冷休克蛋白RBM3对视黄酸诱导的神经母细胞瘤细胞凋亡的影响
发布时间：2018-11-28 11:37
【摘要】：背景全反式视黄酸(all-trans retinoic acid,RA)是维生素A的活性代谢产物,是维生素A生物学功能的主要介导物。RA广泛分布于未成熟组织及成熟组织中,是细胞分化、细胞增殖和程序性死亡的关键调节因子,但过量服用维生素A会对神经系统存在毒副作用。冷休克蛋白RBM3是一种很重要的RNA结合蛋白,广泛参与缺氧-缺血应激、紫外照射应激、细胞增殖、骨骼肌调节、生长发育等多种生理过程,对神经细胞存在显著保护作用。本课题旨在探究RBM3对RA诱导的神经细胞凋亡的影响,并阐明其分子机制。目的1.探索亚低温对RA诱导的神经细胞凋亡的保护作用;2.详细研究RBM3对RA诱导的SH-SY5Y细胞凋亡的影响极其机制。方法1.利用本实验室已经建立起来的神经细胞凋亡模型SH-SY5Y,作为研究对象。利用MTT法和流式细胞术测定不同浓度的RA的细胞存活率。2.亚低温(32℃)预先处理细胞24 h,随后利用RA诱导SH-SY5Y细胞凋亡。借助Western免疫印迹和MTT检测细胞凋亡标志物cleaved PARP和cleaved caspase 3以及细胞存活率,并结合TUNEL染色方法,分析低温是否有助于减小RA的毒性并提高细胞的存活率。3.亚低温预处理后,检测到冷休克蛋白RBM3的表达量显著上升,进而我们推测亚低温介导的神经保护效果与RBM3的大量表达有关。于是,我们在亚低温条件下,利用siRNA沉默RBM3的基因表达,再加入RA处理24 h,与对照组相比,Western印迹分析RBM3干扰效果以及凋亡标志物表达水平的变化,分析RBM3的沉默对亚低温的神经保护作用的影响。4.过表达RBM3后,进行RA处理,MTT法和Western免疫印迹技术检测细胞存活率以及凋亡标志物,分析RBM3过表达对RA诱导的细胞凋亡是否存在影响,并采用DAPI核染色分析细胞凋亡情况。同时,分析多个压力应激信号通路中的关键激酶的磷酸化水平变化,揭示哪些信号通路介导了RBM3的神经保护效果。5.利用这些信号通路中关键激酶的特异性抑制剂或激活剂来阻断或活化上一步检测到的信号通路,利用MTT法和Western免疫印迹进行探究,借此阐明RBM3对神经细胞SH-SY5Y凋亡的保护机制。结果1.研究显示,低剂量的RA可轻微促进SH-SY5Y细胞增殖,高剂量的RA(5~50μM)会诱导细胞凋亡,且呈现浓度依赖性,结果显示,20μM RA可导致50%左右的细胞凋亡,此浓度用于进一步的实验。2.研究发现,亚低温预处理可保护细胞免受RA诱导的细胞凋亡,同时伴随冷休克蛋白RBM3的表达上调,另外,RBM3基因沉默后显著降低亚低温的神经保护效果,凋亡的标志物cleaved PARP的水平亦出现明显升高。3.为进一步证实RBM3在低温保护中的作用,我们研究了RBM3过表达对RA诱发的细胞凋亡的影响。结果显示,RBM3过表达可显著降低RA对SH-SY5Y细胞的凋亡诱导。此外,RBM3过表达能够明显抑制RA对JNK和p38信号通路的活化,并显著降低RA对AMPK信号通路的抑制,加入相应的特异性阻断剂(SP600125,SB203580)以及AMPK激活剂(AICAR)后,与RA处理组相比,细胞的存活率均显著提升。结论1.亚低温可保护SH-SY5Y细胞免受RA诱导的神经细胞凋亡。2.亚低温对神经细胞的保护作用主要通过RBM3蛋白的诱导表达来实现,即RBM3是亚低温保护的关键介导因子。3.RBM3能够通过抑制JNK和p38信号通路以及激活AMPK信号通路来协同保护神经细胞,可为维生素A滥用导致的神经相关疾病提供有意义的参考。
[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.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.4
本文编号：2362718
[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.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.4
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