in hippocampus, striatum derived neural stem cells.

【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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