發(fā)布時間：2018-11-28 11:37

【摘要】：背景全反式視黃酸(all-trans retinoic acid,RA)是維生素A的活性代謝產(chǎn)物,是維生素A生物學(xué)功能的主要介導(dǎo)物。RA廣泛分布于未成熟組織及成熟組織中,是細(xì)胞分化、細(xì)胞增殖和程序性死亡的關(guān)鍵調(diào)節(jié)因子,但過量服用維生素A會對神經(jīng)系統(tǒng)存在毒副作用。冷休克蛋白RBM3是一種很重要的RNA結(jié)合蛋白,廣泛參與缺氧-缺血應(yīng)激、紫外照射應(yīng)激、細(xì)胞增殖、骨骼肌調(diào)節(jié)、生長發(fā)育等多種生理過程,對神經(jīng)細(xì)胞存在顯著保護作用。本課題旨在探究RBM3對RA誘導(dǎo)的神經(jīng)細(xì)胞凋亡的影響,并闡明其分子機制。目的1.探索亞低溫對RA誘導(dǎo)的神經(jīng)細(xì)胞凋亡的保護作用;2.詳細(xì)研究RBM3對RA誘導(dǎo)的SH-SY5Y細(xì)胞凋亡的影響極其機制。方法1.利用本實驗室已經(jīng)建立起來的神經(jīng)細(xì)胞凋亡模型SH-SY5Y,作為研究對象。利用MTT法和流式細(xì)胞術(shù)測定不同濃度的RA的細(xì)胞存活率。2.亞低溫(32℃)預(yù)先處理細(xì)胞24 h,隨后利用RA誘導(dǎo)SH-SY5Y細(xì)胞凋亡。借助Western免疫印跡和MTT檢測細(xì)胞凋亡標(biāo)志物cleaved PARP和cleaved caspase 3以及細(xì)胞存活率,并結(jié)合TUNEL染色方法,分析低溫是否有助于減小RA的毒性并提高細(xì)胞的存活率。3.亞低溫預(yù)處理后,檢測到冷休克蛋白RBM3的表達量顯著上升,進而我們推測亞低溫介導(dǎo)的神經(jīng)保護效果與RBM3的大量表達有關(guān)。于是,我們在亞低溫條件下,利用siRNA沉默RBM3的基因表達,再加入RA處理24 h,與對照組相比,Western印跡分析RBM3干擾效果以及凋亡標(biāo)志物表達水平的變化,分析RBM3的沉默對亞低溫的神經(jīng)保護作用的影響。4.過表達RBM3后,進行RA處理,MTT法和Western免疫印跡技術(shù)檢測細(xì)胞存活率以及凋亡標(biāo)志物,分析RBM3過表達對RA誘導(dǎo)的細(xì)胞凋亡是否存在影響,并采用DAPI核染色分析細(xì)胞凋亡情況。同時,分析多個壓力應(yīng)激信號通路中的關(guān)鍵激酶的磷酸化水平變化,揭示哪些信號通路介導(dǎo)了RBM3的神經(jīng)保護效果。5.利用這些信號通路中關(guān)鍵激酶的特異性抑制劑或激活劑來阻斷或活化上一步檢測到的信號通路,利用MTT法和Western免疫印跡進行探究,借此闡明RBM3對神經(jīng)細(xì)胞SH-SY5Y凋亡的保護機制。結(jié)果1.研究顯示,低劑量的RA可輕微促進SH-SY5Y細(xì)胞增殖,高劑量的RA(5~50μM)會誘導(dǎo)細(xì)胞凋亡,且呈現(xiàn)濃度依賴性,結(jié)果顯示,20μM RA可導(dǎo)致50%左右的細(xì)胞凋亡,此濃度用于進一步的實驗。2.研究發(fā)現(xiàn),亞低溫預(yù)處理可保護細(xì)胞免受RA誘導(dǎo)的細(xì)胞凋亡,同時伴隨冷休克蛋白RBM3的表達上調(diào),另外,RBM3基因沉默后顯著降低亞低溫的神經(jīng)保護效果,凋亡的標(biāo)志物cleaved PARP的水平亦出現(xiàn)明顯升高。3.為進一步證實RBM3在低溫保護中的作用,我們研究了RBM3過表達對RA誘發(fā)的細(xì)胞凋亡的影響。結(jié)果顯示,RBM3過表達可顯著降低RA對SH-SY5Y細(xì)胞的凋亡誘導(dǎo)。此外,RBM3過表達能夠明顯抑制RA對JNK和p38信號通路的活化,并顯著降低RA對AMPK信號通路的抑制,加入相應(yīng)的特異性阻斷劑(SP600125,SB203580)以及AMPK激活劑(AICAR)后,與RA處理組相比,細(xì)胞的存活率均顯著提升。結(jié)論1.亞低溫可保護SH-SY5Y細(xì)胞免受RA誘導(dǎo)的神經(jīng)細(xì)胞凋亡。2.亞低溫對神經(jīng)細(xì)胞的保護作用主要通過RBM3蛋白的誘導(dǎo)表達來實現(xiàn),即RBM3是亞低溫保護的關(guān)鍵介導(dǎo)因子。3.RBM3能夠通過抑制JNK和p38信號通路以及激活A(yù)MPK信號通路來協(xié)同保護神經(jīng)細(xì)胞,可為維生素A濫用導(dǎo)致的神經(jīng)相關(guān)疾病提供有意義的參考。
[Abstract]:Background All-trans retinoic acid (RA) is the active metabolite of vitamin A, which is the main mediator of the biological function of vitamin A. RA is widely distributed in immature tissues and mature tissues, and is a key regulatory factor for cell differentiation, cell proliferation and programmed death. However, excessive administration of vitamin A may have a toxic and side effect on the nervous system. The cold shock protein RBM3 is an important RNA binding protein, which is widely used in various physiological processes such as hypoxia-ischemia stress, ultraviolet irradiation stress, cell proliferation, skeletal muscle regulation, growth and development, and has a significant protective effect on nerve cells. The purpose of this study is to explore the effect of RBM3 on the apoptosis of the nerve cells induced by RA, and to elucidate its molecular mechanism. Purpose 1. to explore the protective effect of sublow temperature on the apoptosis of the nerve cells induced by RA; The effect of RBM3 on the apoptosis of SH-SY5Y cells induced by RA was studied in detail. Method 1. The cell apoptosis model SH-SY5Y, which has been established in this lab, is used as the research object. Cell viability of RA with different concentrations was determined by MTT and flow cytometry. Cells were pre-treated at sublow temperature (32.degree. C.) for 24 h, followed by an RA-induced apoptosis of SH-SY5Y cells. The cell apoptosis was detected by Western immunoblotting and MTT, and the cell survival rate was detected by TUNEL staining. After low-temperature pre-treatment, the expression of RBM3 in cold shock protein was detected to be significantly increased, and it was suggested that the effect of sublow-temperature mediated neuroprotection was related to the significant expression of RBM3. Then, we used siRNA to silence the gene expression of RBM3 under the sublow temperature condition, and then added RA for 24h, and compared with the control group, the effect of RBM3 and the expression level of the apoptosis marker were analyzed by Western blot, and the effect of the silencing of RBM3 on the neuroprotective effect of sublow temperature was analyzed. After the expression of RBM3, the cell survival rate and the apoptosis marker were detected by RA treatment, MTT and Western immunoblotting, and the effect of the overexpression of RBM3 on the apoptosis induced by RA was analyzed, and the cell apoptosis was analyzed by DAPI nuclear staining. At the same time, the level of phosphorylation of key kinases in multiple stress-stress signal pathways was analyzed to reveal which signal pathways mediate the neuroprotective effect of RBM3. Using the specific inhibitor or activator of the key kinase in these signal paths to block or activate the signal path detected in one step, the MTT and Western immunoblotting were used to explore the mechanism of the protection of SH-SY5Y in the nerve cells. Results 1. The results showed that the low dose of RA could promote the proliferation of SH-SY5Y cells, and the high-dose RA (5-50. mu.M) could induce apoptosis and present a concentration-dependent relationship. The results showed that 20. m The results showed that sublow-temperature pre-treatment could protect the cells from RA-induced apoptosis, and the expression of RBM3 was up-regulated with cold shock protein RBM3. To further confirm the role of RBM3 in low temperature protection, we studied the effect of the overexpression of RBM3 on the cell apoptosis induced by RA. The results showed that the overexpression of RBM3 could significantly reduce the apoptosis induced by RA on SH-SY5Y cells. In addition, the overexpression of RBM3 can significantly inhibit the activation of RA on the JNK and p38 signaling pathways, and significantly reduce the inhibition of RA on the AMPK signaling pathway, the addition of the corresponding specific blocker (SP600125, SB203580), and the AMPK activator (AICAR), and the survival rate of the cells is significantly improved compared to the RA treatment group. Conclusion 1. Sublow temperature can protect SH-SY5Y cells from RA-induced apoptosis. The protective effect of sub-low temperature on the nerve cells is mainly realized by the induction expression of the RBM3 protein, that is, the RBM3 is the key-mediated factor of sublow-temperature protection, and the RBM3 can be used for synergistically protecting the nerve cells by inhibiting the JNK and p38 signal paths and activating the AMPK signal path, and can provide a meaningful reference for the neurorelated diseases caused by the abuse of the vitamin A.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.4

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