【摘要】：本研究分為3部分： 1.let-7a在腦缺血再灌注后的表達(dá)及其作用 在本部分研究中，我們通過對正常大鼠、缺血再灌注大鼠腦內(nèi)let-7a的表達(dá)的檢測，發(fā)現(xiàn)let-7a在腦缺血再灌注后，在腦組織內(nèi)高表達(dá)。為進(jìn)一步研究let-7a在體內(nèi)的作用，我們構(gòu)建過表達(dá)let-7a的AAV9質(zhì)粒，通過尾靜脈注射的方式使之在大鼠體內(nèi)過表達(dá)。與普通大鼠相比，過表達(dá)let-7a的大鼠在缺血再灌注后腦組織病理改變嚴(yán)重、神經(jīng)功能缺損評分增高，并且腦組織的內(nèi)炎癥因子IL-6和TNF-α含量高于普通大鼠，細(xì)胞凋亡程度也明顯重于普通大鼠。此結(jié)果提示let-7a在腦缺血再灌注后表達(dá)上調(diào)，并促進(jìn)缺血再灌注后的炎癥反應(yīng)和細(xì)胞凋亡，進(jìn)而加重神經(jīng)損傷。 2.MKP1在腦缺血再灌注后的表達(dá)及其作用 MKP1是一種重要的抗炎、抗凋亡蛋白，通過數(shù)據(jù)庫比對可以發(fā)現(xiàn)MKP1可能是let-7a的下游作用靶點(diǎn)。在本部分研究中，我們對MKP1在腦缺血再灌注后的作用進(jìn)行研究。將大鼠分為假手術(shù)（Sham）組、對照組（Control）、MKP1抑制劑組（MKP1抑制劑-Sanguinarine chloride，血根堿）。其中，Sham組不做任何處理，Control組大鼠經(jīng)尾靜脈注射生理鹽水，抑制組經(jīng)大鼠尾靜脈注射MKP1抑制劑，Sham組大鼠在腦缺血再灌注模型制備時(shí)線栓未阻斷血流。與對照組大鼠相比，MKP1抑制劑組的大鼠在缺血再灌注后出現(xiàn)腦組織病理改變嚴(yán)重、梗死面積大、神經(jīng)功能缺損評分高。同時(shí)，MKP1抑制劑組大鼠腦組織的炎癥因子TNF-α和IL-6含量高于對照組大鼠，Tunel和染色陽性細(xì)胞數(shù)以及小膠質(zhì)細(xì)胞增生也明顯多于對照組大鼠。結(jié)果提示MKP1有抑制炎癥反應(yīng)、細(xì)胞凋亡、神經(jīng)保護(hù)的作用。 3.let-7a對MKP1的調(diào)節(jié) 在本部分研究中，我們對let-7a與MKP1兩者之間的具體作用進(jìn)行研究。通過western blot實(shí)驗(yàn)結(jié)果證實(shí)，let-7a mimic可下調(diào)神經(jīng)元細(xì)胞系PC12細(xì)胞中MKP1蛋白的表達(dá)，而let-7a抑制劑let-7a inhibitor可促進(jìn)其蛋白表達(dá)，但兩者均不影響MKP1mRNA的水平。Luciferase assay顯示，轉(zhuǎn)染let-7a mimic后，含野生型MKP1的3’-UTR段的熒光報(bào)告質(zhì)粒的熒光表達(dá)強(qiáng)度明顯降低，將MKP13’-UTR突變后，let-7a不能降低熒光報(bào)告質(zhì)粒的熒光表達(dá)強(qiáng)度。此結(jié)果表明，let-7a可通過結(jié)合MKP13’-UTR端在轉(zhuǎn)錄后水平下調(diào)MKP1的表達(dá)。本研究還發(fā)現(xiàn)，let-7a可上調(diào)缺氧狀態(tài)下PC12細(xì)胞的凋亡，，過表達(dá)MKP1的PC12細(xì)胞可抑制let-7a促PC12細(xì)胞凋亡的作用。進(jìn)一步研究說明，let-7a可直接靶向作用于重要抗炎、抗凋亡因子MKP1，通過對MAPK信號通路的調(diào)控從而加促進(jìn)神經(jīng)細(xì)胞損傷。
[Abstract]:This study is divided into three parts: the expression of 1.let-7a after cerebral ischemia-reperfusion and its effects in this part of the study, we measured the expression of let-7a in the brain of normal rats and ischemia-reperfusion rats. It was found that let-7a was highly expressed in brain tissue after cerebral ischemia reperfusion. To further study the role of let-7a in vivo, we constructed a AAV9 plasmid expressing let-7a, which was overexpressed in rats by tail vein injection. Compared with the normal rats, the brain tissue pathological changes and neurological deficit score of the rats with overexpression of let-7a were serious, and the contents of IL-6 and TNF- 偽 in the brain tissues were higher than those in the normal rats. The degree of apoptosis was also more severe than that of normal rats. These results suggest that the expression of let-7a is up-regulated after cerebral ischemia-reperfusion, and it also promotes the inflammatory response and apoptosis after ischemia-reperfusion. The expression and effect of 2.MKP1 after cerebral ischemia-reperfusion is an important anti-inflammatory and anti-apoptotic protein. It can be found that MKP1 may be the downstream target of let-7a by database comparison. In this part of the study, we studied the role of MKP1 after cerebral ischemia reperfusion. Rats were divided into sham-operated (Sham) group and control (Control) MKP1 inhibitor group (MKP1 inhibitor -Sanguinarine chloride, root alkaloid). The rats in the control group were injected with normal saline via caudal vein, while the rats in the control group were injected with MKP1 inhibitor, MKP1 inhibitor, through the tail vein of the control group, and the blood flow was not blocked in the model of cerebral ischemia-reperfusion. Compared with the control group, the rats in the MKP1 inhibitor group had severe pathological changes of brain tissue, large infarct area and high neurological deficit score after ischemia-reperfusion. At the same time, the contents of TNF- 偽 and IL-6 in brain tissue of MKP1 inhibitor group were higher than those of control group. The number of Tunel and staining positive cells and the proliferation of microglia in MKP1 inhibitor group were significantly higher than those in control group. The results suggest that MKP1 can inhibit inflammation, apoptosis and neuroprotection. The regulation of MKP1 by 3.let-7a in this part of the study, we study the specific role between let-7a and MKP1. The results of western blot assay confirmed that mimic could down-regulate the expression of MKP1 protein in the neuronal cell line PC12 cells, while let-7a inhibitor, the inhibitor of let-7a, could promote the expression of MKP1 protein, but neither of them could affect the level of MKP1mRNA. Luciferase assay showed that the expression of MKP1 protein was decreased after let-7a mimic transfection. The fluorescence expression intensity of the fluorescent reporter plasmid containing the 3'-UTR segment of wild type MKP1 was significantly decreased, but the fluorescence expression intensity of the fluorescent reporter plasmid could not be reduced after the mutation of MKP13'-UTR. These results suggest that let-7a can down-regulate the expression of MKP1 at post-transcriptional level by binding to the MKP13'-UTR terminal. It was also found that let-7a could up-regulate the apoptosis of PC12 cells under hypoxia, and that PC12 cells over-expressing MKP1 could inhibit the apoptosis of PC12 cells induced by let-7a. It is further demonstrated that let-7a can directly target the important anti-inflammatory and anti-apoptotic factor MKP1 and promote neuronal injury by regulating the MAPK signaling pathway.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R743.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 ;Identification and functional annotation of novel microRNAs in the proximal sciatic nerve after sciatic nerve transection[J];Science China(Life Sciences);2011年09期

2 許建;武治印;于典科;;血清microRNA在腫瘤診斷和預(yù)后評估中的應(yīng)用[J];科學(xué)通報(bào);2010年01期

3 顧蕓;李石營;王星輝;劉倩倩;馮倩;陳褚;顧曉松;;MicroRNA-1對施萬細(xì)胞增殖的調(diào)節(jié)作用[J];交通醫(yī)學(xué);2012年04期

4 李石營;陳褚;王星輝;劉倩倩;馮倩;顧曉松;;Let-7對施萬細(xì)胞凋亡的調(diào)節(jié)作用[J];交通醫(yī)學(xué);2012年06期

5 金吉春;金星林;;microRNA的概述及其研究[J];醫(yī)學(xué)研究生學(xué)報(bào);2013年10期

6 杜燁鴻;徐明亮;賀桂瓊;王凌f^;劉剛;周澤芬;龍志敏;;MKP-1在APP/PS1雙轉(zhuǎn)基因小鼠腦內(nèi)的分布及表達(dá)[J];解放軍醫(yī)學(xué)雜志;2014年11期

7 戴冬偉;趙文元;黃清海;許奕;洪波;楊鵬飛;賈澤軍;張照龍;劉建民;;血清microRNA在煙霧病發(fā)病機(jī)制中的潛在作用[J];中華神經(jīng)外科疾病研究雜志;2013年02期

8 翁鳴;趙波濤;馬中良;金由辛;;LIN28與兩族microRNA在多功能干細(xì)胞誘導(dǎo)中的功能[J];生命的化學(xué);2012年06期

9 胡國艷;李萍;何柯新;徐鵬;羅新妮;鐘笑梅;侯樂;寧玉萍;劉學(xué)軍;;阿爾茨海默病患者腦脊液miRNA檢測及初步分析[J];實(shí)用醫(yī)學(xué)雜志;2013年06期

10 孟金蘭;蘭愛平;楊春濤;楊戰(zhàn)利;王立偉;陳麗新;朱琳燕;陳培熹;馮鑒強(qiáng);;熱休克蛋白90在硫化氫保護(hù)PC12細(xì)胞對抗化學(xué)性缺氧損傷中的作用[J];中國藥理學(xué)通報(bào);2010年01期

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