BMP-2对SVZa神经干细胞向多巴胺能神经元分化及DLX5表达影响的实验研究

发布时间：2019-05-27 15:38

【摘要】： 研究背景和目的: 神经干细胞是近年来神经科学领域的研究热点。神经干细胞的自我更新和多向分化潜能为许多神经系统疾病如帕金森氏病等的治疗带来了新的希望。但关键问题是诱导神经干细胞定向分化为所需的神经元的机制尚不明确。如何实现神经干细胞定向诱导分化是神经发育研究的核心问题。目前研究表明决定神经干细胞定向分化的机制有两种:一种是细胞发育、分化的表观遗传学机制,另一种是以各类细胞因子为主的外来信号的调控,即微环境。其中关键基因的调控对神经干细胞的增殖分化至关重要,它决定着神经干细胞向特定神经元分化的方向。目前主要通过以下2个途径展开研究: (1)在体研究,通过基因转导与敲除,检测这些基因在促进神经干细胞分化方向上的作用; (2)体外研究,通过各种细胞因子诱导特定的基因,检测在诱导刺激下基因对神经干细胞的增殖与分化的影响。但是目前有关基因调控机制仍未完全清楚。因此对神经干细胞基因调控机制的研究显得十分重要。室管膜前下区(Anterior subventricular zone,SVZa)是公认的神经干细胞较为集中的地方之一。从胚胎期到成年阶段,此区的神经干细胞在体内背-腹信号的控制下,不断沿着嘴侧迁移流(Rostral migratory stream,RSM)向嗅球(Olfactory bulb,OB)迁移,最后在OB分化为具有OB表型特征的中间神经元,其中就包括多巴胺能神经元(dopamine neurons,DA neurons )。SVZa神经干细胞除具有一般神经干细胞的自我更新和多向分化潜能外,还具有以下特点:(1)自产生起即具备发育为神经元的潜能;(2)迁移路径固定,高度局限化,长距离迁移;(3)在迁移过程中始终维持增殖状态和神经元分化潜能而不进一步分化。故SVZa神经干细胞成为研究神经干细胞增殖、迁移和分化调控机制的较佳模型,并成为神经干细胞研究的重点。 SVZa神经干细胞在向OB迁移、分化的过程中,需要体内外复杂的环境信号来调控,其中包括DLX5及骨形成蛋白-2(Bone morphogenetic protein-2,BMP-2)等。研究表明,DLX(distall-less homeobox)基因家族中的DLX5基因与OB中间神经元的发育密切相关。既往通过基因敲除鼠发现DLX5基因在SVZa神经干细胞向嗅球多巴胺能中间神经元分化过程中起着重要的作用,是出生后神经发生的必须基因。虽然在体实验的研究证实DLX5基因是生后OB多巴胺能中间神经元的必须基因,但DLX5具体调节神经干细胞分化机制目前还不清楚。而且由于DLX5基因敲除鼠在围生期就死亡,以后的研究就集中在体外实验研究。 BMP-2作为骨形成蛋白家族(Bone morphogenetic proteins ,BMPs)中重要成员,是干细胞所处局部微环境中非常重要的一种细胞因子。发现其对不同发育阶段和不同部位的神经干细胞的作用并不相同,既可以促进其向神经元分化也可以促进其向星形胶质细胞分化。而在胚胎鼠SVZa ,BMP-2可以促进神经干细胞向神经元分化,并且明显促进其向多巴胺能神经元分化。研究已经证实DLX5基因是OB多巴胺能中间神经元分化的必须基因,那么什么基因可以调节DLX5对SVZa神经干细胞向多巴胺能神经元分化呢?结合既往研究:BMP-2及DLX5在SVZa神经干细胞向OB多巴胺能神经元分化中的重要作用; BMP-2明显促进骨组织DLX5的表达。那么在SVZa神经干细胞向多巴胺能神经元分化中,BMP-2是否作为DLX5的上游调节因子,通过调节DLX5的表达从而促进SVZa神经干细胞向多巴胺能神经元分化呢?因此,本研究首先分离培养出SVZa神经干细胞作为研究对象,观察BMP-2及其拮抗剂Noggin对SVZa神经干细胞向多巴胺能神经元分化的影响,并分别用免疫荧光染色和RT-PCR检测这个过程中DLX5表达水平的变化,证明BMP-2通过DLX5途径诱导SVZa神经干细胞向多巴胺能神经元分化,希望为神经干细胞的定向分化研究提供一些实验资料。方法: 1、运用细胞培养技术对SVZa神经干细胞进行分离培养,并将收获的细胞克隆球行免疫细胞学的Nestin鉴定,然后将细胞克隆球分化后行NSE、GFAP、Oligo-2的免疫细胞学鉴定,以明确其具有干细胞特性和多向分化潜能。 2、将同批次体外培养所得的SVZa神经干细胞,分别以对照组、BMP-2组、BMP-2+Noggin组和Noggin组诱导,分别用免疫荧光染色和RT PCR检测不同处理组DLX5蛋白和DLX5 mRNA表达情况。 3、将同批次体外培养所得的SVZa神经干细胞,分别以对照组、BMP-2组、BMP-2+Noggin组和Noggin组诱导,通过免疫荧光染色和流式细胞技术检测不同处理组SVZa神经干细胞分化为多巴胺能神经元的比例。结果: 1、在体外无血清培养的条件下,可以培养出大量悬浮生长的细胞克隆球,Nestin免疫荧光染色阳性,分化后的NSE、GFAP、Oligo-2的免疫细胞学鉴定反应也呈阳性反应。 2、经对照组、BMP-2组、BMP-2+Noggin组和Noggin组诱导72小时后,分别行免疫荧光染色和RT PCR检测,结果显示BMP-2组中镜下可见DLX5表达明显增强,而对照组、BMP-2+Noggin组和Noggin组的DLX5表达无明显变化;BMP-2组中DLX5 mRNA表达量也明显高于对照组,p0.05,差异有统计学意义,而对照组、BMP-2+Noggin组和Noggin组DLX5mRNA表达量组间两两比较,差异无显著性,p0.05。 3、经对照组、BMP-2组、BMP-2+Noggin组和Noggin组诱导分化72小时后,分别行免疫荧光染色及流式细胞仪检测,结果显示BMP-2组可见较多TH+细胞,而对照组、BMP-2 +Noggin组和Noggin组的TH +细胞明显较少;流式细胞结果也提示BMP-2组TH+神经元分化比例较对照组明显增高,p0.05,差异有统计学意义;而对照组、BMP-2+Noggin组和Noggin组TH +神经元分化比例组间两两比较,差异无显著性,p0.05。结论: 1、从E15天Wistar大鼠SVZa脑组织中成功分离得到具有不断增殖和自我更新能力,并可以分化为神经元、星形胶质细胞和少突胶质细胞的神经干细胞。 2、BMP-2明显促进SVZa神经干细胞DLX5蛋白和DLX5mRNA表达,而且这种作用能被其拮抗剂Noggin所拮抗。 3、BMP-2明显促进SVZa神经干细胞分化为多巴胺能神经元,并且这种作用同样能被其拮抗剂Noggin所拮抗。在SVZa,BMP-2可能通过上调DLX5表达从而促进神经干细胞向TH+神经元分化。
[Abstract]:Background and purpose of the study: Neural stem cells are the field of neuroscience in recent years The self-renewal and multiple-to-differentiation potential of neural stem cells bring new treatment to many nervous system diseases, such as Parkinson's disease Hopefully, the key issue is that the mechanism for inducing the directional differentiation of neural stem cells into the desired neurons is not yet It is clear how to realize the directional induction and differentiation of neural stem cells is the core of the neurodevelopmental study. The present study indicates that there are two mechanisms to determine the directional differentiation of neural stem cells: one is the epigenetic mechanism of cell development and differentiation, and the other is the regulation of external signals dominated by various cytokines. The regulation of the key genes is crucial to the proliferation and differentiation of neural stem cells, which determines the differentiation of neural stem cells to specific neurons. direction. The study was conducted mainly by (1) in vivo studies, by gene transduction and knock-out, to detect the role of these genes in promoting the differentiation of neural stem cells, (2) in vitro studies, by the induction of specific cytokines by various cytokines, Gene, to detect the proliferation and differentiation of the gene to neural stem cells under the induction of stimulation However, the current regulation mechanism of the gene is still incomplete. It is clear that the research on the regulation mechanism of neural stem cell gene is very important It is important that the anterior inferior zone (SVZa) of the ependymal membrane is a well-recognized neural stem cell. One of the places from the embryonic stage to the adult stage, the neural stem cells of this region, under the control of the in vivo back-abdominal signal, migrate continuously along the mouth-side migration flow (RSM) to the olfactory bulb (OB), and finally, in the OB differentiation to have the OB phenotype characteristic the intermediate neurons, including the dopaminergic neurons (dopamine netrons, da neu, The SVZa neural stem cells have the following characteristics in addition to the self-renewal and multi-directional differentiation potential of general neural stem cells: (1) the potential for development as a neuron is self-generated; (2) the migration path is fixed and highly localized, Long-distance migration; (3) always maintain the proliferative state and the potential of the neuron differentiation during the migration, and not The SVZa neural stem cell is a better model for the study of the mechanism of the proliferation, migration and differentiation of neural stem cells and becomes a neural stem cell. The focus of the study was that the SVZa neural stem cells, in the process of migration and differentiation to OB, needed to be regulated by a complex in-vivo environment signal, including DLX5 and bone morphogenetic protein-2 (Bone morphogenetic protein-2). and BMP-2, etc. The study shows that the DLX5 gene in the DLX (distall-less homoerox) gene family and the OB middle nerve It is closely related to the development of meta. In the past, the gene knockout mice have found that the DLX5 gene plays an important role in the differentiation of the dopaminergic neurons of the olfactory bulb in the SVZa neural stem cell, and is the postnatal god. Although the study of in vivo experiments confirmed that the DLX5 gene is a necessary gene for OB dopaminergic intermediate neurons, the DLX5 specifically regulates the differentiation of neural stem cells. The mechanism is not yet clear. And since the DLX5 knockout mice died during the perinatal period, the later study set In vitro, BMP-2 is an important member of bone morphogenetic protein (BMPs) and is the local microenvironment of stem cells. It is a very important cytokine. It is found that its effects on the neural stem cells at different developmental stages and in different parts are not the same, which can promote the differentiation of the neurons to the neurons. In embryonic rat SVZa, BMP-2 can promote the differentiation of neural stem cells into neurons and promote the differentiation of neural stem cells. The study has confirmed that the DLX5 gene is an essential gene for OB dopaminergic neuron differentiation, so what gene can regulate the DLX5 to the SVZa nerve The differentiation of stem cells to dopaminergic neurons? Combined with previous studies: BMP-2 and DLX5 play an important role in the differentiation of the neural stem cells of SVZa to OB dopaminergic neurons; BMP-2 The expression of the DLX5 in the bone tissue is obviously promoted. In the differentiation of the SVZa neural stem cell into the dopaminergic neuron, whether the BMP-2 is the upstream regulatory factor of the DLX5, and the expression of the DLX5 is adjusted to promote the SVZa nerve. The effect of BMP-2 and its antagonist Noggin on the differentiation of the dopaminergic neurons of the SVZa neural stem cells was observed and the effect of the antagonist Noggin on the differentiation of the dopaminergic neurons was observed, and this was detected by immunofluorescent staining and RT-PCR, respectively. The changes of the expression level of the DLX5 in the process demonstrated that the BMP-2 induces the differentiation of the SVZa neural stem cells to the dopaminergic neurons through the DLX5 pathway and is expected to be a neural stem cell. directional point Methods:1. The SVZa neural stem cells were isolated and cultured by cell culture, and the harvested cells were identified by Nestin, and then the cells were cloned into NSE. The Immune Cytological Evaluation of GFAP and Oligo-2 (2) The SVZa neural stem cells obtained from in-vitro culture of the same batch were cultured in the control group, the BMP-2 group, the BMP-2 + Noggin group and the Noggin group, respectively. The expression of DLX5 and DLX5 mRNA in the same batch was compared with that of the control group, the BMP-2 group, the BMP-2 + Noggin group and the Noggin group. co-processing The ratio of the group of SVZa neural stem cells to the dopaminergic neurons was determined. The results were as follows:1. Under the condition of no serum culture in vitro, a large number of cell clone balls of suspended growth could be cultured, and the Nestin immunofluorescence staining was positive and differentiated. The results showed that the expression of DLX5 in BMP-2 group was significantly enhanced by RT-PCR, and the results showed that the expression of DLX5 was significantly enhanced in BMP-2 group. There was no significant change in the expression of DLX5 in the group, BMP-2 + Noggin group and Noggin group, and the amount of DLX5 mRNA in the BMP-2 group was significantly higher than that of the control group, p0.05, the difference was of statistical significance, and the control group, BMP-2 + Noggin group and No. The expression of DLX5mRNA in ggin group was not significant between the two groups. The difference was not significant, p0.05.3. After 72 hours of induction and differentiation of the control group, the BMP-2 group, the BMP-2 + Noggin group and the Noggin group, the results showed that the BMP-2 group showed more TH + fine. In the control group, the TH + cells of the control group, the BMP-2 + Noggin group and the Noggin group were significantly less. The flow cytometry also suggested that the proportion of TH + neurons in the BMP-2 group was significantly higher than that in the control group. Group and N There was no significant difference between the ratio of TH and neuron in the goggin group, and the difference was not significant, p0.05. Conclusion:1. The successful separation of the brain of the SVZa from the Wistar rats from E15 days. neural stem cells with constant proliferation and self-renewal and can be differentiated into neurons, astrocytes, and oligodendrocytes. BMP-2 significantly promotes S VZa neural stem cell DLX5 protein and DLX5mRNA expression, and this effect can be antagonized by its antagonist Noggin.3, BM P-2 significantly promotes the differentiation of SVZa neural stem cells into dopaminergic neurons, and this effect can also be used as an antagonist, N,
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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