【摘要】：缺氧是最重要的應(yīng)激反應(yīng)之一,是也臨床最常見的病理生理過程,與心血管疾病和高原性肺水腫等的發(fā)生密切相關(guān)。糖皮質(zhì)激素作為體內(nèi)最重要的應(yīng)激激素,在機(jī)體對(duì)低氧適應(yīng)中發(fā)揮重要作用。人工合成的糖皮質(zhì)激素-地塞米松(Dex)在臨床上被廣泛用于治療肺水腫和肺損傷,它的這種作用是通過降低血管內(nèi)皮細(xì)胞的通透性、加速肺泡腔中液體的清除以及增強(qiáng)肺上皮屏障功能來實(shí)現(xiàn)的。 Stomatin是一個(gè)重要的脂筏標(biāo)志蛋白,它廣泛表達(dá)在人和小鼠的各組織細(xì)胞中,參與脂筏結(jié)構(gòu)的裝配,囊泡運(yùn)輸、物質(zhì)的跨膜轉(zhuǎn)運(yùn)、以及對(duì)離子通道和細(xì)胞骨架的調(diào)節(jié)等生理過程。但是迄今對(duì)stomatin的功能了解的有限,對(duì)其在生理和病理?xiàng)l件下的調(diào)節(jié)研究的更少。我們前期研究了低氧和地塞米松(Dex)單獨(dú)及聯(lián)合作用對(duì)大鼠肺臟和人肺腺癌A549細(xì)胞stomatin表達(dá)的影響。結(jié)果發(fā)現(xiàn)低氧和Dex在體內(nèi)和體外均可以上調(diào)stomatin的表達(dá)。本課題將從細(xì)胞和整體水平進(jìn)一步確定糖皮質(zhì)激素和低氧單獨(dú)或共同作用對(duì)stomatin基因表達(dá)的影響,在此基礎(chǔ)上,重點(diǎn)研究低氧和地塞米松上調(diào)stomatin表達(dá)的機(jī)制,并初步探討stomatin表達(dá)上調(diào)的生物學(xué)意義。 采用Real time-PCR和Western Blot的方法,我們首先在大鼠肺臟和原代培養(yǎng)的肺泡上皮細(xì)胞水平進(jìn)一步證實(shí)了地塞米松和低氧不僅單獨(dú)可誘導(dǎo)stomatin的表達(dá),二者還能聯(lián)合上調(diào)stomatin表達(dá)。接下來我們研究了低氧和地塞米松上調(diào)stomatin的機(jī)制。研究發(fā)現(xiàn),stomatin mRNA有一個(gè)相當(dāng)長的半衰期,約為32小時(shí),低氧和地塞米松處理都能夠增強(qiáng)stomatin mRNA的穩(wěn)定性,使它的半衰期分別延長至原來的1.22倍和1.5倍。我們用熒光素酶報(bào)告基因?qū)嶒?yàn)研究發(fā)現(xiàn),低氧不能誘導(dǎo)stomatin啟動(dòng)子的轉(zhuǎn)錄活性,與低氧不同的是,地塞米松能夠以時(shí)間和濃度依賴性的方式誘導(dǎo)stomatin啟動(dòng)子的活性,這個(gè)結(jié)果表明地塞米松也能夠直接在轉(zhuǎn)錄水平上誘導(dǎo)stomatin mRNA的表達(dá)。為了進(jìn)一步定位stomatin啟動(dòng)子對(duì)糖皮質(zhì)激素誘導(dǎo)反應(yīng)的位點(diǎn),我們構(gòu)建了一系列含stomatin啟動(dòng)子5’端不同截短形式的報(bào)告基因載體,熒光素酶活性分析試驗(yàn)表明,啟動(dòng)子的-162至+244區(qū)域是對(duì)糖皮質(zhì)激素誘導(dǎo)反應(yīng)所不可缺少的,而且這一區(qū)域在多種細(xì)胞類型中均貢獻(xiàn)了stomatin基因的絕大部分基礎(chǔ)轉(zhuǎn)錄活性,因此我們推測這一區(qū)域是它的核心啟動(dòng)子區(qū)。隨后,我們用軟件預(yù)測了這一區(qū)域所包含的可能介導(dǎo)糖皮質(zhì)激素誘導(dǎo)反應(yīng)的轉(zhuǎn)錄因子結(jié)合位點(diǎn),并且構(gòu)建了這些位點(diǎn)的相應(yīng)突變型報(bào)告基因載體。結(jié)果發(fā)現(xiàn),其中一個(gè)位點(diǎn)GRE3,突變之后能夠使stomatin啟動(dòng)子失去對(duì)地塞米松的誘導(dǎo)反應(yīng),這說明GRE3介導(dǎo)了地塞米松對(duì)stomatin的誘導(dǎo)反應(yīng)。因此我們認(rèn)為,地塞米松在轉(zhuǎn)錄水平上對(duì)stomatin的誘導(dǎo)是通過激活的糖皮質(zhì)激素受體與這個(gè)GRE3位點(diǎn)結(jié)合來實(shí)現(xiàn)的。 最后,我們用激光共聚焦顯微鏡的方法觀察了A549細(xì)胞中stomatin的表達(dá)與actin細(xì)胞骨架的關(guān)系。結(jié)果發(fā)現(xiàn),stomatin在膜周與actin細(xì)胞骨架存在共定位。低氧暴露或Dex處理均能使膜周actin增加,干擾內(nèi)源性stomatin表達(dá)后低氧暴露或Dex處理A549細(xì)胞都能使actin細(xì)胞骨架在膜周分布顯著減少,表明低氧和Dex能通過上調(diào)stomatin表達(dá),增加與細(xì)胞膜連接的細(xì)胞骨架,從而增強(qiáng)肺泡上皮細(xì)胞膜的穩(wěn)定性。這可能是低氧和GC增強(qiáng)肺泡上皮細(xì)胞屏障的功能,從而使得機(jī)體產(chǎn)生對(duì)低氧適應(yīng)性反應(yīng)的機(jī)制之一。
[Abstract]:Hypoxia is one of the most important stress responses, and is also the most common pathophysiological process in the clinic, which is closely related to the occurrence of cardiovascular disease and high altitude pulmonary edema. Glucocorticoid, as the most important stress hormone in the body, plays an important role in the adaptation of hypoxia. The synthetic glucocorticoid-dexamethasone (Dex) is widely used in the treatment of pulmonary edema and lung injury, which is achieved by reducing the permeability of the vascular endothelial cells, accelerating the removal of the liquid in the alveolar cavity, and enhancing the pulmonary epithelial barrier function. Stomatin is an important protein of the lipid raft, which is widely expressed in the tissue cells of human and mouse, is involved in the assembly of the lipid raft structure, the transport of the vesicles, the transport of the substance, and the regulation of the ion channel and the cytoskeleton, and so on. Process. But to date, there is a limited understanding of the function of stomatin, which has been studied under physiological and pathological conditions. Less. We studied the expression of stomatin in lung and human lung adenocarcinoma A549 cells by hypoxia and dexamethasone (Dex) alone and in combination. The results showed that both hypoxia and Dex could be up-regulated in vivo and in vitro. The effect of glucocorticoid and hypoxia on the expression of the statin gene is further determined from the cell and the whole level. On the basis of this, the mechanism of up-regulation of the expression of stomatin by hypoxia and dexamethasone is mainly studied. Meantime, with the method of Real time-PCR and Western Blot, we first confirmed that the expression of dexamethasone and hypoxia not only can induce the expression of the statin in the lung and the primary cultured alveolar epithelial cells of the rat, but can also be used to upregulate the staoma. Tin's expression. Next we studied hypoxia and dexamethasone up-regulation of stomat. In this study, it was found that the stomatin mRNA has a long half-life, which is about 32 hours, and the hypoxia and dexamethasone treatment can enhance the stability of the stomatin mRNA and prolong the half-life of the statin mRNA to the original 1.22-fold. We found that hypoxia could not induce the transcriptional activity of the statostatin promoter, and it was different from the hypoxia that the dexamethasone can induce stomatin in a time-and concentration-dependent manner. The results of the activity of the mover indicate that the dexamethasone can also directly induce the statin mR at the level of transcription. in order to further position the site of the stomatin promoter to the glucocorticoid response, a series of reporter vector, luciferase activity, The analysis shows that the region of-162 to + 244 of the promoter is indispensable to the response of the glucocorticoid, and the region contributes most of the basic transcriptional activity of the stomatin gene in a variety of cell types, so we assume that this region is its nucleus. The core promoter region. Subsequently, we used the software to predict the transcription factor binding site that could mediate the glucocorticoid-mediated response contained in this region, and the corresponding mutant reports of these sites were constructed The results showed that GRE3 in one of the sites, after the mutation, can cause the stomatin promoter to lose the response to dexamethasone, which indicates that the GRE3 mediates the effect of dexamethasone on the stomatin. It is therefore believed that the induction of the statin at the level of transcription by dexamethasone is by activating the glucocorticoid receptor with this GRE3 site. Finally, we used a laser confocal microscope to observe the expression of stomatin in A549 cells and acti. The relationship of the cytoskeleton of n cells was found. The results showed that the statin was fine in the membrane and actin. In the presence of a co-localization of the cytoskeleton, the hypoxia exposure or the Dex treatment can increase the expression of actin cytoskeleton in the membrane around the membrane cycle, and it is shown that the hypoxia and Dex can increase the expression, increase and the cells of the actin cytoskeleton by up-regulation of the statin expression. The membrane is attached to the cytoskeleton to enhance the alveoli. The stability of the skin cell membrane. This may be a function of hypoxia and GC to enhance the barrier of the alveolar epithelial cells, making it possible for the body to adapt to hypoxia
【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】：2011
【分類號(hào)】：R363

【共引文獻(xiàn)】

相關(guān)期刊論文 前5條

1 王雁;崔宇輝;張金濤;;Stomatin在哺乳動(dòng)物機(jī)械感覺傳導(dǎo)中的作用[J];東南國防醫(yī)藥;2010年02期

2 常棟;王天佑;劉芝華;;stomatin家族與惡性腫瘤[J];中華臨床醫(yī)師雜志(電子版);2011年02期

3 田啟宇;陳永輝;范志勇;李鐵軍;賀建華;鄧近平;;炎熱氣候條件下母豬妊娠后期不同組織中GRα mRNA的表達(dá)規(guī)律研究[J];中國農(nóng)學(xué)通報(bào);2013年29期

4 林雪彩;孫紅英;;GR亞型α和β在口腔扁平苔蘚中的表達(dá)及臨床分析[J];中華臨床醫(yī)師雜志(電子版);2013年12期

5 高亞s，

本文編號(hào)：2438714