少突胶质谱系细胞发育过程中S-100β的表达及易损性的研究
发布时间：2018-10-20 12:37
【摘要】： O-2A祖细胞是双潜能干细胞,既能分化为少突胶质细胞(oligodendrocyte,OL),又能分化为2型星形胶质细胞。从O-2A祖细胞发育到成熟的OL这个过程中往往伴随着一些抗原的消失和出现。本文研究在OL发育过程中S-100β表达的变化。S-100β属于S100家族的一个钙离子结合蛋白,它广泛的分布在脊椎动物的神经系统,沉积在哺乳动物的脑中S-100β,参与细胞类型的分化。本研究在分离、纯化和体外培养O-2A祖细胞的基础上,检测S-100β在O-2A祖细胞分化至成熟OL的过程中S-100β表达的变化,探讨S-100β是否在OL发生与分化过程中具有调控作用。脑室周围白质的软化(Periventricular leukomalacia,PVL)主要是发生在早产儿的一种白质的损伤,发育中的少突胶质谱系细胞的损伤是PVL的一个标志性特征。据临床和动物实验报道促炎症因子TNF-α在PVL中发挥作用,因此研究其对发育中少突胶质谱系细胞的作用非常重要。 第一部分少突胶质谱系细胞发育过程中S-100β的表达 目的探讨新生大鼠大脑皮质来源的少突胶质谱系细胞中S-100β是否表达,如若表达始于少突胶质谱系细胞发育过程中的何种阶段。 方法取新生大鼠大脑皮质进行混合胶质细胞的原代培养,根据各种胶质细胞黏附性及生长时间的差异,通过机械振荡法和差速贴壁法获得纯化的O-2A祖细胞,并结合使用生长因子获得一系列的少突胶质谱系细胞,且通过细胞特异性抗体进行鉴定。采用免疫荧光双重标记方法,检测S-100β在少突胶质谱系细胞中的表达情况。 结果纯化的O-2A祖细胞在含有bFGF和PDGF的培养液中培养48h,不表达S-100β。换成含3′碘甲状腺原氨酸(triiodothyronine,T3)培养液继续培养24 h,开始表达S-100β。原少突胶质细胞和成熟的OL中S-100β免疫反应呈现阳性。 结论S-100β表达始于O-2A祖细胞分化为原少突胶质细胞的阶段,可能与O-2A祖细胞从双潜能阶段分化成OL过程中的形态学变化有关。 第二部分少突胶质谱系细胞易损性的研究 目的研究TNF-α对少突胶质谱系细胞的三个不同发育阶段的易损性。 方法(1)5 ng/ml、50 ng/ml、500 ng/ml TNF-α分别刺激O-2A祖细、原少突胶质细胞、未成熟的OL48 h,同时设立对照组(未加TNF-α处理)。利用MTT法以酶联免疫检测仪测定各孔光吸收值(OD),计算细胞的存活率。存活率((?)±s)%=实验组细胞OD/对照组细胞OD×100%。(2)50 ng/ml TNF-α刺激O-2A祖细、原少突胶质细胞、未成熟的OL12 h、24 h、48 h,以未加TNF-α处理的为对照组,利用MTT法计算细胞的存活率。存活率((?)±s)%=实验组细胞OD/对照组细胞OD×100%。(3)50 ng/ml TNF-α刺激O-2A祖细、原少突胶质细胞、未成熟的OL48 h,caspase-3的免疫细胞化学染色检测细胞的凋亡。 结果(1)TNF-α降低细胞的存活率具有剂量依赖性和时间依赖性,随着TNF-α剂量的增加和刺激时间的延长O-2A祖细胞、原少突胶质细胞、未成熟OL的存活率降低。(2)随着TNF-α剂量的增加,O-2A祖细胞与未成熟OL相比较细胞存活率明显降低(P＜0.01),原少突胶质细胞与未成熟OL相比较细胞存活率也是降低的(P＜0.05),O-2A祖细胞存活率与原少突胶质细胞存活率相比较差别没有统计学意义;50 ng/ml TNF-α刺激少突胶质谱系细胞48 h以内,0.2A祖细胞的存活率与未成熟OL存活率相比较差别没有统计学意义,O-2A祖细胞的存活率与原少突胶质细胞的存活率相比较也不存在差别,而原少突胶质细胞的存活率与未成熟OL存活率相比较细胞的存活率明显降低(P＜0.05)。(3)50 ng/ml TNF-α分别刺激O-2A祖细胞、原少突胶质细胞、未成熟OL48 h,caspase-3免疫细胞化学染色显示在这些细胞的胞浆和胞核有大量的特异性棕黄色的着色而对照组这三个发育阶段的细胞未见阳性表达。 结论TNF-α降低少突胶质谱系细胞的存活率具有细胞成熟程度的依赖性;caspase-3参与TNF-α诱导的O-2A祖细胞、原少突胶质细胞及未成熟OL的凋亡。
[Abstract]:The O-2A progenitor cells are potent stem cells that can differentiate into oligodendrocyte (OL) and differentiate into 2 astrocytes. It is often accompanied by the disappearance and appearance of some antigens from the development of O-2A progenitor cells to mature OL. In this paper, the changes of S-100 gene expression during the development of OL were studied. S-100 is a calcium ion binding protein belonging to the S100 family, which is widely distributed in the nervous system of vertebrates, deposited in the brain of a mammal, S-100 kcal, participating in the differentiation of cell types. On the basis of isolation, purification and in vitro culture of O-2A progenitors, this study examined the changes of S-100 gene expression in the process of differentiation of O-2A progenitors into mature OL, and discussed the regulation of S-100 CCR5 in the development and differentiation of OL-2A progenitor cells. Periventricular leukomalacia (PVL) is mainly a white matter damage occurring in premature infants, and the damage of oligodendrocyte in development is a characteristic feature of PVL. According to clinical and animal experiments, we reported the role of pro-inflammatory factor TNF-7721 in PVL, and therefore it was very important to study its role in the development of oligodendrocyte. S-100 in the development of oligodendrocytes in the first part Objective To investigate the expression of S-100 gene in oligodendrocyte of newborn rat cerebral cortex, if the expression begins with oligodendrocyte development. Methods: The primary culture of mixed glial cells in the cerebral cortex of neonatal rats was carried out by means of mechanical oscillation method and differential attachment method. The purified O-2A progenitor cells are obtained and a series of oligodendrocyte lineage cells are obtained using growth factors, and by Cell-specific antibodies were identified. Immunofluorescence double labeling was used to detect S-100 in oligodendrocytes. Expression of purified O-2A progenitor cells in a culture medium containing bFGF and PDGF It was cultured for 48h without expression of S-100 kcal. It was replaced with triiodothyronine (T3). 24 h, starting to express S-100 kcal. oligodendrocytes and mature OL Conclusion The expression of S-100 may begin with the differentiation of O-2A progenitor cells into oligodendrocytes, which may be related to the potential of O-2A progenitors from double potential. Morphological Changes in the Process of Energy Differentiation into OL A study on the study of the cytoskeleton of oligodendrocytes in the second part Methods (1) 5ng/ ml, 50ng/ ml, 500ng/ ml TNF-gal stimulated O-2A progenitor cells and oligodendrocytes, respectively. Cells, immature OL48 h, and control group (not treated with TNF-MAA). Use MT T-method is free from enzyme-linked The light absorption value (OD) of each hole was measured by the disease detector and the survival rate of cells was calculated. The survival rate ((?)% s)% = OD of the OD/ control group of the experimental group was 100%. (2) 50 ng/ ml of TNF-MAA stimulated the O-2A progenitor thin, oligodendrocyte, immature OL12h, 24h, 48h in the absence of TN The survival rate of cells was calculated by MTT method. The survival rate ((?)% s)% = OD of the OD/ control group of the experimental group was 100%. (3) 50 ng/ ml of TNF-MAA stimulated the O-2A progenitor cell thin, oligodendrocyte and immature. The results showed that (1) TNF-7721 decreased the survival rate of cells with dose-dependent and time-dependent, and increased with the increase of TNF-tau dose. At the same time, the survival rate of O-2A progenitor cells and immature L-cells decreased with the increase of the dose of TNF-7721, and the survival rate of O-2A progenitor cells compared with the immature OL decreased significantly (P <0.01). Compared with the immature OL, the survival rate was also decreased (P <0.05). The survival rate of O-2A progenitor cells was significantly different from that of oligodendrocyte. The survival rate of O-2A progenitor cells was not statistically significant compared with the survival rate of oligodendrocytes, but the survival rate of O-2A progenitors did not differ from the survival rate of oligodendrocytes. The survival rate of the cells was significantly lower than that of the immature OL (P <0.05). (3) 50 ng/ ml of TNF-7721 stimulated O-2A progenitors, oligodendrocytes, immature OL48 h, caspase-3 immune cells chemically stained the cytoplasm of these cells. There was a large number of specific brown-yellow coloration in the nucleus, and no positive expression was found in the three developmental stages of the control group. Conclusion TNF-7721 reduced the survival rate of oligodendrocytes with the dependence on the degree of cell maturation; casp
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
本文编号：2283201
[Abstract]:The O-2A progenitor cells are potent stem cells that can differentiate into oligodendrocyte (OL) and differentiate into 2 astrocytes. It is often accompanied by the disappearance and appearance of some antigens from the development of O-2A progenitor cells to mature OL. In this paper, the changes of S-100 gene expression during the development of OL were studied. S-100 is a calcium ion binding protein belonging to the S100 family, which is widely distributed in the nervous system of vertebrates, deposited in the brain of a mammal, S-100 kcal, participating in the differentiation of cell types. On the basis of isolation, purification and in vitro culture of O-2A progenitors, this study examined the changes of S-100 gene expression in the process of differentiation of O-2A progenitors into mature OL, and discussed the regulation of S-100 CCR5 in the development and differentiation of OL-2A progenitor cells. Periventricular leukomalacia (PVL) is mainly a white matter damage occurring in premature infants, and the damage of oligodendrocyte in development is a characteristic feature of PVL. According to clinical and animal experiments, we reported the role of pro-inflammatory factor TNF-7721 in PVL, and therefore it was very important to study its role in the development of oligodendrocyte. S-100 in the development of oligodendrocytes in the first part Objective To investigate the expression of S-100 gene in oligodendrocyte of newborn rat cerebral cortex, if the expression begins with oligodendrocyte development. Methods: The primary culture of mixed glial cells in the cerebral cortex of neonatal rats was carried out by means of mechanical oscillation method and differential attachment method. The purified O-2A progenitor cells are obtained and a series of oligodendrocyte lineage cells are obtained using growth factors, and by Cell-specific antibodies were identified. Immunofluorescence double labeling was used to detect S-100 in oligodendrocytes. Expression of purified O-2A progenitor cells in a culture medium containing bFGF and PDGF It was cultured for 48h without expression of S-100 kcal. It was replaced with triiodothyronine (T3). 24 h, starting to express S-100 kcal. oligodendrocytes and mature OL Conclusion The expression of S-100 may begin with the differentiation of O-2A progenitor cells into oligodendrocytes, which may be related to the potential of O-2A progenitors from double potential. Morphological Changes in the Process of Energy Differentiation into OL A study on the study of the cytoskeleton of oligodendrocytes in the second part Methods (1) 5ng/ ml, 50ng/ ml, 500ng/ ml TNF-gal stimulated O-2A progenitor cells and oligodendrocytes, respectively. Cells, immature OL48 h, and control group (not treated with TNF-MAA). Use MT T-method is free from enzyme-linked The light absorption value (OD) of each hole was measured by the disease detector and the survival rate of cells was calculated. The survival rate ((?)% s)% = OD of the OD/ control group of the experimental group was 100%. (2) 50 ng/ ml of TNF-MAA stimulated the O-2A progenitor thin, oligodendrocyte, immature OL12h, 24h, 48h in the absence of TN The survival rate of cells was calculated by MTT method. The survival rate ((?)% s)% = OD of the OD/ control group of the experimental group was 100%. (3) 50 ng/ ml of TNF-MAA stimulated the O-2A progenitor cell thin, oligodendrocyte and immature. The results showed that (1) TNF-7721 decreased the survival rate of cells with dose-dependent and time-dependent, and increased with the increase of TNF-tau dose. At the same time, the survival rate of O-2A progenitor cells and immature L-cells decreased with the increase of the dose of TNF-7721, and the survival rate of O-2A progenitor cells compared with the immature OL decreased significantly (P <0.01). Compared with the immature OL, the survival rate was also decreased (P <0.05). The survival rate of O-2A progenitor cells was significantly different from that of oligodendrocyte. The survival rate of O-2A progenitor cells was not statistically significant compared with the survival rate of oligodendrocytes, but the survival rate of O-2A progenitors did not differ from the survival rate of oligodendrocytes. The survival rate of the cells was significantly lower than that of the immature OL (P <0.05). (3) 50 ng/ ml of TNF-7721 stimulated O-2A progenitors, oligodendrocytes, immature OL48 h, caspase-3 immune cells chemically stained the cytoplasm of these cells. There was a large number of specific brown-yellow coloration in the nucleus, and no positive expression was found in the three developmental stages of the control group. Conclusion TNF-7721 reduced the survival rate of oligodendrocytes with the dependence on the degree of cell maturation; casp
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
【参考文献】
相关期刊论文 前1条
1 李春鹏;张晔;夏春林;沈慧;张静;陆志方;;巢蛋白和阶段特异性胚胎抗原-1在大鼠2型星形胶质细胞中的表达[J];解剖学报;2007年02期
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