本文選題：神經(jīng)干細(xì)胞 + 谷氨酸　； 參考：《新鄉(xiāng)醫(yī)學(xué)院》2014年碩士論文

【摘要】：背景腦梗死的治療一直是醫(yī)學(xué)界的難題,傳統(tǒng)的治療無(wú)法修復(fù)受損的腦組織。神經(jīng)干細(xì)胞(neural stem cells)的發(fā)現(xiàn)為解決該難題帶來(lái)了新的切入點(diǎn),但神經(jīng)干細(xì)胞在存活增殖等諸多方面的復(fù)雜機(jī)制使得神經(jīng)干細(xì)胞的應(yīng)用研究仍無(wú)突破性進(jìn)展。腦梗死后神經(jīng)干細(xì)胞在原位被激活增殖,表現(xiàn)為梗死區(qū)谷氨酸(glutamate)濃度升高,神經(jīng)干細(xì)胞內(nèi)Shh(sonic hedgehog)蛋白表達(dá)量也顯著增加；聯(lián)系到Shh信號(hào)通路在胚胎干細(xì)胞增殖中的重要作用,我們推測(cè)Glu可能經(jīng)由Shh信號(hào)通路介導(dǎo)了神經(jīng)干細(xì)胞的增殖。因此本實(shí)驗(yàn)擬通過(guò)體外實(shí)驗(yàn)探索Shh表達(dá)與Glu刺激神經(jīng)干細(xì)胞增殖的關(guān)系。本實(shí)驗(yàn)分三部分： 第一部分成年SD大鼠神經(jīng)干細(xì)胞分離培養(yǎng)和鑒定的改良 目的改良成年SD大鼠神經(jīng)干細(xì)胞分離、培養(yǎng)和鑒定方法,觀察神經(jīng)干細(xì)胞的增殖和分化特點(diǎn),為后續(xù)實(shí)驗(yàn)提供細(xì)胞。 方法從成年SD大鼠分離出完整海馬,采用機(jī)械吹打法獲得原代細(xì)胞,用accutase消化傳代,利用CCK-8法檢測(cè)神經(jīng)干細(xì)胞的增殖情況,應(yīng)用多重標(biāo)記免疫熒光細(xì)胞化學(xué)方法鑒定神經(jīng)干細(xì)胞及其誘導(dǎo)分化后細(xì)胞。 結(jié)果機(jī)械吹打法可獲得原代神經(jīng)干細(xì)胞,accutase消化傳代有利于神經(jīng)干細(xì)胞的傳代培養(yǎng),CCK-8法能夠測(cè)定神經(jīng)干細(xì)胞的增殖,多重免疫熒光展現(xiàn)了成年神經(jīng)干細(xì)胞經(jīng)胎牛血清誘導(dǎo)后的誘導(dǎo)分化過(guò)程。 結(jié)論改良后的培養(yǎng)方法可以更加簡(jiǎn)單高效的獲得和培養(yǎng)出大量細(xì)胞,經(jīng)多重免疫熒光鑒定所分離和培養(yǎng)的細(xì)胞是神經(jīng)干細(xì)胞。 第二部分谷氨酸刺激神經(jīng)干細(xì)胞增殖的最適濃度探索 目的用不同濃度的谷氨酸刺激成體神經(jīng)干細(xì)胞增殖,尋求最適合的谷氨酸濃度,為后續(xù)試驗(yàn)做準(zhǔn)備。 方法取傳代3代以后的神經(jīng)干細(xì)胞,在含有不同濃度谷氨酸的去生長(zhǎng)因子培養(yǎng)基中培養(yǎng)24h、48h、72h,采用CCK-8法檢測(cè)不同谷氨酸濃度下神經(jīng)干細(xì)胞的增殖情況。 結(jié)果0μmol/L～1OOμmol/L濃度范圍的谷氨酸對(duì)成體神經(jīng)干細(xì)胞有增殖作用,20μmol/L谷氨酸濃度最適合成體神經(jīng)干細(xì)胞的增殖,當(dāng)谷氨酸濃度大于100μmol/L時(shí)將抑制成體神經(jīng)干細(xì)胞的增殖,并對(duì)成體神經(jīng)干細(xì)胞具有一定的細(xì)胞毒性。 結(jié)論在體外培養(yǎng)條件下,谷氨酸在一定濃度范圍內(nèi)的(Oμmol/L～100μmol/L)可以刺激成體神經(jīng)干細(xì)胞的增殖,刺激成體神經(jīng)干細(xì)胞增殖的最適濃度為20μmol/L；當(dāng)谷氨酸濃度大于100μmol/L時(shí)將抑制成體神經(jīng)干細(xì)胞的增殖,并對(duì)成體神經(jīng)干細(xì)胞具有一定的細(xì)胞毒性。 第三部分谷氨酸經(jīng)Shh信號(hào)途徑對(duì)神經(jīng)干細(xì)胞的增殖作用 目的用2Oμmol/L谷氨酸刺激成體神經(jīng)干細(xì)胞的增殖,探索Shh表達(dá)與谷氨酸刺激成體神經(jīng)干細(xì)胞增殖的關(guān)系。 方法取傳代3代以后的神經(jīng)干細(xì)胞,在終濃度為20μmol/L谷氨酸的除生長(zhǎng)因子的培養(yǎng)基中分別培養(yǎng)24h、48h、72h,設(shè)為Glu刺激24h、48h、72h3組,在未加谷氨酸的除生長(zhǎng)因子培養(yǎng)基中培養(yǎng)72h,設(shè)為空白對(duì)照組；按上述分組方法另設(shè)一組在相應(yīng)時(shí)間分別加入胎牛血清誘導(dǎo)分化培養(yǎng)1周,觀察神經(jīng)干細(xì)胞及誘導(dǎo)分化后細(xì)胞增殖情況,采用westemblot法檢測(cè)nestin蛋白、Shh蛋白、GFAP蛋白及βⅢ-tubulin蛋白的表達(dá)情況,采用實(shí)時(shí)熒光定量法檢測(cè)nestin mRNA、Shh mRNA、 GFAP mRNA及βⅢ-tubulin mRNA表達(dá)情況。 結(jié)果在谷氨酸刺激成體神經(jīng)干細(xì)胞的增殖過(guò)程中,nestin表達(dá)量變化與Shh表達(dá)量變化相一致。誘導(dǎo)分化后1周神經(jīng)干細(xì)胞大部分分化為神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞。 結(jié)論Shh信號(hào)通路可能參與了谷氨酸刺激成體神經(jīng)干細(xì)胞增殖的過(guò)程；谷氨酸刺激神經(jīng)干細(xì)胞增殖不影響成體神經(jīng)干細(xì)胞的分化潛能。
[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.

【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743.3

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