胚胎干细胞分化神经细胞特性及分化机制的研究

发布时间：2018-06-12 21:41

本文选题：胚胎干细胞 + 体外培养　；参考：《浙江大学》2005年硕士论文

【摘要】：干细胞研究在人类生命科学中具有重要的基础研究意义,在人类健康和生命质量领域具有广泛的应用前景。胚胎干细胞来源于受精卵发育早期胚胎的内细胞团,具有发育的全能性,可以分化为体内所有类型的成熟细胞。在发育生物学、细胞治疗、组织器官工程等研究和开发中具有巨大的潜力。为了全面认识ES细胞的特性并为今后进行转基因动物、组织工程研究做好铺垫,我们进行了小鼠ES细胞的建系研究,以ICR种和昆明种小鼠为实验对象,采用超数排卵的方法获得孕3.5d小鼠囊胚,培养在用丝裂霉素C处理过的MEF细胞饲养层上,选取适当时间离散增殖的ICM和类ES细胞集落,分析了在小鼠ES细胞建系过程中,饲养层细胞、消化液及不同品系来源的胚胎的影响。结果,ICR小鼠和昆明小鼠两种来源的饲养层细胞对胚胎贴壁、ICM增殖无影响;消化液浓度和作用时间对ICM的影响明显,以较低浓度的胰酶和EDTA消化为宜;另外,血清质量、培养板质量等因素可能影响建系。为了探讨小鼠胚胎干细胞分化神经细胞的功能特性,及其分化过程中N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体的表达情况,我们利用全反式维甲酸(RA)诱导胚胎干细胞拟胚体定向分化为神经细胞,用摄像相差显微镜观察分化神经细胞的形态,用免疫细胞化学方法鉴定分化神经细胞表达特异性抗原,用逆转录—聚合酶链反应(RT-PCR)检测分化神经细胞Calbindin D28K和NMDA受体(NR)亚型mRNA的表达情况,用荧光共聚焦显微镜检测分化神经细胞在NMDA刺激下胞内游离Ca~(2+)浓度的变化。结果显示,1×10~(-6)mol/LRA的培养基诱导培养ES细胞拟胚体4天,在拟胚体周围出现大量神经样细胞,分化神经细胞的比率与拟胚体的大小有关;撤除RA继续培养4d后可以检测到NR2B mRNA的转录,NR2C有弱的转录;在第6d后NR2A有转录;在第8d及其之后NR1的转录出现。撤除RA继续培养8天后在拟胚体周围出现大量神经样细胞;RT-PCR检测显示有Calbindin D28和NR1、NR2A、NR2B、NR2C mRNA的转录;在NMDA刺激下胞内游离Ca~(2+)浓度明显升高(P0.01)。提示,用RA诱导ES细胞分化的神经细胞NMDA受体亚型的表达与中枢神经发育过程中神经细胞NMDA受体亚型的表达具有一定的相似性。用RA诱导ES细胞分化的神
[Abstract]:Stem cell research has important basic significance in human life science and has a wide application prospect in the field of human health and quality of life. Embryonic stem cells (ESCs) are derived from the inner cell clusters of the early embryos of fertilized eggs and have the developmental totipotency to differentiate into all types of mature cells in the body. It has great potential in developmental biology, cell therapy, tissue and organ engineering and so on. In order to fully understand the characteristics of es cells and pave the way for future transgenic animals and tissue engineering research, we studied the establishment of mouse es cells. ICR and Kunming mice were used as experimental objects. Mouse blastocysts were obtained by superovulation and cultured on MEF cell feeder layer treated with mitomycin C. ICM and es like cell colonies were selected in appropriate time to analyze the process of establishing mouse es cell line. Effects of feeder cells, digestive fluids and embryos from different strains. Results the feeder layer cells from ICR mice and Kunming mice had no effect on the proliferation of ICM, the concentration of digestive fluid and the time of action had obvious effects on ICM digestion, and the lower concentrations of trypsin and EDTA were suitable for digestion. The quality of culture plate may affect the establishment of the system. To investigate the functional characteristics of mouse embryonic stem cell differentiation neurons and the expression of N-methyl-D-aspartate NMDA-receptor during differentiation. We used all-trans retinoic acid (RAA) to induce the directional differentiation of embryonic stem cells into neuronal cells. The morphology of differentiated neurons was observed by camera phase contrast microscope, and the expression of specific antigen was identified by immunocytochemistry. The expression of Calbindin D28K and NMDA receptor NR- subtype mRNA in differentiated neurons was detected by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that a large number of neuron-like cells appeared around the embryoid body for 4 days after induction on 1 脳 10 ~ (-6) mol / L LRA medium, and the ratio of differentiated nerve cells was related to the size of the embryoid body. NR2C, NR2A and NR1 transcripts of NR2B mRNA and NR2a were detected after 4 days and 8 days, respectively, after the removal of RA for 4 d, NR2C was weakly transcribed, NR2A was transcripted at the 6th day, and NR1 was transcripted at and after the 8th day. After the removal of RA for 8 days, a large number of neuron-like cells appeared around the embryoid body. RT-PCR results showed that there were transcription of Calbindin D28 and NR1NR2ANR2BU NR2C mRNA, and that the concentration of intracellular free Caan2) increased significantly under the stimulation of NMDA. These results suggest that the expression of NMDA receptor subtypes in neural cells induced by RA is similar to that of NMDA receptor subtypes in neural cells during the development of central nervous system. Differentiation of es cells induced by RA
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329

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