本文關(guān)鍵詞： 神經(jīng)干細胞 氧化應(yīng)激 自噬　出處：《吉林大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：神經(jīng)干細胞（neural stem cells，NSCs）是當(dāng)今生命科學(xué)研究的熱點之一。NSCs治療應(yīng)用主要體現(xiàn)在：細胞移植治療、細胞替代和基因治療及其自我修復(fù)治療。隨著NSCs研究的深入，發(fā)現(xiàn)NSCs移植后在炎癥反應(yīng)、氧化應(yīng)激較重的微環(huán)境下NSCs的存活率均較低。 實驗?zāi)康模河^察NSCs在氧化應(yīng)激狀態(tài)下是否有自噬的發(fā)生，為研究神經(jīng)干細胞在氧化應(yīng)激狀態(tài)下的生存機制提供新的依據(jù)。 實驗方法：分離、培養(yǎng)Wistar孕鼠的神經(jīng)干細胞，對生長良好的神經(jīng)干細胞球進行Nestin免疫細胞化學(xué)染色。應(yīng)用免疫細胞化學(xué)技術(shù)對神經(jīng)干細胞分化情況進行鑒定。應(yīng)用MTT法檢測不同濃度H_2O_2對NSCs生存率的影響，計算神經(jīng)干細胞的生存率。通過吖啶橙(AO)染色觀察細胞內(nèi)酸性自吞噬空泡的積聚。應(yīng)用透射電子顯微鏡對比觀察各組NSCs超微結(jié)構(gòu)的變化。通過Western blot技術(shù)檢測各組LC3蛋白的表達情況。 實驗結(jié)果：我們成功的分離、培養(yǎng)了NSCs；MTT比色法結(jié)果顯示，不同劑量H_2O_2作用于NSCs12h或24h后，隨著H_2O_2濃度的增加，細胞生存率逐漸降低，并呈劑量時間相關(guān)性，而0.5mM、1mM H_2O_2組與對照組相比具有統(tǒng)計學(xué)意義。與對照組相比，0.5mM H_2O_2組AO染色結(jié)果可見紅色熒光陽性結(jié)果顯著增強，細胞酸性自吞噬空泡產(chǎn)生明顯增多。0.5mM H_2O_2作用于NSCs12h后，應(yīng)用透射電子顯微鏡檢測自吞噬空泡的形成。結(jié)果顯示，對照組NSCs表現(xiàn)為正常細胞形態(tài)，細胞核呈規(guī)則圓形。0.5mM H_2O_2作用后細胞體積變大，，細胞膨脹，有明顯的自吞噬空泡生成。0.5mM H_2O_2作用于NSCs12h后，通過Western Blot檢測LC3蛋白表達。結(jié)果表明，與對照組相比，0.5mM H_2O_2作用后LC3-Ⅱ蛋白表達明顯增強。結(jié)論：氧化應(yīng)激可以通過一些通路來誘導(dǎo)神經(jīng)干細胞自噬的發(fā)生，這為探討神經(jīng)干細胞生存與死亡機制的研究提供了新的理論依據(jù)。
[Abstract]:Neural stem cells (NSCs) is one of the hotspots in life sciences. The therapeutic applications of NSCs are mainly reflected in cell transplantation, cell substitution, gene therapy and self-repair therapy. It was found that the survival rate of NSCs was lower in microenvironment with severe oxidative stress and inflammatory response after NSCs transplantation. Objective: to observe whether autophagy occurs in NSCs under oxidative stress, and to provide a new basis for studying the survival mechanism of neural stem cells under oxidative stress. Methods: neural stem cells of Wistar pregnant mice were isolated and cultured. The differentiation of neural stem cells was identified by immunocytochemistry. The effect of different concentrations of H _ 2O _ 2 on the survival rate of NSCs was detected by MTT method. The survival rate of neural stem cells was calculated. Acridine orange (AOO) staining was used to observe the accumulation of acridine autophagocytic vacuoles. The ultrastructural changes of NSCs were observed by transmission electron microscopy (TEM). The ultrastructure of NSCs was detected by Western blot technique. Expression of LC3 protein. The results showed that the cell survival rate decreased with the increase of H _ 2O _ 2 concentration in NSCs12h or 24 h after different doses of H _ (2) O _ (2) were treated with NSCs12h or 24 h, and the cell survival rate decreased with the increase of H _ 2O _ (2) concentration, and the cell survival rate decreased with the increase of H _ 2O _ 2 concentration. In comparison with the control group, the AO staining results of 0.5 mm M H _ 2O _ 2 group and 0.5 mm M H _ 2O _ 2 group showed that the red fluorescence positive results were significantly increased, and the vacuoles produced by acid-induced autophagocytosis increased significantly. 0.5 mm H _ 2O _ 2 significantly increased the effect of NSCs12h. The formation of autophagocytic vacuoles was detected by transmission electron microscope. The results showed that the NSCs of the control group showed normal cell morphology, the nucleus of the control group was regular round, 0.5mm H _ 2O _ 2, the cell volume became larger and the cells expanded. The expression of LC3 protein was detected by Western Blot, and the expression of LC3 protein was detected by Western Blot. Compared with the control group, the expression of LC3- 鈪

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