BMP2及ACM对SVZa神经干细胞诱导分化为多巴胺能神经元作用的实验研究
发布时间：2019-03-20 16:15
【摘要】： 研究背景和目的: 神经干细胞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、少突胶质细胞,能为中枢神经系统功能重建和神经再生提供新的供体源,而且能较好地解决神经移植治疗的伦理道德及供体不足等问题,为神经系统的许多疾病如帕金森氏病的治疗带来了新希望。 室管膜前下区(Anterior subventriculor zone, SVZa)是公认的神经干细胞富集区,该区的神经干细胞在体内背-腹信号控制下,不断沿着一条局限化的迁移通道-吻侧迁移流(Rostral migratory stream,RMS)向嗅球(Olfactory bulb,OB)迁移,最后在嗅球分化为具有OB表型特征的中间神经元,包括:多巴胺能神经元和γ-氨基丁酸能神经元。并且SVZa神经干细胞除具有一般干细胞的自我更新能力和多分化潜能外,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)能长距离定向迁移;(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、迁移和分化的最佳模型。 神经干细胞的分化主要取决于两个方面:一是干细胞的内在遗传基因,二是干细胞所处的外部微环境。相同来源的干细胞处于不同的微环境中可以分化为不同类型不同谱系的细胞,因此目前对于干细胞分化的研究除了其自身基因的调控外,多侧重于外来信号的调控。BMP2作为转化生长因子-β(TGF-β)超家族成员,是干细胞所处局部微环境中非常重要的一种细胞因子。其在体外条件下对室管膜前下区神经干细胞分化为多巴胺能神经元作用如何还需要进一步的实验研究。 另外,既往研究表明,单一因子诱导神经干细胞分化为多巴胺能神经元作用非常有限,这就要求探求多种因子联合作用从而使分化为多巴胺能神经元比例增加。ACM作为星形胶质细胞体外培养时的细胞外液,其内含有胶质细胞分泌的丰富的可溶性生物活性物质,如:神经生长因子(NGF)、胶质细胞源性神经营养因子(GDNF)、脑源性神经营养因子(BDNF)等,且与神经干细胞所处的体内环境较为接近,其促分化作用是单一神经营养因子所无法比拟的。因此,本研究首先分离培养出SVZa神经干细胞作为研究对象,然后再以不同浓度的BMP2诱导其分化,并分别检测其分化为多巴胺能神经元的比例。最后再以BMP2和ACM诱导SVZa神经干细胞分化,研究它们在SVZa神经干细胞分化为多巴胺能神经元中的作用,希望为SVZa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NF、GFAP、CNP的免疫细胞学鉴定,以明确其具有干细胞特性和多分化潜能。 2、将体外培养所得的SVZa神经干细胞,分别以0、0.1、1、10、20、100(ng/ml)浓度的BMP2诱导其分化,通过免疫组化和流式细胞仪技术检测不同浓度情况下SVZa神经干细胞分化为TH+(酪氨酸羟化酶)神经元的比例。 3、将体外培养所得的SVZa神经干细胞,分为对照组、BMP2组、ACM组、BMP2+ACM组分别予以诱导分化,通过免疫组化和流式细胞仪技术检测各组中SVZa神经干细胞分化为TH+细胞的比例。 结果: 1、在体外无血清培养条件下,可以收获大量悬浮生长的细胞克隆球,Nestin免疫细胞学反应阳性,分化后的NF、GFAP、CNP免疫细胞学反应也呈阳性反应。 2、SVZa神经干细胞分别以0、0.1、1、10、20、100(ng/ml)浓度的BMP2诱导分化后,经检测0.1～100(ng/ml)组的TH+细胞比例均明显高于0ng/ml组,10ng/ml组达最高峰,各组间差异均有统计学意义(P㩳0.05)。 3、SVZa神经干细胞分为对照组、BMP2组、ACM组、BMP2+ACM组诱导分化后,免疫组化和流式细胞仪检测结果提示:各实验组的TH+细胞比例均明显高于对照组,其中BMP2+ACM组分化比例最高,差异有统计学意义(P㩳0.05) 结论: 1、从孕16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、BMP2可以促进SVZa神经干细胞分化为多巴胺能神经元,但不完全是剂量依赖性的,而是有一个最佳浓度,即10ng/ml的BMP2作用最明显。 3、ACM可以促进SVZa神经干细胞向多巴胺能神经元分化,而且BMP2与ACM具有正协同作用。
[Abstract]:Background and purpose of the study: Neural stem cells have the ability to proliferate and self-update and can be differentiated into neurons, astrocytes, oligodendrocytes, which can provide a new way for the functional reconstruction and nerve regeneration of the central nervous system The donor source can better solve the problems of the ethics and the donor deficiency of the nerve graft treatment, and brings the treatment of a plurality of diseases of the nervous system such as Parkinson's disease The new hope is that the anterior inferior zone (SVZa) of the ependymal membrane is a well-established neural stem cell-rich region, and the neural stem cells in the region are under the control of the in vivo back-and-abdominal signals, and the olfactory bulb (Olfactory bul) is continuously transported along a localized migration channel-side migration stream (RMS) under the control of the in vivo back-abdominal signal. b, OB) migration, and finally, differentiation of the olfactory bulb into an intermediate neuron having an OB phenotypic characteristic, including: a dopaminergic neuron and a HCO3-ammonia; The SVZa neural stem cell has the following characteristics in addition to the self-renewal capacity and the multi-differentiation potential of the general stem cells: (1) the self-generation of the neural stem cells has the following characteristics: (1) the self-generation of the neural stem cells has the potential to develop into the neuron; (2) the SVZa neural stem cell can Long-distance directional migration; (3) always maintain the proliferative state and the potential of neuronal differentiation during the migration Therefore, the SVZa neural stem cells become the study of the proliferation and migration of neural stem cells. The differentiation is the best model. The differentiation of neural stem cells depends on two aspects: one is the inner genetic gene of the stem cells, and the other is the second. The stem cells of the same origin are in different microenvironment and can be differentiated into different types of cells, so the present study on stem cell differentiation, in addition to its own gene regulation, Multi-focus on the regulation of external signals. BMP2 is a member of the superfamily of the transformation growth factor-1 (TGF-1), which is the local microenvironment of the stem cells. A cytokine that is very important. How can the differentiation of neural stem cells in the anterior inferior region of the ependytomembrane into dopaminergic neurons under in vitro conditions Further experimental studies are also required. In addition, previous studies have shown that the differentiation of a single factor-induced neural stem cell into dopaminergic neurons is very limited, which requires a combination of multiple factors. AACM, as the extracellular fluid in the in vitro culture of astrocytes, contains abundant soluble bioactive substances, such as nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), brain, The derived neurotrophic factor (BDNF) and the like are close to the in-vivo environment in which the neural stem cells are located, and the differentiation effect In this study, the SVZa neural stem cells were isolated and cultured as the research object, then the differentiation was induced by BMP2 at different concentrations. In the end, the differentiation of SVZa neural stem cells was induced by BMP2 and ACM, and the role of them in the differentiation of the SVZa neural stem cells into dopaminergic neurons was studied, and it was desired to be SVZa neural stem cells orientation Methods:1. The SVZa neural stem cells were isolated and cultured by a cell culture technique, and the harvested cells were identified by the Nestin identification of the immunocytological cell line, and then the cells were cloned into the cells. The immunity of NF, GFAP and CNP after differentiation The SVZa neural stem cells obtained from in-vitro culture were induced by BMP2 at 0, 0.1,1,10,20,100 (ng/ ml), respectively, and SV was detected by immunohistochemistry and flow cytometry. Za neural stem cells were differentiated into TH + (tyrosine hydroxylase) neurons.3. The SVZa neural stem cells obtained in vitro were divided into control group, BMP2 group, ACM group and BMP2 + ACM group, respectively. cell The ratio of SVZa neural stem cells to TH + cells in each group was detected by the instrument technique. Results:1. In the absence of serum culture in vitro, a large number of cell clone balls of suspension growth could be harvested, Nesti The positive and differentiated NF, GFAP and CNP were positive. The ratio of TH + cells in 0.1 to 100 (ng/ ml) group was significantly higher after the differentiation of the SVZa neural stem cells at 0, 0.1,1,10,20,100 (ng/ ml). Compared with the control group, the control group, the BMP2 group, the ACM group and the BMP2 + ACM group were divided into the control group, the BMP2 group, the ACM group and the BMP2 + ACM group to induce the differentiation. Tip of the cytometric test: TH of each experimental group + cells The ratio of BMP2 + ACM group was higher than that of control group, and the difference was significant (P? 0.05). the successful isolation of Za brain tissue has the ability to continuously proliferate and self-update, and can be differentiated into neural stem cells of neurons, astrocytes and oligodendrocytes. BMP2 can promote S VZa neural stem cells are differentiated into dopaminergic neurons, but are not completely dose-dependent, but have an optimal concentration, i.e.,10 ng
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
本文编号：2444380
[Abstract]:Background and purpose of the study: Neural stem cells have the ability to proliferate and self-update and can be differentiated into neurons, astrocytes, oligodendrocytes, which can provide a new way for the functional reconstruction and nerve regeneration of the central nervous system The donor source can better solve the problems of the ethics and the donor deficiency of the nerve graft treatment, and brings the treatment of a plurality of diseases of the nervous system such as Parkinson's disease The new hope is that the anterior inferior zone (SVZa) of the ependymal membrane is a well-established neural stem cell-rich region, and the neural stem cells in the region are under the control of the in vivo back-and-abdominal signals, and the olfactory bulb (Olfactory bul) is continuously transported along a localized migration channel-side migration stream (RMS) under the control of the in vivo back-abdominal signal. b, OB) migration, and finally, differentiation of the olfactory bulb into an intermediate neuron having an OB phenotypic characteristic, including: a dopaminergic neuron and a HCO3-ammonia; The SVZa neural stem cell has the following characteristics in addition to the self-renewal capacity and the multi-differentiation potential of the general stem cells: (1) the self-generation of the neural stem cells has the following characteristics: (1) the self-generation of the neural stem cells has the potential to develop into the neuron; (2) the SVZa neural stem cell can Long-distance directional migration; (3) always maintain the proliferative state and the potential of neuronal differentiation during the migration Therefore, the SVZa neural stem cells become the study of the proliferation and migration of neural stem cells. The differentiation is the best model. The differentiation of neural stem cells depends on two aspects: one is the inner genetic gene of the stem cells, and the other is the second. The stem cells of the same origin are in different microenvironment and can be differentiated into different types of cells, so the present study on stem cell differentiation, in addition to its own gene regulation, Multi-focus on the regulation of external signals. BMP2 is a member of the superfamily of the transformation growth factor-1 (TGF-1), which is the local microenvironment of the stem cells. A cytokine that is very important. How can the differentiation of neural stem cells in the anterior inferior region of the ependytomembrane into dopaminergic neurons under in vitro conditions Further experimental studies are also required. In addition, previous studies have shown that the differentiation of a single factor-induced neural stem cell into dopaminergic neurons is very limited, which requires a combination of multiple factors. AACM, as the extracellular fluid in the in vitro culture of astrocytes, contains abundant soluble bioactive substances, such as nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), brain, The derived neurotrophic factor (BDNF) and the like are close to the in-vivo environment in which the neural stem cells are located, and the differentiation effect In this study, the SVZa neural stem cells were isolated and cultured as the research object, then the differentiation was induced by BMP2 at different concentrations. In the end, the differentiation of SVZa neural stem cells was induced by BMP2 and ACM, and the role of them in the differentiation of the SVZa neural stem cells into dopaminergic neurons was studied, and it was desired to be SVZa neural stem cells orientation Methods:1. The SVZa neural stem cells were isolated and cultured by a cell culture technique, and the harvested cells were identified by the Nestin identification of the immunocytological cell line, and then the cells were cloned into the cells. The immunity of NF, GFAP and CNP after differentiation The SVZa neural stem cells obtained from in-vitro culture were induced by BMP2 at 0, 0.1,1,10,20,100 (ng/ ml), respectively, and SV was detected by immunohistochemistry and flow cytometry. Za neural stem cells were differentiated into TH + (tyrosine hydroxylase) neurons.3. The SVZa neural stem cells obtained in vitro were divided into control group, BMP2 group, ACM group and BMP2 + ACM group, respectively. cell The ratio of SVZa neural stem cells to TH + cells in each group was detected by the instrument technique. Results:1. In the absence of serum culture in vitro, a large number of cell clone balls of suspension growth could be harvested, Nesti The positive and differentiated NF, GFAP and CNP were positive. The ratio of TH + cells in 0.1 to 100 (ng/ ml) group was significantly higher after the differentiation of the SVZa neural stem cells at 0, 0.1,1,10,20,100 (ng/ ml). Compared with the control group, the control group, the BMP2 group, the ACM group and the BMP2 + ACM group were divided into the control group, the BMP2 group, the ACM group and the BMP2 + ACM group to induce the differentiation. Tip of the cytometric test: TH of each experimental group + cells The ratio of BMP2 + ACM group was higher than that of control group, and the difference was significant (P? 0.05). the successful isolation of Za brain tissue has the ability to continuously proliferate and self-update, and can be differentiated into neural stem cells of neurons, astrocytes and oligodendrocytes. BMP2 can promote S VZa neural stem cells are differentiated into dopaminergic neurons, but are not completely dose-dependent, but have an optimal concentration, i.e.,10 ng
【学位授予单位】：第三军医大学
【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3条
1 高国一,卢亦成,江基尧,李莉,张玲珍,朱诚;免疫磁珠法分选胚胎大鼠脑神经干细胞的初步研究[J];第二军医大学学报;2000年11期
2 严稽文;黄其林;;星形胶质细胞条件培养液对缺氧损伤神经元的保护作用[J];神经解剖学杂志;2007年03期
3 高方友;杨辉;张治元;何家全;刘仕勇;邱克军;;Dlx5 mRNA在室管膜前下区神经干细胞中的表达规律及其对神经元分化的影响[J];中华创伤杂志;2006年05期
，本文编号：2444380
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2444380.html
最近更新
教材专著
