eEF1A1在脑缺血中的保护作用研究

发布时间：2018-05-28 06:21

本文选题：脑缺血 + 真核翻译延长因子1A1（eEF1A1）　；参考：《第二军医大学》2014年硕士论文

【摘要】：研究背景：脑卒中是一种常见的急性脑血管疾病，是目前全世界三大死亡原因之一，包括缺血性和出血性卒中两大类，在全世界具有较高的发病率和致残率。随着我国人口老龄化进程不断加快，脑缺血给患者家庭及社会健康资源带来了日益严重的负担，针对脑缺血发病的相关靶点研发预防及治疗药物已成为迫切需要。当脑组织出现供血供氧不足时，会通过细胞毒性损伤、氧化应激、炎症反应等多种机制导致缺血部位细胞的凋亡。其中，炎症反应在脑缺血进程中发挥着十分重要的作用。缺血部位细胞分泌的炎性细胞因子IL-1β、TNF-α、E-选择素、P-选择素、细胞间粘附分子（ICAM-1）以及趋化因子等促进了炎性细胞向缺血部位的浸润，并导致缺血部位血脑屏障通透性的改变，进一步加重缺血部位血管阻塞，最终出现“无复流”（No-reflow）现象。因此，抑制缺血部位的炎症反应对于缺血部位损伤保护意义重大。本课题组前期研究发现一种具有抗炎活性的五肽MLIF可以降低脑缺血模型大鼠、小鼠脑梗死面积，并发现MLIF在脑微血管内皮细胞中作用于真核翻译延长因子1A1（eukaryotic elongation factor1A1，eEF1A1），可以升高eNOS表达从而降低脑缺血导致的ICAM-1、VCAM-1水平的升高，减少缺血部位细胞的损伤。本课题拟在前期研究基础上对eEF1A1在脑缺血中的保护作用进行进一步研究，为脑缺血的预防及治疗提供新的思路。方法与结果： 1、采用免疫共沉淀方法找到在bEnd3细胞内与eEF1A1存在相互作用的蛋白，经质谱鉴定并与蛋白质数据库比对，发现相互作用的蛋白为HSC70。 2、通过Western Blot方法与免疫荧光法对免疫共沉淀结果进行验证。免疫共沉淀得到的蛋白通过Western Blot方法杂交HSC70抗体，ECL显色并用胶片显影后发现71kD处有清晰条带；用免疫荧光方法也观察到eEF1A1与HSC70在胞浆中存在共定位，确证了bEnd3细胞内与eEF1A1相互作用的蛋白为HSC70。 3、采用RNA干扰技术分别抑制eEF1A1与HSC70蛋白表达，Western Blot方法检测另一蛋白的表达，结果显示分别抑制eEF1A1与HSC70蛋白的表达后，另一蛋白的表达并未受到影响。而采用免疫共沉淀技术观察缺氧前后这两个蛋白的结合情况，发现eEF1A1与HSC70结合随着缺氧刺激时间的增加呈现先降低后升高的趋势，表明缺氧可能影响eEF1A1与HSC70的结合。说明eEF1A1与HSC70可能以分子伴侣形式在缺血性脑卒中进程中发挥保护作用。其确切机制尚待进一步研究。 4、分别将eEF1A1与HSC70基因敲减后，采用Annexin V-FITC/PI双染法染色细胞,流式细胞仪检测细胞的凋亡情况。结果显示，HSC70siRNA转染48h后细胞凋亡率高达50%，明显高于阴性对照组（P＜0.01）；虽然eEF1A1基因敲减的正常细胞未出现明显凋亡，但是缺氧处理8h后RNAi组凋亡率明显高于对照组。表明eEF1A1与HSC70具有抑制缺氧诱导的细胞凋亡作用。 5、Western Blot方法检测缺氧诱导bEnd3细胞中凋亡相关蛋白变化，我们发现缺氧刺激细胞后，JNK相关信号传导通路中磷酸化JNK、磷酸化c-JUN（Ser63、Ser73）、cleaved caspase-9、cleaved caspase-3蛋白水平均会升高，这一现象可以被JNK抑制剂sp600125抑制，，从而证明缺氧刺激可以激活JNK相关信号通路，导致细胞凋亡。 6、向细胞内分别转染eEF1A1siRNA和HSC70siRNA48h后用Western Blot方法检测JNK信号通路中相关蛋白变化。结果表明，HSC70基因敲减组中磷酸化JNK、磷酸化c-JUN（Ser63、Ser73）、cleaved caspase-9、cleaved caspase-3表达均明显高于阴性对照组（P＜0.01）；eEF1A1基因敲减细胞在给予缺氧刺激后上述蛋白水平高于阴性对照组（P＜0.01）。表明eEF1A1与HSC70可以通过抑制JNK信号通路的激活发挥脑缺血保护作用。 7、基于文献报道，在对bEnd3细胞给予不同时间缺氧刺激后，观察分子伴侣介导的自噬相关蛋白HSC70以及LAMP-2A蛋白水平变化，发现不同缺氧刺激时间下HSC70与LAMP-2A蛋白表达无明显差异，eEF1A1RNAi组与对照组相比两者表达也无明显差异，表明eEF1A1与HSC70未通过分子伴侣介导的自噬发挥脑缺血保护作用。结论： 1、在bEnd3细胞中，eEF1A1与HSC70之间存在分子伴侣形式的相互作用。 2、eEF1A1与HSC70可以通过抑制JNK信号传导通路的激活在缺血性脑卒中进程中发挥保护作用。
[Abstract]:Research background:
Cerebral apoplexy is a common acute cerebrovascular disease. It is one of the three major causes of death in the world, including two major categories of ischemic and hemorrhagic stroke. It has high morbidity and disability in the world. With the rapid population aging process in our country, the family and social health resources of the patients with cerebral ischemia have become more and more serious. It is an urgent need to develop the prevention and treatment drugs for the related targets of cerebral ischemia. When the brain tissue is deficient in supply of blood and oxygen, the apoptosis of the cells is caused by cytotoxic damage, oxidative stress, and inflammatory reaction. Among them, the inflammatory reaction plays a very important role in the process of cerebral ischemia. The inflammatory cytokines IL-1 beta, TNF- a, E- selectin, P- selectin, P- selectin, ICAM-1, and chemokines promote the infiltration of inflammatory cells to the ischemic site, and lead to the changes in the permeability of the blood brain barrier at the ischemic site, further aggravating the vascular obstruction at the ischemic site, and eventually "no recovery". "No-reflow" phenomenon, therefore, inhibiting the inflammatory reaction of ischemic sites is of great significance for the protection of ischemic sites.
In our previous study, we found that a five peptide MLIF with anti-inflammatory activity could reduce the area of cerebral ischemia model rats and cerebral infarction, and found that MLIF plays a role in eukaryotic translation prolongation factor 1A1 (eukaryotic elongation factor1A1, eEF1A1) in cerebral microvascular endothelial cells, which can increase the expression of eNOS and thus reduce ICAM caused by cerebral ischemia. The elevation of -1 and VCAM-1 levels reduces the damage of cells in the ischemic site. This topic is to further study the protective effect of eEF1A1 on cerebral ischemia on the basis of earlier research, and provide a new way of thinking for the prevention and treatment of cerebral ischemia.
Methods and results:
1, the proteins interacting with eEF1A1 in bEnd3 cells were found by the immunoprecipitation method. The proteins interacting with the protein database were identified by mass spectrometry and compared with the protein database. The proteins interacting with the protein were found to be HSC70.
2, the results of CO immunoprecipitation were verified by Western Blot and immunofluorescence.
The protein obtained by the immunoprecipitation was hybridized with HSC70 antibody by Western Blot method. The ECL color was found and the clear strip was found at 71kD after the film was developed. The co localization of eEF1A1 and HSC70 in the cytoplasm was also observed by immunofluorescence, and the protein used in bEnd3 cells with eEF1A1 was confirmed to be HSC70..
3, RNA interference technique was used to inhibit the expression of eEF1A1 and HSC70 protein, and the Western Blot method was used to detect the expression of another protein. The results showed that the expression of the other protein was not affected after the inhibition of the expression of eEF1A1 and HSC70 protein, and the combination of the two proteins before and after anoxia was observed by immunoprecipitation. EEF1A1 was found. HSC70 combined with the increase of hypoxia stimulation time first decreased and then increased, indicating that hypoxia may affect the combination of eEF1A1 and HSC70. It shows that eEF1A1 and HSC70 may play a protective role in the process of ischemic stroke in the form of molecular chaperone. The exact mechanism remains to be further studied.
4, after the knockout of eEF1A1 and HSC70 genes, the cell apoptosis was detected by Annexin V-FITC/PI double staining and flow cytometry. The results showed that the apoptosis rate was up to 50% after HSC70siRNA transfection of 48h, obviously higher than that of the negative control group (P < 0.01). Although the normal cells of eEF1A1 gene knockout did not appear obvious apoptosis, but lack of apoptosis, but lack of eEF1A1 gene knockout. After 8h treatment, the apoptotic rate of RNAi group was significantly higher than that of the control group, indicating that eEF1A1 and HSC70 could inhibit hypoxia induced apoptosis.
5, Western Blot method was used to detect the changes of apoptosis related proteins in bEnd3 cells induced by hypoxia. We found that after the hypoxia stimulated the cells, the phosphorylated JNK, the phosphorylated c-JUN (Ser63, Ser73), cleaved caspase-9, cleaved caspase-3 protein levels in the JNK related signal transduction pathways all increased. This phenomenon could be inhibited by the inhibitor. Hypoxia stimulation can activate JNK related signaling pathways, leading to apoptosis.
6, after transfection of eEF1A1siRNA and HSC70siRNA48h into the cells, the changes in the related proteins in the JNK signaling pathway were detected by Western Blot method. The results showed that the phosphorylation of JNK, phosphorylated c-JUN (Ser63, Ser73), cleaved caspase-9 in the HSC70 gene knockout group were significantly higher than that of the negative control group (< 0.01). The protein level of the cells was higher than that of the negative control group (P < 0.01) after the stimulation of hypoxia (P < 0.01). The results showed that eEF1A1 and HSC70 could inhibit the protection of cerebral ischemia by inhibiting the activation of the JNK signaling pathway.
7, based on the literature report, the changes of the molecular chaperone mediated autophagy related protein HSC70 and the level of LAMP-2A protein were observed after the bEnd3 cells were given anoxic stimulation at different time. It was found that there was no significant difference in the expression of HSC70 and LAMP-2A protein in the time of different hypoxia stimulation, and there was no significant difference in the expression between the eEF1A1RNAi and the control groups, indicating that e was not significantly different from the control group. EF1A1 and HSC70 do not play the role of cerebral ischemia protection through molecular chaperone mediated autophagy.
Conclusion:
1, in bEnd3 cells, there is a molecular chaperone interaction between eEF1A1 and HSC70.
2, eEF1A1 and HSC70 can play a protective role in the process of ischemic stroke by inhibiting the activation of JNK signaling pathway.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

【参考文献】