谷氨酸经Shh信号转导通路介导成体神经干细胞增殖作用的研究

发布时间：2018-05-02 22:50

本文选题：神经干细胞 + 谷氨酸　；参考：《新乡医学院》2014年硕士论文

【摘要】：背景脑梗死的治疗一直是医学界的难题,传统的治疗无法修复受损的脑组织。神经干细胞(neural stem cells)的发现为解决该难题带来了新的切入点,但神经干细胞在存活增殖等诸多方面的复杂机制使得神经干细胞的应用研究仍无突破性进展。脑梗死后神经干细胞在原位被激活增殖,表现为梗死区谷氨酸(glutamate)浓度升高,神经干细胞内Shh(sonic hedgehog)蛋白表达量也显著增加；联系到Shh信号通路在胚胎干细胞增殖中的重要作用,我们推测Glu可能经由Shh信号通路介导了神经干细胞的增殖。因此本实验拟通过体外实验探索Shh表达与Glu刺激神经干细胞增殖的关系。本实验分三部分：第一部分成年SD大鼠神经干细胞分离培养和鉴定的改良目的改良成年SD大鼠神经干细胞分离、培养和鉴定方法,观察神经干细胞的增殖和分化特点,为后续实验提供细胞。方法从成年SD大鼠分离出完整海马,采用机械吹打法获得原代细胞,用accutase消化传代,利用CCK-8法检测神经干细胞的增殖情况,应用多重标记免疫荧光细胞化学方法鉴定神经干细胞及其诱导分化后细胞。结果机械吹打法可获得原代神经干细胞,accutase消化传代有利于神经干细胞的传代培养,CCK-8法能够测定神经干细胞的增殖,多重免疫荧光展现了成年神经干细胞经胎牛血清诱导后的诱导分化过程。结论改良后的培养方法可以更加简单高效的获得和培养出大量细胞,经多重免疫荧光鉴定所分离和培养的细胞是神经干细胞。第二部分谷氨酸刺激神经干细胞增殖的最适浓度探索目的用不同浓度的谷氨酸刺激成体神经干细胞增殖,寻求最适合的谷氨酸浓度,为后续试验做准备。方法取传代3代以后的神经干细胞,在含有不同浓度谷氨酸的去生长因子培养基中培养24h、48h、72h,采用CCK-8法检测不同谷氨酸浓度下神经干细胞的增殖情况。结果0μmol/L～1OOμmol/L浓度范围的谷氨酸对成体神经干细胞有增殖作用,20μmol/L谷氨酸浓度最适合成体神经干细胞的增殖,当谷氨酸浓度大于100μmol/L时将抑制成体神经干细胞的增殖,并对成体神经干细胞具有一定的细胞毒性。结论在体外培养条件下,谷氨酸在一定浓度范围内的(Oμmol/L～100μmol/L)可以刺激成体神经干细胞的增殖,刺激成体神经干细胞增殖的最适浓度为20μmol/L；当谷氨酸浓度大于100μmol/L时将抑制成体神经干细胞的增殖,并对成体神经干细胞具有一定的细胞毒性。第三部分谷氨酸经Shh信号途径对神经干细胞的增殖作用目的用2Oμmol/L谷氨酸刺激成体神经干细胞的增殖,探索Shh表达与谷氨酸刺激成体神经干细胞增殖的关系。方法取传代3代以后的神经干细胞,在终浓度为20μmol/L谷氨酸的除生长因子的培养基中分别培养24h、48h、72h,设为Glu刺激24h、48h、72h3组,在未加谷氨酸的除生长因子培养基中培养72h,设为空白对照组；按上述分组方法另设一组在相应时间分别加入胎牛血清诱导分化培养1周,观察神经干细胞及诱导分化后细胞增殖情况,采用westemblot法检测nestin蛋白、Shh蛋白、GFAP蛋白及βⅢ-tubulin蛋白的表达情况,采用实时荧光定量法检测nestin mRNA、Shh mRNA、 GFAP mRNA及βⅢ-tubulin mRNA表达情况。结果在谷氨酸刺激成体神经干细胞的增殖过程中,nestin表达量变化与Shh表达量变化相一致。诱导分化后1周神经干细胞大部分分化为神经元和神经胶质细胞。结论Shh信号通路可能参与了谷氨酸刺激成体神经干细胞增殖的过程；谷氨酸刺激神经干细胞增殖不影响成体神经干细胞的分化潜能。
[Abstract]:The treatment of background cerebral infarction has been a difficult problem in the medical field. The traditional treatment can not repair the damaged brain tissue. The discovery of neural stem cells has brought new entry points to solve this problem, but the complex mechanism of neural stem cells in many aspects, such as survival and proliferation, makes the application of neural stem cells still unbreakable. Progress. After cerebral infarction, neural stem cells are activated in situ, showing an increase in the concentration of glutamate (glutamate) in the infarct area and a significant increase in the expression of Shh (Sonic hedgehog) protein in the neural stem cells, which is associated with the important role of the Shh signaling pathway in the proliferation of embryonic stem cells. We speculate that Glu may be mediated by the Shh signaling pathway. The proliferation of neural stem cells, therefore, we intend to explore the relationship between Shh expression and the proliferation of neural stem cells stimulated by Glu in vitro. This experiment is divided into three parts.
Part one: isolation, culture and identification of neural stem cells from adult SD rats
Objective to improve the isolation, culture and identification of neural stem cells from adult SD rats, and to observe the proliferation and differentiation characteristics of neural stem cells, so as to provide cells for subsequent experiments.
Methods the intact hippocampus was isolated from adult SD rats. The primary cells were obtained by mechanical blowing. Accutase was used to digest the passages. The proliferation of neural stem cells was detected by CCK-8. The multiple labeled immunofluorescent cytochemical method was used to identify the neural stem cells and their induced cells after differentiation.
Results the primary neural stem cells could be obtained by mechanical blowing. Accutase digestion was beneficial to the generation of neural stem cells, and the CCK-8 method could determine the proliferation of neural stem cells. Multiple immunofluorescence showed the induced differentiation of adult neural stem cells after fetal bovine serum induction.
Conclusion the improved culture method can be more simple and efficient to obtain and cultivate a large number of cells. The cells isolated and cultured through multiple immunofluorescence identification are neural stem cells.
The second part explores the optimal concentration of glutamate stimulating the proliferation of neural stem cells.
Objective to stimulate the proliferation of adult neural stem cells with different concentrations of glutamic acid, and seek the most suitable glutamic acid concentration, so as to prepare for subsequent experiments.
Methods the neural stem cells from 3 generations after passage were cultured, and 24h, 48h, 72h were cultured in the growth factor medium containing different concentrations of glutamic acid. The proliferation of neural stem cells under different glutamic acid concentrations was detected by CCK-8 method.
Results the glutamic acid of 0 mol/L to 1OO mu mol/L has a proliferation effect on adult neural stem cells. The concentration of 20 mu mol/L is the most suitable for the proliferation of adult neural stem cells. When the concentration of glutamic acid is greater than 100 u mol/L, it will inhibit the proliferation of adult neural stem cells and have a certain cytotoxicity to adult stem cells.
Conclusion in vitro, glutamic acid can stimulate the proliferation of adult neural stem cells in a certain concentration range (O mu mol/L ~ 100 mu mol/L), and the optimum concentration of the proliferation of adult neural stem cells is 20 mu mol/L. When the concentration of glutamic acid is greater than 100 u mol/L, the proliferation of adult neural stem cells will be inhibited and the adult neural stem is fine. The cell has a certain cytotoxicity.
The third part is the effect of glutamate on the proliferation of neural stem cells through Shh signaling pathway.
Objective to stimulate the proliferation of adult neural stem cells stimulated by 2O (mol/L) glutamate, and explore the relationship between Shh expression and the proliferation of neural stem cells stimulated by glutamate.
Methods after 3 generations of neural stem cells were passed, 24h, 48h, 72h were cultured in the medium with a final concentration of 20 mu mol/L glutamic acid, and Glu stimulated 24h, 48h, 72h3 group. The expression of nestin protein, Shh protein, GFAP protein and beta III -tubulin protein were detected by westemblot method, and the expression of nestin mRNA, Shh mRNA, GFAP mRNA and beta III -tubulin were detected by westemblot method.
Results during the proliferation of adult neural stem cells stimulated by glutamic acid, the expression of nestin was in accordance with the changes in the expression of Shh. Most of the neural stem cells differentiated into neurons and glial cells at 1 weeks after differentiation.
Conclusion Shh signaling pathway may be involved in the proliferation of adult neural stem cells stimulated by glutamic acid, and the proliferation of neural stem cells stimulated by glutamic acid does not affect the differentiation potential of adult neural stem cells.

【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

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