诱导小鼠胚胎干细胞分化为神经元样细胞的研究

发布时间：2018-08-25 07:51

【摘要】： 前言胚胎干细胞(Embryonic stem cells,ESCs)来自囊胚内细胞团(Inner cell mass,ICM),在体外可长期自我复制,并且具有分化成内、中、外三个胚层细胞的多潜能性,研究表明其可在体外分化为神经细胞。而神经细胞被认为是终末分化细胞,其损伤后很难再由自体的神经干细胞补充,研究ESCs向神经细胞的分化不仅可以作为研究哺乳动物神经系统发育的体外模型,了解神经分化过程中基因表达调控的变化机制;还可以应用于临床,为神经系统疾病,如帕金森症、脑卒中、癫痫,及脊髓损伤等患者进行细胞移植,使其尽快康复。但是,目前人们对胚胎干细胞向神经细胞分化的确切机制仍不够了解。本课题组前期已建立了将小鼠胚胎干细胞定向诱导分化为神经元样细胞的“序贯诱导法”,本研究在优化“序贯诱导法”诱导条件的基础上,通过应用免疫荧光法和蛋白质免疫印记法对分化后细胞进行神经细胞标志物鉴定,并用流式细胞仪测定分化比率,为进一步研究胚胎干细胞向神经细胞分化的作用机理打下基础,以期揭示出胚胎干细胞分化的确切机制。方法 1、饲养层细胞制备、ESCs培养及诱导分化使用原代小鼠胚胎成纤维细胞(PMEF)第2-4代状态好的作为饲养层细胞,mESCs(小鼠ES细胞系AB2.1,由美国南加州大学医学院惠赠)接种培养于其上。诱导分化时去饲养层,重新接种于无包被的培养皿中,使用NSC培养液,逐渐更换为无血清NC培养液。 2、“序贯诱导法”分化条件的优化 (1)诱导分化时ESCs接种的密度梯度 (2)首次换用NSC培养液时培养液的浓度梯度 (3)逐渐更换为无血清NC培养液的时间梯度 3、鉴定诱导分化后细胞的特异性标志物 (1)免疫荧光法检测神经干细胞及成熟神经细胞特异性抗原Nestin、NeuN、TUJ1、NCAM1和GFAP (2)蛋白质免疫印记法(Western Blotting)检测成熟神经元特异性标志物TUJ1 4、流式细胞仪测定NeuN阳性细胞百分比,计算诱导分化比率。实验结果 1、优化了“序贯诱导法”的诱导条件优化后“序贯诱导法”在分化时首次接种所用培养液的血清浓度为12.5%,mESCs最佳接种密度为1.0×10~5/ML,完全使用无血清NC培养液的时间应为诱导第5天。 2、使用优化的“序贯诱导法”,可简便、高效、稳定的将ESCs诱导为神经元样细胞未分化的ESCs聚集成团状生长,细胞排列紧密,集落边界清楚。使用优化的“序贯诱导法”可将ESCs大比例诱导分化为神经元样细胞,光镜下观察细胞形态均一,胞体圆亮,突起细长。 3、鉴定分化后细胞的神经细胞特异性抗原免疫荧光法检测神经干细胞及成熟神经元特异性抗原Nestin、NSE、NeuN、TUJ1和NCAM1表达阳性,神经胶质细胞标志物GFAP表达阴性;蛋白质免疫印记法检测到成熟神经细胞特异性标志物TUJ1表达阳性。 4、流式细胞仪检测NeuN阳性细胞百分比,计算出诱导分化比率为89.91%±2.03%。结论根据本研究所得的实验结果,可得出以下结论: 1、优化的“序贯诱导法”可稳定诱导ESCs高比率分化神经元样细胞,并经过鉴定具有了成熟神经元特异性标志物,而可能正是这些标志物基因的程序性表达,与定向分化的机制相关。 2、应用流式细胞仪将分化结果作了定量分析,明确得出诱导分化比率可达89.91%±2.03%。
[Abstract]:Preface
Embryonic stem cells (ESCs) are derived from the intrablastocyst mass (ICM) and can reproduce themselves for a long time in vitro, and have the multipotential to differentiate into three embryonic layers: inner, middle and outer. Studies have shown that ESCs can differentiate into nerve cells in vitro. Neural cells are considered terminal differentiated cells and are difficult to differentiate after injury. The study of ESCs differentiation into neural cells supplemented by autologous neural stem cells can not only be used as an in vitro model for studying the development of mammalian nervous system to understand the mechanism of gene expression regulation during neural differentiation, but also be used in clinic for nervous system diseases such as Parkinson's disease, stroke, epilepsy, and spinal cord injury. However, the exact mechanism by which embryonic stem cells differentiate into neural cells is still poorly understood.
Our research group has established a sequential induction method to induce mouse embryonic stem cells to differentiate into neuron-like cells. On the basis of optimizing the induction conditions of the sequential induction method, the differentiated cells were identified by immunofluorescence and protein immunoblotting, and then the neuronal markers were identified by flow cytometry. The differentiation ratio was measured by a cell analyzer, which laid a foundation for further study on the mechanism of embryonic stem cell differentiation into neural cells, in order to reveal the exact mechanism of embryonic stem cell differentiation.
Method
1, feeder layer cell preparation, ESCs culture and induced differentiation.
The second to fourth passages of primary mouse embryonic fibroblasts (PMEF) were used as feeder layer cells, and mESCs (mouse ES cell line AB2.1, a gift from USC Medical College) were inoculated and cultured on them. Liquid.
2, the optimization of differentiation conditions by sequential induction method.
(1) density gradient of ESCs inoculation when inducing differentiation.
(2) the concentration gradient of culture medium for the first time when NSC culture medium was replaced.
(3) the time gradient of gradually replacing serum free NC medium.
3, identify specific markers of differentiated cells.
(1) Nestin, NeuN, TUJ1, NCAM 1 and GFAP were detected by immunofluorescence assay.
(2) protein immunoblotting (Western Blotting) was used to detect mature neuron specific marker TUJ1.
4, the percentage of NeuN positive cells was measured by flow cytometry, and the ratio of induced differentiation was calculated.
experimental result
1, optimize the induction condition of sequential induction method.
The optimum concentration of serum and the optimum density of mESCs were 12.5% and 1.0 65507
2, the optimized sequential induction method can be used to induce ESCs into neuron like cells in a simple, efficient and stable manner.
The undifferentiated ESCs grew in clumps with compact cell arrangement and clear colony boundary. ESCs could be induced to differentiate into neuron-like cells in large proportion by the optimized sequential induction method. The morphology of the cells was uniform, the soma was round and the processes were slender under light microscope.
3, identify the neural cell specific antigen of differentiated cells.
Nestin, NSE, NeuN, TUJ1 and NCAM1 were positive by immunofluorescence assay, but GFAP was negative by immunoblotting, and TUJ1 was positive by immunoblotting.
4, the percentage of NeuN positive cells was detected by flow cytometry, and the induced differentiation rate was 89.91% + 2.03%..
conclusion
Based on the experimental results obtained in this study, the following conclusions can be drawn:
1. The optimized sequential induction method can stably induce ESCs to differentiate into neuron-like cells at high rates, and has identified specific markers of mature neurons, which may be the procedural expression of these markers genes and may be related to the mechanism of directional differentiation.
2. The results of differentiation were analyzed quantitatively by flow cytometry. The induction rate was 89.91%+2.03%.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【相似文献】