维甲酸及其衍生物诱导胚胎干细胞定向分化为神经细胞及其应用研究
发布时间：2019-03-24 20:57
【摘要】：胚胎干细胞(embryonic stem cell,ES cell)是一种具有自我更新能力和多种分化潜能的细胞,可以分化为三个胚层的细胞。作为一种独特的体外生物系统,胚胎干细胞可以用来发现和阐明生物发育过程中的奥秘,也是疾病模拟、药物筛选和细胞治疗等非常有用的材料。 胚胎干细胞经体外诱导可以分化为外胚层组织,并进一步分化为神经元和神经胶质细胞。利用全反式维甲酸(alltrans retinoic acid,RA/ATRA)作为神经诱导剂,已经成功从人或者小鼠拟胚体(embryoid bodies,EBs)中分化得到多种神经细胞。然而,由于RA作用的广泛性和复杂性及所用拟胚体分化系统的复杂性和难以调控性,RA对胚胎干细胞向神经细胞方向诱导分化的作用机制尚未明确阐释。为了简化诱导体系,我们采用了单层贴壁培养作为分化模式,以无血清培养的方式,用不同浓度的RA诱导胚胎干细胞,观察了RA对胚胎干细胞定向分化为神经细胞的作用,并通过干预与神经细胞分化相关的多条信号通路,探讨了RA诱导胚胎干细胞分化为神经细胞的机制。实验表明,在无血清单层贴壁培养条件下,RA可以促进神经细胞的分化、加速胚胎干细胞标志性分子的丢失;维甲酸相关核受体及代谢酶类也发生动念变化;维甲酸核受体RAR alpha的特异性拮抗剂、ERK(extracellular signal-regulated protein kinase)信号通路的抑制剂、GSK3(glycogen synthase kinase 3)的抑制剂均可以有效阻断RA的诱导作用;FGF(fibroblast growth factor)信号通路的抑制剂不能有效阻断RA的作用。实验阐明了,RA促进胚胎干细胞分化为神经细胞的作用是不依赖FGF信号通路的;维甲酸核受体RAR alpha在诱导分化过程中扮演重要角色;RA通过与ERK及Wnt信号通路之间的联系而发挥诱导分化作用。 维脯酰胺(N-all-trans-retinoyl-L-proline,ATRP)是基于全反式维甲酸和芬维A胺的结构合成的新化合物。有研究表明该化合物具有抑制肝癌细胞侵袭的作用,而在胚胎干细胞定向分化为神经细胞中的作用还缺少研究。我们的实验证明了ATRP可以促进胚胎干细胞向神经细胞的分化,其诱导分化得到的神经前体细胞比率要高于RA;与此同时,ATRP加速了胚胎干细胞标志性分子的丢失;ATRP发挥诱导分化作用的机制与RA有一定相似之处。值得注意的是,先前的报道认为ATRP不是通过与维甲酸相关核受体结合而发挥作用的,而本研究观察到,阻断维甲酸核受体RAR alpha的信号通路也可以有效阻断ATRP的诱导分化作用。ATRP与维甲酸信号通路之间的关系还需要进一步地阐释;ATRP的代谢情况也需进一步研究,其促进分化的作用有可能是代谢过程中转化为RA的结果。 阿尔茨海默病(Alzheimer's disease,AD)是一种进行性发展的致死性神经退行性疾病,临床表现为认知障碍和记忆功能不断恶化,日常生活能力进行性减退,并有各种神经精神症状和行为异常。目前,阿尔茨海默病的药物治疗主要集中在减轻病人胆碱能系统障碍而出现的功能紊乱和疾病症状上,没能从根本上解决问题,而且病人在服用相关药物后还会产生多种不良反应。我们应用RA诱导胚胎干细胞定向分化得到神经干细胞、以侧脑室为移植靶位,进行了胚胎干细胞来源的神经干细胞治疗AD模型小鼠(Mo/Hu APPswe PS1dE9双转基因小鼠)的初步探索。实验表明,经RA诱导后,小鼠胚胎干细胞分化为表达特异性分子标志Nestin且具有分化为神经元和神经胶质细胞能力的神经干细胞。该神经干细胞经体外纯化、扩增及标记后,通过立体定位注射移植入双转基因AD模型小鼠的一侧脑室。手术一周后,动物侧脑室壁可以见到移植的细胞;手术六周后,Morris水迷宫测试显示治疗动物的空间记忆能力得到显著提高;手术八周后,在治疗动物的皮层发现部分移植细胞呈神经元特异性标志MAP2阳性和胆碱能神经元特异性标志ChAT阳性。研究显示,经侧脑室移植胚胎干细胞来源的外源性神经干细胞可以有效提高AD模型动物的认知能力。这为临床应用胚胎干细胞治疗AD等中枢神经系统退行性疾病提供了一定理论和实践基础。
[Abstract]:Embryonic stem cell (ES cell) is a kind of cell with self-renewal ability and multiple differentiation potential, and can be divided into three germ cells. As a unique in vitro biological system, embryonic stem cells can be used to identify and elucidate the mysteries of the process of biological development, as well as very useful materials such as disease simulation, drug screening and cell therapy. In-vitro induction of embryonic stem cells can be differentiated into ectodermal tissue and further differentiated into neurons and glial cells. The use of all-trans retinoic acid (RA/ ATRA) as a nerve inducing agent has been successfully differentiated from human or mouse embryonic bodies (EBs) to obtain a variety of nerves. However, the mechanism of the mechanism of RA to induce differentiation of embryonic stem cells in the direction of nerve cells is not clear due to the wide and complex nature of RA and the complexity and difficulty of the differentiation system of the embryoid used. In order to simplify the induction system, a single-layer adherent culture was used as the differentiation mode, and the embryonic stem cells were induced with different concentrations of RA in the form of serum-free culture, and the orientation and differentiation of the embryonic stem cells into the nerve cells were observed. The effects of RA on the differentiation of embryonic stem cells into nerve cells were discussed by means of multiple signal pathways related to the differentiation of neural cells. Mechanism. The experiment shows that RA can promote the differentiation of nerve cells and accelerate the loss of the symbolic molecules of embryonic stem cells under the condition of serum-free monolayer adherent culture, and the related nuclear receptors and the metabolic enzymes of the retinoic acid also change; the specificity of the retinoic acid nuclear receptor RAR alpha The inhibitor of the signal pathway of the anti-agent, ERK, and the inhibitor of GSK3 can effectively block the induction of RA, and the inhibitor of the signal pathway of the fibroblast growth factor can not block the RA effectively. The role of RA in promoting the differentiation of embryonic stem cells into nerve cells is not dependent on the signaling pathway of the FGF, and the retinoic acid nuclear receptor RAR alpha plays an important role in the induction of differentiation, and RA induces differentiation through the association with the ERK and Wnt signaling pathways. The function of N-all-trans-retinol-L-proline (ATRP) is based on the structural synthesis of all-trans-retinoic acid and finiveramine. The compound has the effect of inhibiting the invasion of the liver cancer cells, and the directional differentiation of the embryonic stem cells into the nerve cells There is also a lack of research. Our experiments have shown that ATRP can promote the differentiation of embryonic stem cells into nerve cells, and the ratio of neural precursor cells induced by the induction of differentiation is higher than that of RA; at the same time, ATRP accelerates the signature of embryonic stem cells The mechanism of ATRP to induce differentiation and RA has one. The similarities are noted. It is worth noting that the previous report found that ATRP did not function by binding to the retinoic acid-related nuclear receptor, while the study observed that the signal path blocking the RAR alpha of the retinoic acid nuclear receptor could also effectively block the induction of the ATRP. The relationship between the ATRP and the signal pathway of the retinoic acid also needs to be further explained; the metabolism of the ATRP is further studied, and the effect of promoting differentiation is likely to be a transformation in the process of metabolism. As a result of RA, Alzheimer's disease (AD) is a progressive, fatal neurodegenerative disease characterized by a worsening of cognitive and memory functions, progressive deterioration of the ability of daily life, and various neuroses. The symptoms and behavior of the people are abnormal. At present, the drug treatment of Alzheimer's disease is mainly focused on the function disorder and the disease symptom which are caused by the disorder of the cholinergic system of the patient, and the problem can not be solved fundamentally, A variety of adverse reactions were produced. We applied RA to induce the directional differentiation of embryonic stem cells to obtain neural stem cells. The neural stem cells derived from embryonic stem cells were used to treat AD model mice (Mo/ Hu APPswe PS1dE9 double transgenes). The results indicated that after RA induction, the mouse embryonic stem cells were differentiated into the expression-specific molecular marker, Nestin, and differentiated into neurons and glial cells. the neural stem cell is purified, expanded and marked in vitro, and then transplanted into the double-transgenic AD mould through a three-dimensional positioning injection, One week after the operation, the wall of the lateral ventricle of the animal can see the transplanted cells; after 6 weeks of operation, the Morris water maze test shows that the spatial memory capacity of the treated animals is significant increased; after 8 weeks of operation, partial transplantation cells were found to be neuron-specific marker MAP2-positive and cholinergic neuron-specific in the cortex of the treated animals The marker ChAT is positive. The study shows that the exogenous neural stem cells derived from the embryonic stem cell in the lateral ventricle can effectively improve the AD model. The cognitive ability of a type of animal is provided for clinical application of embryonic stem cells to treat the degenerative diseases of the central nervous system such as AD.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329
本文编号：2446672
[Abstract]:Embryonic stem cell (ES cell) is a kind of cell with self-renewal ability and multiple differentiation potential, and can be divided into three germ cells. As a unique in vitro biological system, embryonic stem cells can be used to identify and elucidate the mysteries of the process of biological development, as well as very useful materials such as disease simulation, drug screening and cell therapy. In-vitro induction of embryonic stem cells can be differentiated into ectodermal tissue and further differentiated into neurons and glial cells. The use of all-trans retinoic acid (RA/ ATRA) as a nerve inducing agent has been successfully differentiated from human or mouse embryonic bodies (EBs) to obtain a variety of nerves. However, the mechanism of the mechanism of RA to induce differentiation of embryonic stem cells in the direction of nerve cells is not clear due to the wide and complex nature of RA and the complexity and difficulty of the differentiation system of the embryoid used. In order to simplify the induction system, a single-layer adherent culture was used as the differentiation mode, and the embryonic stem cells were induced with different concentrations of RA in the form of serum-free culture, and the orientation and differentiation of the embryonic stem cells into the nerve cells were observed. The effects of RA on the differentiation of embryonic stem cells into nerve cells were discussed by means of multiple signal pathways related to the differentiation of neural cells. Mechanism. The experiment shows that RA can promote the differentiation of nerve cells and accelerate the loss of the symbolic molecules of embryonic stem cells under the condition of serum-free monolayer adherent culture, and the related nuclear receptors and the metabolic enzymes of the retinoic acid also change; the specificity of the retinoic acid nuclear receptor RAR alpha The inhibitor of the signal pathway of the anti-agent, ERK, and the inhibitor of GSK3 can effectively block the induction of RA, and the inhibitor of the signal pathway of the fibroblast growth factor can not block the RA effectively. The role of RA in promoting the differentiation of embryonic stem cells into nerve cells is not dependent on the signaling pathway of the FGF, and the retinoic acid nuclear receptor RAR alpha plays an important role in the induction of differentiation, and RA induces differentiation through the association with the ERK and Wnt signaling pathways. The function of N-all-trans-retinol-L-proline (ATRP) is based on the structural synthesis of all-trans-retinoic acid and finiveramine. The compound has the effect of inhibiting the invasion of the liver cancer cells, and the directional differentiation of the embryonic stem cells into the nerve cells There is also a lack of research. Our experiments have shown that ATRP can promote the differentiation of embryonic stem cells into nerve cells, and the ratio of neural precursor cells induced by the induction of differentiation is higher than that of RA; at the same time, ATRP accelerates the signature of embryonic stem cells The mechanism of ATRP to induce differentiation and RA has one. The similarities are noted. It is worth noting that the previous report found that ATRP did not function by binding to the retinoic acid-related nuclear receptor, while the study observed that the signal path blocking the RAR alpha of the retinoic acid nuclear receptor could also effectively block the induction of the ATRP. The relationship between the ATRP and the signal pathway of the retinoic acid also needs to be further explained; the metabolism of the ATRP is further studied, and the effect of promoting differentiation is likely to be a transformation in the process of metabolism. As a result of RA, Alzheimer's disease (AD) is a progressive, fatal neurodegenerative disease characterized by a worsening of cognitive and memory functions, progressive deterioration of the ability of daily life, and various neuroses. The symptoms and behavior of the people are abnormal. At present, the drug treatment of Alzheimer's disease is mainly focused on the function disorder and the disease symptom which are caused by the disorder of the cholinergic system of the patient, and the problem can not be solved fundamentally, A variety of adverse reactions were produced. We applied RA to induce the directional differentiation of embryonic stem cells to obtain neural stem cells. The neural stem cells derived from embryonic stem cells were used to treat AD model mice (Mo/ Hu APPswe PS1dE9 double transgenes). The results indicated that after RA induction, the mouse embryonic stem cells were differentiated into the expression-specific molecular marker, Nestin, and differentiated into neurons and glial cells. the neural stem cell is purified, expanded and marked in vitro, and then transplanted into the double-transgenic AD mould through a three-dimensional positioning injection, One week after the operation, the wall of the lateral ventricle of the animal can see the transplanted cells; after 6 weeks of operation, the Morris water maze test shows that the spatial memory capacity of the treated animals is significant increased; after 8 weeks of operation, partial transplantation cells were found to be neuron-specific marker MAP2-positive and cholinergic neuron-specific in the cortex of the treated animals The marker ChAT is positive. The study shows that the exogenous neural stem cells derived from the embryonic stem cell in the lateral ventricle can effectively improve the AD model. The cognitive ability of a type of animal is provided for clinical application of embryonic stem cells to treat the degenerative diseases of the central nervous system such as AD.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329
【参考文献】
相关期刊论文 前1条
1 ;Transplanted neuronal precursors migrate and differentiate in the devel-oping mouse brain[J];Cell Research;2002年Z1期
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