脑源性神经营养因子对神经干细胞增殖与分化的影响

发布时间：2019-02-15 21:39

【摘要】： 神经干细胞(neural stem cells, NSCs)是一种具有自我更新能力的细胞,可增殖分化为中枢神经系统内的3种细胞:神经元、星形胶质细胞和少突胶质细胞。因其具有一定的自我更新和分化能力,从而在临床上有广阔的应用前景。新近研究表明,脊髓损伤后,在损伤局部移植NSCs能促进成年动物模型的功能恢复,但移植的NSCs主要分化为胶质细胞,形成胶质瘢痕,阻碍了神经功能进一步恢复。脑源性神经营养因子(brain -derived neurotrophic factor BDNF)是Barde等1982年从猪脑中提取的含量较低的碱性蛋白,是神经营养因子家族中最具有代表性的成员之一,许多的研究显示,脑源性神经营养因子能够促进神经干细胞向神经元方向分化。因此我们选择神经干细胞和脑源性神经营养因子作为研究对象,主要探讨了脑源性神经营养因子对神经干细胞增殖及其分化的影响,寻找合适的诱导时间和诱导浓度。同时研究了脑源性神经营养因子的两个受体TrkB和P75在脑源性神经营养因子促进神经干细胞向神经元方向分化中的作用。本文主要分为两部分第一部分神经干细胞的体外培养、分化及其鉴定目的掌握新生SD大鼠神经干细胞体外分离培养的方法,并对其生长特性和光、电镜结构进行观察。方法 1从出生1-3d的SD大鼠脑组织中分离神经干细胞进行原代培养。 2采用不同的接种密度、不同的传代方法,在体外培养神经干细胞,观察细胞的生长情况。 3 Nestin、Brdu、NSE、GFAP免疫细胞化学对神经干细胞进行鉴定。 4透射电子显微镜观察神经球的超微结构。结果和结论 1培养的细胞Nestin阳性,具有多向分化和自我增殖能力(Brdu、NSE、GFAP阳性),是神经干细胞。本方法简单、易操作,可行性强。 2体外培养神经干细胞接种密度为1×106/ml时,细胞增殖速度快,克隆球生成的数量多。传代的方法应视情况而定,传代时间为7d左右。 3透镜结果:未分化的NSCs,细胞核浆比例比较大,核圆形或椭圆形。胞浆内的细胞器比较少,核糖体丰富。分化的NSCs,核浆比例缩小,胞浆内细胞器开始增多,可见到高尔基复合体、线粒体、微丝微管等。第二部分脑源性神经营养因子对神经干细胞增殖和分化的影响目的探讨BDNF对NSCs增殖与分化的影响,寻找合适的诱导时间和诱导浓度,同时研究BDNF两个受体TrkB和P75在促NSCs分化为神经元中的作用。方法 1 BDNF对NSCs增殖的影响。实验分为4个组,各组按照BDNF的浓度不同分为对照组0ng/ml实验组2ng/ml、20ng/ml、200ng/ml。MTT法检测实验组和对照组在不同的时间点(1d、3d、5d、7d)对细胞的增殖效应,并绘制生长曲线。 2观察BDNF对NSCs分化的影响,主要是采用免疫细胞化学技术。在体外诱导分化NSCs,分别在诱导后的1d、3d、5d、7d取出细胞,行免疫细胞化学染色,并计算阳性率。对比分析各组在各个时间段的细胞分化为神经元的能力,探索促神经元分化的最佳浓度和最佳时间。实验分组同上MTT测定。 3使用RT-PCR技术检测TrkB、p75 mRNA水平的变化。实验分为三组:未分化组,分化后未加干预组和分化后加入20ng/mlBDNF组。未分化组在传代后第三天,分化组在诱导分化后的第1 d、3d、5d分别检测上述三个指标的变化。探讨在BDNF的干预下,NSCs分化为神经元过程中,BDNF的两个受体的表达。结果 1 MTT的统计结果表明:不同浓度的BDNF在不同的时间,所测得490nm处的OD值与对照组相比均无显著性差异,P0.05。 2随着BDNF浓度的增加,神经干细胞分化为神经元的比率也呈增高的趋势,差异显著,P0.05。在分化的时间上,各组均是在第3天时达到最高,其中,实验组最高接近80%,而对照组仅为35%。 3 RT-PCR的结果显示:BDNF的两个受体TrkB和p75在未分化的NSCs均有所表达。BDNF在促NSCs分化的过程中,在分化后的1d、3d、5d,TrkB表达与对照组相比均有所上升,差异显著P0.05。p75表达与对照组相比则无显著性差异P0.05。结论 1 BDNF并不能明显的促进NSCs的增殖。 2 BDNF能显著的促进NSCs向神经元方向分化,其分化的比率呈剂量依赖性,并且在分化后的第三天达到最高。 3 BDNF在促NSCs向神经元方向分化过程中,主要是通过上调TrkB受体发挥作用,p75无明显作用。
[Abstract]:Neural stem cells (NSCs) are a kind of cell with self-renewal ability, and can proliferate and differentiate into three kinds of cells in the central nervous system: neurons, astrocytes and oligodendrocytes. because of its self-renewal and differentiation ability, it has a wide application prospect in clinic. Recent studies have shown that, after spinal cord injury, NSCs can promote the functional recovery of adult animal models after spinal cord injury, but the transplanted NSCs mainly differentiate into the glial cells to form the glial scar, which is an obstacle to the further recovery of the neurological function. Brain-derived neurotrophic factor (BDNF) is a low-content basic protein extracted from pig brain in 1982, and is one of the most representative members in the family of neurotrophin. The brain-derived neurotrophic factor can promote the differentiation of neural stem cells into the neurons. So we selected the neural stem cells and the brain-derived neurotrophic factor as the research object, mainly discussed the effects of the brain-derived neurotrophic factor on the proliferation and differentiation of the neural stem cells, and looked for the appropriate induction time and induction. At the same time, the two receptors, TrkB and P75 of the brain-derived neurotrophic factor, were studied to promote the differentiation of the neural stem cells to the neuron in the brain-derived neurotrophic factor. The present invention The paper is divided into two parts: the first part of the neural stem cells body Method for culturing and culturing new SD rat neural stem cells in vitro for external culture, differentiation and identification purposes and The growth characteristics and the structure of light and electron microscope were observed in this paper. primary culture of isolated neural stem cells from SD rat brain tissue of 1-3d with different inoculation Density, different passage methods, cultured neural stem cells in vitro to observe the growth of the cells. estin, Brdu, NSE, GFAP immunocytochemical the identification of neural stem cells. The ultrastructures of the neurospheres were observed by a transmission electron microscope. Nestin-positive, with multiple-orientation The method is simple and easy to operate, strong feasibility. In vitro culture of neural stem cells. In the case of 1-106/ ml, the rate of cell proliferation is fast, and the number of cloned balls is much higher. The method shall be determined according to the condition and the passage time is 7days left. The results of the right. 3 lens: the non-differentiated NSCs, the proportion of the cytoplasm of the nucleus is large, the nucleus is circular or elliptical, and the organelles in the cytoplasm The number of NSCs and the cytoplasm of the differentiated NSCs were reduced, and the cytoplasm and the cytoplasm of the NSCs were reduced. inner fine The effects of the second part of the brain-derived neurotrophic factor on the proliferation and differentiation of neural stem cells are discussed in this paper. The NF The effect of the proliferation and differentiation of NSCs and the finding The effect of the two receptors TrkB and P75 on the proliferation of NSCs was studied. The effect of BDNF on the proliferation of NSCs was studied. The experiment was divided into 4 groups. Test group: 2ng/ ml, 20ng/ ml, 200ng/ ml. MTT assay the proliferation of the cells was observed at different time points (1d, 3d, 5d, 7d) in the experimental group and the control group, and the growth curve was plotted. The effect of DNF on the differentiation of NSCs is mainly the use of immunocytochemistry. after the guide 1d, 3d, 5d, 7. The cells were taken out, the immunocytochemical staining was performed, and the positive rate was calculated. The cell differentiation of each time period is the ability of the neuron to explore the optimal concentration and the best time for the differentiation of the neuron. The changes of TrkB and p75 mRNA levels were detected by RT-PCR. The experiment was divided into three groups: non-differentiation group, no intervention group after differentiation, and after differentiation, 20ng/ ml of BDNF group was added. Not divided In the third day after passage, the differentiation group detected the changes of the three indices in the first day, the third day and the third day after induction of differentiation. In the process of neurons, the expression of the two receptors of BDNF. At different time, the OD value at 490nm was not significantly different from that of the control group, P <0.05. 2. With the increase of the concentration of BDNF, the ratio of the neural stem cells to the neurons was also increased, the difference was significant, P The results of RT-PCR showed that the two receptors, TrkB and p, were the highest in the experimental group and 35% in the control group. 75 Expression of BDNF in undifferentiated NSCs. In the process of NSCs differentiation, the expression of TrkB in the differentiated 1d, 3d, 5d, and TrkB was significantly higher than that of the control group (P0.05). The expression of p75 was no significant difference between the control group and the control group (P <0.05). Conclusion 1BDNF does not significantly promote the NS.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

【相似文献】