本文選題：腦缺血 + 真核翻譯延長(zhǎng)因子1A1（eEF1A1）��； 參考：《第二軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：研究背景： 腦卒中是一種常見的急性腦血管疾病，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兩大類，在全世界具有較高的發(fā)病率和致殘率。隨著我國(guó)人口老齡化進(jìn)程不斷加快，腦缺血給患者家庭及社會(huì)健康資源帶來了日益嚴(yán)重的負(fù)擔(dān)，針對(duì)腦缺血發(fā)病的相關(guān)靶點(diǎn)研發(fā)預(yù)防及治療藥物已成為迫切需要。當(dāng)腦組織出現(xiàn)供血供氧不足時(shí)，會(huì)通過細(xì)胞毒性損傷、氧化應(yīng)激、炎癥反應(yīng)等多種機(jī)制導(dǎo)致缺血部位細(xì)胞的凋亡。其中，炎癥反應(yīng)在腦缺血進(jìn)程中發(fā)揮著十分重要的作用。缺血部位細(xì)胞分泌的炎性細(xì)胞因子IL-1β、TNF-α、E-選擇素、P-選擇素、細(xì)胞間粘附分子（ICAM-1）以及趨化因子等促進(jìn)了炎性細(xì)胞向缺血部位的浸潤(rùn)，并導(dǎo)致缺血部位血腦屏障通透性的改變，進(jìn)一步加重缺血部位血管阻塞，最終出現(xiàn)“無復(fù)流”（No-reflow）現(xiàn)象。因此，抑制缺血部位的炎癥反應(yīng)對(duì)于缺血部位損傷保護(hù)意義重大。 本課題組前期研究發(fā)現(xiàn)一種具有抗炎活性的五肽MLIF可以降低腦缺血模型大鼠、小鼠腦梗死面積，并發(fā)現(xiàn)MLIF在腦微血管內(nèi)皮細(xì)胞中作用于真核翻譯延長(zhǎng)因子1A1（eukaryotic elongation factor1A1，eEF1A1），可以升高eNOS表達(dá)從而降低腦缺血導(dǎo)致的ICAM-1、VCAM-1水平的升高，減少缺血部位細(xì)胞的損傷。本課題擬在前期研究基礎(chǔ)上對(duì)eEF1A1在腦缺血中的保護(hù)作用進(jìn)行進(jìn)一步研究，為腦缺血的預(yù)防及治療提供新的思路。 方法與結(jié)果： 1、采用免疫共沉淀方法找到在bEnd3細(xì)胞內(nèi)與eEF1A1存在相互作用的蛋白，經(jīng)質(zhì)譜鑒定并與蛋白質(zhì)數(shù)據(jù)庫(kù)比對(duì)，發(fā)現(xiàn)相互作用的蛋白為HSC70。 2、通過Western Blot方法與免疫熒光法對(duì)免疫共沉淀結(jié)果進(jìn)行驗(yàn)證。 免疫共沉淀得到的蛋白通過Western Blot方法雜交HSC70抗體，ECL顯色并用膠片顯影后發(fā)現(xiàn)71kD處有清晰條帶；用免疫熒光方法也觀察到eEF1A1與HSC70在胞漿中存在共定位，確證了bEnd3細(xì)胞內(nèi)與eEF1A1相互作用的蛋白為HSC70。 3、采用RNA干擾技術(shù)分別抑制eEF1A1與HSC70蛋白表達(dá)，Western Blot方法檢測(cè)另一蛋白的表達(dá)，結(jié)果顯示分別抑制eEF1A1與HSC70蛋白的表達(dá)后，另一蛋白的表達(dá)并未受到影響。而采用免疫共沉淀技術(shù)觀察缺氧前后這兩個(gè)蛋白的結(jié)合情況，發(fā)現(xiàn)eEF1A1與HSC70結(jié)合隨著缺氧刺激時(shí)間的增加呈現(xiàn)先降低后升高的趨勢(shì)，表明缺氧可能影響eEF1A1與HSC70的結(jié)合。說明eEF1A1與HSC70可能以分子伴侶形式在缺血性腦卒中進(jìn)程中發(fā)揮保護(hù)作用。其確切機(jī)制尚待進(jìn)一步研究。 4、分別將eEF1A1與HSC70基因敲減后，采用Annexin V-FITC/PI雙染法染色細(xì)胞,流式細(xì)胞儀檢測(cè)細(xì)胞的凋亡情況。結(jié)果顯示，HSC70siRNA轉(zhuǎn)染48h后細(xì)胞凋亡率高達(dá)50%，明顯高于陰性對(duì)照組（P＜0.01）；雖然eEF1A1基因敲減的正常細(xì)胞未出現(xiàn)明顯凋亡，但是缺氧處理8h后RNAi組凋亡率明顯高于對(duì)照組。表明eEF1A1與HSC70具有抑制缺氧誘導(dǎo)的細(xì)胞凋亡作用。 5、Western Blot方法檢測(cè)缺氧誘導(dǎo)bEnd3細(xì)胞中凋亡相關(guān)蛋白變化，我們發(fā)現(xiàn)缺氧刺激細(xì)胞后，JNK相關(guān)信號(hào)傳導(dǎo)通路中磷酸化JNK、磷酸化c-JUN（Ser63、Ser73）、cleaved caspase-9、cleaved caspase-3蛋白水平均會(huì)升高，這一現(xiàn)象可以被JNK抑制劑sp600125抑制，，從而證明缺氧刺激可以激活JNK相關(guān)信號(hào)通路，導(dǎo)致細(xì)胞凋亡。 6、向細(xì)胞內(nèi)分別轉(zhuǎn)染eEF1A1siRNA和HSC70siRNA48h后用Western Blot方法檢測(cè)JNK信號(hào)通路中相關(guān)蛋白變化。結(jié)果表明，HSC70基因敲減組中磷酸化JNK、磷酸化c-JUN（Ser63、Ser73）、cleaved caspase-9、cleaved caspase-3表達(dá)均明顯高于陰性對(duì)照組（P＜0.01）；eEF1A1基因敲減細(xì)胞在給予缺氧刺激后上述蛋白水平高于陰性對(duì)照組（P＜0.01）。表明eEF1A1與HSC70可以通過抑制JNK信號(hào)通路的激活發(fā)揮腦缺血保護(hù)作用。 7、基于文獻(xiàn)報(bào)道，在對(duì)bEnd3細(xì)胞給予不同時(shí)間缺氧刺激后，觀察分子伴侶介導(dǎo)的自噬相關(guān)蛋白HSC70以及LAMP-2A蛋白水平變化，發(fā)現(xiàn)不同缺氧刺激時(shí)間下HSC70與LAMP-2A蛋白表達(dá)無明顯差異，eEF1A1RNAi組與對(duì)照組相比兩者表達(dá)也無明顯差異，表明eEF1A1與HSC70未通過分子伴侶介導(dǎo)的自噬發(fā)揮腦缺血保護(hù)作用。 結(jié)論： 1、在bEnd3細(xì)胞中，eEF1A1與HSC70之間存在分子伴侶形式的相互作用。 2、eEF1A1與HSC70可以通過抑制JNK信號(hào)傳導(dǎo)通路的激活在缺血性腦卒中進(jìn)程中發(fā)揮保護(hù)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【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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