神经干细胞向神经元方向的诱导和分化的研究

发布时间：2018-10-29 15:51

【摘要】： 目的：探讨无血清环境下联合应用bFGF、PDGF、Ra、HRG、Forskolin对大鼠神经干细胞诱导分化的作用。方法：用无血清培养技术从14-16天孕龄胚鼠脑中分离、培养神经干细胞；用免疫细胞化学方法对神经干细胞进行鉴定，并通过流式细胞仪检测神经干细胞的增殖能力；用传2-3代的神经干细胞接种于多聚赖氨酸包被的玻片，分以下四组，分别为两步法组、一步法组、血清组和神经干细胞培养组(bFGF组)；通过免疫细胞化学技术，用Map2和S100抗体鉴定分化的细胞；通过图像分析系统来测量神经元样细胞的最长突起长度、胞体周长、胞体面积，神经元细胞计数并计算各组Map2阳性的细胞比例。用全细胞膜片钳技术记录分化后细胞的钠电流；并且用统计分析软件对数据进行统计分析。结果：1、神经干细胞的分离、培养、观察和鉴定：从孕14—15天的胎鼠大脑皮层分离的细胞呈圆形，折光性好；培养6—7天后可以传代，细胞球边界清晰，折光性好，立体感强；免疫细胞化学方法鉴定细胞球内有较多Nestin阳性细胞。流式细胞仪检测各代细胞球内均有较多细胞处于G1／M和S期。2．神经干细胞的诱导分化：Map2的免疫荧光技术显示一步法组Map2阳性神经元样细胞的比例最高，为43.35±13.75μm，，且一步法诱导出的神经元样细胞中有相当多细胞表达TH阳性。图像分析结果显示：一步法组与两步法组细胞胞体较大，胞体面积分别为136.22±33.08μm~2、131.45±24.58μm~2，胞体周长分别为48.63±5.5μm、49.3±7.71μm，细胞突起长，最长突起分别为123.63±31.48μm、116.51±38.34μm。统计结果显示：①Map2阳性神经元样细胞所占比例：一步法组与血清组有显著差异(P＜0.05)，其它各组之间没有显著差异。②胞体面积及周长：一步法组与两步法组之间没有显著差异，但它们和其余各组之间都有显著差异。③突起的长度：一步法组与血清组、bFGF组有显著差异，与两步法组之间没有显著差异：两步法组与bFGF组有显著差异。电生理检测显示：一步法组中大部分形态成熟的神经元样细胞(80％)能诱发电压依赖性的Na~+通道电流，两步法仅有少量细胞(20％)能诱发电压依赖性的Na~+通道电流，而未分化细胞及其它两组细胞均未记录到内向电流。结论：1．培养的原代及传代后的大鼠胚胎皮层NSCs表达Nestin，并具有自我更新和多向分化的能力。2．在体外无血清条件下联合应用bFGF、PDGF、RA、HRG、Forskolin对大鼠NSCs向神经元定向诱导分化是可行的，能促进NSCs分化成神经元。3．在体外无血清条件下，联合应用5种因子的一步法和分两步进行诱导的两步法相比，一步法既能促进NSCs分化成更多的神经元，也可促进分化后神经元的成熟
[Abstract]:Aim: to investigate the effect of combined use of bFGF,PDGF,Ra,HRG,Forskolin in serum-free environment on differentiation of neural stem cells (NSCs) in rats. Methods: the neural stem cells were isolated from the brains of 14-16 days gestational embryos by serum-free culture technique, the neural stem cells were identified by immunocytochemistry, and the proliferative ability of neural stem cells was detected by flow cytometry. Two to three generations of neural stem cells were inoculated on the polylysine coated slides. They were divided into the following four groups: two-step group, one-step group, serum group and neural stem cell culture group (bFGF group). The differentiated cells were identified by Map2 and S100 antibodies by immunocytochemistry. The longest process length, cell body perimeter, cell body area, neuronal cell count and Map2 positive cell proportion were measured by image analysis system. The sodium currents of differentiated cells were recorded by whole-cell patch clamp technique, and the data were analyzed by statistical analysis software. Results: 1. Isolation, culture, observation and identification of neural stem cells: the cells isolated from fetal rat cerebral cortex were round and good refraction; After 6-7 days of culture, the boundary of the cell ball was clear, the refractive index was good, the stereosensitivity was strong, and many Nestin positive cells were identified by immunocytochemistry. Flow cytometry analysis showed that more cells were in G 1 / M and S phase in each generation. 2. The differentiation of neural stem cells: the proportion of Map2 positive neuron-like cells in Map2 group was the highest (43.35 卤13.75 渭 m). Many of the neuron-like cells induced by one step method expressed TH positive. The results of image analysis showed that the cell bodies in the one-step group and the two-step group were larger, the cell body area was 136.22 卤33.08 渭 m ~ 2131.45 卤24.58 渭 m ~ (-2), the cell body circumference was 48.63 卤5.5 渭 m ~ (-1) and 49.3 卤7.71 渭 m, respectively. The longest protuberances were 123.63 卤31.48 渭 m and 116.51 卤38.34 渭 m respectively. The results showed that the proportion of 1Map2 positive neuron-like cells in the one-step group was significantly different from that in the serum group (P < 0. 05). There was no significant difference in cell body area and perimeter between other groups. 2 there was no significant difference between one-step group and two-step group, but there was significant difference between them and other groups. There was significant difference between bFGF group and two-step group. There was significant difference between two-step group and bFGF group. Electrophysiological examination showed that most of the mature neuron-like cells (80%) in the one-step group could induce voltage-dependent Na~ channel currents, and only a few cells (20%) could induce voltage-dependent Na~ channel currents in the two-step method. However, no inward currents were recorded in undifferentiated cells and in other two groups. Conclusion: 1. Primary and passage rat embryonic cortex NSCs express Nestin, and have the ability of self-renewal and multi-differentiation. 2. Combined use of bFGF,PDGF,RA,HRG, in serum-free condition in vitro It is feasible to induce the differentiation of rat NSCs into neurons by Forskolin, which can promote the differentiation of NSCs into neurons. 3. Under the condition of serum-free in vitro, the one-step method of five kinds of factors was used in combination with the two-step method of two-step induction. One-step method can not only promote the differentiation of NSCs into more neurons, but also promote the maturation of differentiated neurons.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.1

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