RA序贯诱导小鼠胚胎干细胞向神经细胞分化及对4.1蛋白表达的影响

发布时间：2018-03-26 10:21

  本文选题：胚胎干细胞　切入点：分化　出处：《郑州大学》2008年硕士论文

【摘要】：胚胎干细胞(embryonic stem cells,ESCs)是从早期囊胚的内细胞团(innercell mass,ICM)分离出来的一种多能细胞系;它能在体外长期不断自我更新,并保持多向分化潜能,可以分化为内、中、外三胚层来源的几乎所有类型的细胞。已有研究表明,ESCs在特定的诱导培养体系中,可被定向诱导分化为神经细胞,为ESCs移植治疗神经系统疾病提供了实践依据。迄今为止,体外诱导法主要有4-/4+法、五步法、基质细胞诱导法、单层粘附培养法和基因修饰法等。其中4-/4+法是最经典的诱导法,但该方法使用维甲酸(retinoic acid,RA)诱导前需形成类胚体(embryonic bodies,EB),使分化较难控制和分析,且培养时间长、操作复杂,对细胞密度、营养、设备等培养要求较高。探索新的细胞诱导方法用以减少诱导时间、简化操作将促进ESCs体外培养分化的研究。目前对ESCs分化为神经细胞的机制仍不清。有报道称在ESCs向神经细胞分化过程中出现某些蛋白的表达增高(如PKC_δ和PKC_ε)或者降低(如notch1)。在对大鼠嗜铬神经瘤PC12细胞分化和小鼠小脑神经元发育的研究中均发现细胞骨架蛋白4.1蛋白的表达量、剪切形式和细胞定位会随着神经细胞的分化而改变。但4.1蛋白在ESCs向神经细胞分化过程中的改变尚有待研究。 目的: 1.建立一种新的体外定向诱导ESCs分化为神经细胞的方法,从而缩短诱导分化时间、简化体外操作过程。 2.研究在ESCs分化为神经细胞过程中4.1N和4.1B的表达情况。 方法: 采用郑州大学干细胞中心培育的小鼠胚胎干细胞系。组织块原代培养法制备小鼠胚胎成纤维细胞(MEF)。ESCs复苏后直接接种到经丝裂霉素C(MMC)处理的MEF上,使用含白血病抑制因子(LIF)的MEF条件化培养基(CM)进行培养和传代。 待ESCs生长稳定,以10~5个/ml接种于培养皿中。分三组:(1)对照组:用EB培养基重悬后贴壁培养,不加入任何诱导剂,自然分化。(2)4-/4+诱导组:用EB培养基重悬,悬浮培养4d,每2-3 h摇晃1次,防止EB贴壁,第5d收集细胞,用诱导培养基(含10~(-6) mol/L RA)重悬,接种至明胶包被的培养皿,4 d后换为EB培养基继续培养。(3)RA序贯诱导组:用诱导培养基(含10~(-6) mol/L RA的EB培养基)重悬,贴壁培养48 h后,每个培养皿加入3 ml神经干细胞培养基(含20μg/L bFGF、2%B27、1%N2的无血清DMEM/F12培养基)培养2 d,此后每天加入1 ml神经细胞培养基(含2%B27、15%FBS的DMEM/F12培养基),直到第7 d用神经细胞培养基换液,继续培养。 倒置显微镜观察分化过程中细胞形态;免疫细胞化学法检测分化第10 d各组成熟神经元标志性抗原神经元特异性烯醇化酶(NSE)和星形胶质细胞标志性抗原胶质纤维酸性蛋白(GFAP),计算NSE和GFAP的阳性细胞率;Western-blot检测分化前后4.1N和4.1B的表达量。 结果: 倒置显微镜下滋养层细胞MEF表现为100%融合的贴壁生长的梭形细胞。分化前ESCs呈圆形或椭圆形克隆样生长,折光性强,细胞排列紧密。对照组自然分化10 d后可见克隆团边缘几乎均为圆形高亮的上皮样细胞,外周为少量梭形细胞;4-/4+诱导组分化第5 d贴壁12 h后即可见梭形细胞爬出;分化第10 d神经样细胞突起细长并形成网络;RA序贯诱导组分化第5 d出现神经细胞样改变,细胞突起细长;神经元样细胞逐渐增多,分化第7 d呈双极性或多极性,形成网络。 免疫细胞化学法检测分化第10 d对照组、4-/4+诱导组和RA序贯诱导组的NSE阳性细胞率分别是11.8%±1.8%、50.6%±2.7%和57.6%±1.4%;GFAP阳性细胞率分别是19.0%±2.9%、36.0%±2.5%和33.0%±2.0%。单因素方差分析显示对照组和两组诱导组之间有显著性差异(P＜0.01),4-/4+诱导组和RA序贯诱导组之间无显著性差异(P＞0.05)。 Western-blot检测显示:未分化ESCs中表达4.1N和4.1B蛋白,诱导分化后两者表达量都增加。 结论 1.RA序贯诱导法可在ESCs分化后第7d获得神经元样细胞,较4-/4+诱导法提前3d,且两者的NSE和GFAP阳性细胞率无差异,因此RA序贯诱导法可作为经典的4-/4+诱导法的替代方法。 2.4.1N和4.1B蛋白的表达量随着ESCs分化为神经细胞而增加。
[Abstract]:Embryonic stem cells (embryonic stem cells, ESCs) is from the inner cell mass of the early blastocyst (innercell mass ICM) is a kind of pluripotent cell lines isolated in vitro; it can continuously self renew and maintain multilineage differentiation potential and can differentiate into inside, outside the three germ layers of almost all sources cell types. Studies have shown that ESCs in the specific induction culture system, can be induced to differentiate into neural cells and provide a practical basis for ESCs transplantation for the treatment of diseases of the nervous system. So far, the main method in vitro by 4-/4+ method, five step, stromal cells induced by adhesion, monolayer culture and gene modification method. The 4-/4+ method is induced by the most classical method, but this method is the use of retinoic acid (retinoic, acid, RA) before induction to the formation of embryoid bodies (embryonic, bodies, EB), the differentiation is difficult to control and analysis, and the training time is long, complex operation of Cell density, nutrition, equipment and other training requirements higher. To explore new cells induced to reduce the induction time, simplify the operation will promote the differentiation of cultured ESCs in vitro. The mechanism of ESCs differentiation into neural cells is still unclear. Some reports said the expression of some proteins to neural cells during the differentiation process of ESCs. (such as PKC_ PKC_ and delta epsilon) or lower (such as Notch1). The rats addicted to the expression of protein 4.1 were found on PC12 cell differentiation and neuron development of mouse cerebellum chromium neuroma and cell form, shear position will change with the differentiation of neural cells. But 4.1 proteins in ESCs the process of neural cell differentiation changes need to be studied.
Objective:
1. a new method of inducing ESCs to differentiate into nerve cells in vitro is established, thus shortening the induction time and simplifying the operation process in vitro.
2. the expression of 4.1N and 4.1B during the differentiation of ESCs into neural cells was studied.
Method:
The Zhengzhou University Center for stem cell mouse embryonic stem cell lines. Tissue cultured preparation of mouse embryonic fibroblasts (MEF).ESCs after resuscitation to direct inoculation of mitomycin C (MMC) treatment on MEF with leukemia inhibitory factor (LIF) MEF of culture medium (CM) were cultured and passaged.
寰匛SCs鐢熼暱绋冲畾,浠，

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