IL-1β、AA诱导鼠胚中脑与皮质神经干细胞向TH阳性神经元分化的实验研究

发布时间：2018-05-18 23:11

本文选题：中脑/皮质 + 神经干细胞　；参考：《武汉大学》2005年硕士论文

【摘要】：目的在己成功分离培养皮质神经干细胞的基础上探索小鼠胚胎中脑区神经干细胞体外培养的方法,并观察比较二者在生长和分化性状方面的差异,以及它们在IL-1β、AA作用下向TH阳性神经元的诱导分化,为神经干细胞的基础理论和帕金森病细胞移植治疗的应用研究提供一定的实验资料。方法 1.无菌条件下分离E12-E13天小鼠胚脑腹侧中脑曲,胰酶消化和机械吹打制成单细胞悬液,在bFGF和B27存在的无血清培养基中培养扩增,机械分离法传代,免疫细胞化学染色方法鉴定神经干细胞及其子代细胞的分化方向。 2.无菌条件下同时分离培养胚鼠大脑皮质与腹侧中脑神经干细胞,倒置显微镜观察比较二者生长状况;含10%血清的DMEM/F12培养基接种分化,观察比较它们分化子代细胞中TH阳性神经元的情况。 3.体外培养中脑神经干细胞和皮质神经干细胞,分别传代各分为四组,在有血清条件下分别予以AA、IL-1β及AA+IL-1β诱导分化,并设空白对照,DAPI细胞核标记,抗酪氨酸羟化酶(TH)免疫细胞化学鉴定分化结果并细胞计数阳性细胞比例。结果 1.从E12-E13鼠胚脑腹侧中脑曲分离的小部分细胞在B27和bFGF存在的无血清培养基中可以在体外培养和传代,这些细胞表达神经干细胞特异性抗原nestin,在撤除B27和bFGF的有血清培养基中能够分化为神经元和神经胶质细胞。 2.小鼠胎脑大脑皮质部位所含神经干细胞明显多于中脑,体外相同条件下皮质神经干细胞也更宜成球;有血清条件下分化,中脑神经干细胞可少量分化出TH阳性神经元;皮质神经干细胞则未见分化出TH阳性神经元。 3.在诱导分化实验中,AA、IL-1β及AA+IL-1β作用下中脑神经干细胞分化出TH阳性神经元比例分别约为10%、9%和11%,明显高于对照组(约1%,P0.01);三实验组间相对比较无统计学差异(P0.05);加AA诱导组中分化出的TH阳性神经元细胞形态更成熟。皮质神经干细胞各实验组均未见分化出TH阳性神经元。
[Abstract]:Objective to explore the method of in vitro culture of neural stem cells from mouse embryonic mesencephalic region on the basis of successful isolation and culture of cortical neural stem cells, and to observe and compare the differences of growth and differentiation between them. And their induction and differentiation into th positive neurons under the action of IL-1 尾 -AA provide some experimental data for the basic theory of neural stem cells and the application of transplantation therapy for Parkinson's disease. Method 1. Single cell suspension was isolated from the ventral midbrain of E12-E13 day mouse embryo under aseptic conditions, digested by trypsin and mechanically blown. The suspension was cultured and amplified in serum-free medium of bFGF and B27, and was subcultured by mechanical separation. Immunocytochemical staining was used to identify the differentiation of neural stem cells and their progenies. 2. Embryonic rat cerebral cortex and ventral mesencephalic neural stem cells were isolated and cultured simultaneously under aseptic condition. The growth status of the two cells was observed and compared by inverted microscope. DMEM/F12 medium containing 10% serum was inoculated and differentiated. The th positive neurons in the differentiated progenies were observed and compared. 3. Neural stem cells (NSCs) and cortical neural stem cells (NSCs) were cultured in vitro and were subcultured into four groups respectively. Under the condition of serum, they were induced to differentiate by AA-IL-1 尾 and AA IL-1 尾, respectively. Antityrosine hydroxylase (THH) immunocytochemistry was used to identify the differentiation and the percentage of positive cells counted. Result 1. A small number of cells isolated from the ventral midbrain of E12-E13 mouse embryos could be cultured and subcultured in serum-free medium containing B27 and bFGF in vitro. These cells express the specific antigen of neural stem cells, Neisin, and can differentiate into neurons and glial cells in serum-containing medium that removes B27 and bFGF. 2. The neural stem cells contained more neural stem cells in the cortex of the fetal brain than in the mesencephalon, and in the same conditions in vitro, the neural stem cells in the cortex were more suitable for forming spheres, and in the presence of serum, the neural stem cells in the mesencephalon could differentiate into a small amount of th positive neurons. No th positive neurons were found in cortical neural stem cells. 3. The percentage of th positive neurons induced by IL-1 尾 and AA IL-1 尾 were 10 9% and 11%, respectively, which were significantly higher than those in the control group (P 0.01); there was no significant difference among the three experimental groups (P 0.05); The differentiated th-positive neurons were more mature in morphology. No th-positive neurons were found in all experimental groups of cortical neural stem cells.
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329

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