本文選題：燒傷　切入點(diǎn)：腸屏障　出處：《中國(guó)人民解放軍醫(yī)學(xué)院》2014年博士論文　論文類型：學(xué)位論文

【摘要】：目的 腸粘膜上皮屏障功能障礙是嚴(yán)重?zé)齻蟮某Ｒ姴l(fā)癥，也是誘發(fā)膿毒癥和多器官功能障礙的重要危險(xiǎn)因素。嚴(yán)重?zé)齻鹉c粘膜上皮屏障功能障礙的具體機(jī)制尚未完全清楚，但越來越多的研究提示，缺氧誘導(dǎo)因子1（hypoxia-inducible factor-1，HIF-1）及其下游靶基因血管內(nèi)皮生長(zhǎng)因子（vascular endothelial growth factor，VEGF）和肌球蛋白輕鏈激酶（myosinlight chain kinase，MLCK）的表達(dá)及功能在缺氧引起的細(xì)胞間屏障損害中起著關(guān)鍵作用。近年來研究表明，組蛋白去乙酰化酶抑制劑（histone deacetylaseinhibitor，HDACI）丙戊酸鈉能提高細(xì)胞對(duì)缺氧、炎癥等打擊的耐受能力，保護(hù)機(jī)體各重要臟器功能，延長(zhǎng)失血、燒傷及膿毒性休克動(dòng)物的生存時(shí)間。此外，最近的研究還發(fā)現(xiàn)，丙戊酸鈉能夠抑制多種細(xì)胞間緊密連接（tight junction）蛋白的降解，，保護(hù)細(xì)胞屏障，減輕大鼠脊髓損傷或腦缺血-再灌注損傷后血腦和血脊髓屏障的通透性和組織水腫。本論文旨在研究和分析丙戊酸鈉對(duì)嚴(yán)重?zé)齻竽c屏障的保護(hù)作用及其機(jī)制。 方法 1.建立大鼠55%TBSAⅢ度燙傷模型并隨機(jī)分為假燙+鹽水組（Sham+NS）、假燙+丙戊酸鈉組（Sham+VPA）、燙傷+鹽水組（Scald+NS）、燙傷+丙戊酸鈉組（Scald+VPA）。大鼠燙傷后即刻給予皮下注射丙戊酸鈉（300mg/Kg）或等體積的生理鹽水，于傷后2小時(shí)、6小時(shí)采用異硫氰酸熒光素標(biāo)記葡聚糖檢測(cè)腸上皮屏障的通透性變化；根據(jù)Chiu提出的腸屏障損害評(píng)分系統(tǒng)評(píng)價(jià)腸粘膜組織形態(tài)學(xué)變化；利用免疫熒光和（或）免疫印跡技術(shù)檢測(cè)腸組織中組蛋白H3第9位賴氨酸的乙�；ˋc-H3K9）水平以及HIF-1α、帶狀閉合蛋白1（zonaoccludens-1，ZO-1）、MLCK蛋白表達(dá)的變化；用酶聯(lián)免疫吸附法檢測(cè)腸組織VEGF蛋白表達(dá)的變化。 2.建立體外培養(yǎng)的單層Caco2腸上皮細(xì)胞模型，應(yīng)用缺氧模擬劑氯化鈷（1mM）刺激腸上皮細(xì)胞24小時(shí)，觀察丙戊酸鈉（2mM）對(duì)HIF-1α、VEGF、MLCK及ZO-1蛋白表達(dá)的影響；通過轉(zhuǎn)染針對(duì)HIF-1α的siRNA特異性地抑制HIF-1α的表達(dá)，研究HIF-1α對(duì)VEGF、MLCK及ZO-1的影響，并分析丙戊酸鈉保護(hù)腸上皮屏障功能的具體機(jī)制。 結(jié)果 1.燙傷+鹽水組大鼠傷后2小時(shí)腸屏障通透性、Chiu’s腸屏障損害評(píng)分、HIF-1α、VEGF及MCLK蛋白表達(dá)均明顯增加，分別為假燙+鹽水組的2.61倍、7.76倍、3.88倍、1.94倍和1.71倍（P均小于0.05）；而Ac-H3K9及ZO-1蛋白表達(dá)則明顯減少，僅為燙傷+鹽水組的0.73倍和0.65倍（P均小于0.05）；上述變化在傷后6小時(shí)更為明顯，燙傷+鹽水組上述指標(biāo)分別為假燙+鹽水組的4.80倍、17.33倍、4.42倍、2.43倍、2.80倍、0.48倍和0.43倍（P均小于0.05）。 2.給予丙戊酸鈉治療后，大鼠燙傷后2小時(shí)腸粘膜HIF-1α的蛋白水平即開始降低，而Ac-H3K9則有所升高，這些變化在傷后6小時(shí)更為明顯（P均小于0.05）。丙戊酸鈉治療還能下調(diào)大鼠傷后6小時(shí)腸粘膜VEGF、MLCK的蛋白水平，同時(shí)抑制ZO-1降解，降低腸屏障通透性并減輕腸粘膜損害（P均小于0.05）。 3.氯化鈷刺激24h后，腸上皮細(xì)胞HIF-1α、VEGF及MLCK的蛋白水平明顯升高，而ZO-1蛋白水平則顯著降低，分別為正常對(duì)照組的5.84倍、9.97倍，3.49倍和0.42倍（P均小于0.05）；丙戊酸鈉治療能顯著降低腸上皮細(xì)胞HIF-1α、VEGF及MLCK的蛋白表達(dá)，同時(shí)增加ZO-1的蛋白水平（P均小于0.05）。 4.采用siRNA干擾技術(shù)特異性抑制HIF-1α能明顯減少氯化鈷刺激后腸上皮細(xì)胞VEGF及MLCK的蛋白表達(dá)及ZO-1蛋白的降解（P均小于0.05）。 結(jié)論 嚴(yán)重?zé)齻竽c粘膜組織組蛋白乙酰化及ZO-1水平降低，并引起腸屏障功能損害；丙戊酸鈉能提高組蛋白乙�；癦O-1水平，保護(hù)腸上皮屏障功能，其作用機(jī)制與丙戊酸鈉對(duì)HIF-1α及其下游靶基因VEGF和MLCK的抑制作用有關(guān)。
[Abstract]:objective
Intestinal mucosal barrier dysfunction is a common complication after severe burns, is an important risk induced by sepsis and multiple organ dysfunction caused by severe burn factors. Specific mechanism of intestinal mucosal barrier dysfunction is not entirely clear, but more and more studies suggest that hypoxia inducible factor 1 (hypoxia-inducible, factor-1, HIF-1) and its downstream target genes vascular endothelial growth factor (vascular endothelial, growth factor, VEGF) and myosin light chain kinase (myosinlight chain, kinase, MLCK) plays a key role in the expression and function in hypoxia induced cell damage between the barrier. Recent studies show that histone deacetylase inhibitors (histone, deacetylaseinhibitor, HDACI) can enhance the cell of sodium valproate hypoxia, inflammation and other attack tolerance, protect the important viscera function, prolong blood loss, burn and sepsis Toxic shock animal survival time. In addition, recent studies also found that sodium valproate can inhibit a variety of intercellular tight junctions (tight junction) protein degradation, cell protection barrier, reduce rat spinal cord injury or cerebral ischemia reperfusion injury after cerebral blood and blood spinal cord barrier permeability and tissue edema. The aim of this paper is to protect the role of the research and analysis of sodium valproate on intestinal barrier and its mechanism.
Method
1. establishment of rat 55%TBSA model and scald were randomly divided into sham + saline group (Sham+NS), sham group (Sham+VPA), sodium valproate + saline group (Scald+NS), scald + scald + sodium valproate group (Scald+VPA). The scalded rats given immediately after subcutaneous injection of sodium valproate (300mg /Kg) or saline etc. the volume, in 2 hours after injury, 6 hours by the permeability of fluorescein isothiocyanate dextran for detecting intestinal epithelial barrier; intestinal barrier damage according to the Chiu's scoring system to evaluate the morphological changes of intestinal mucosa by immunofluorescence; and (or) acetylation detected by immunoblotting technique in intestinal tissue of histone H3 lysine ninth acid (Ac-H3K9) and the level of HIF-1 alpha, zona occluden 1 (zonaoccludens-1, ZO-1), the changes of the expression of MLCK protein; adsorption changes in expression of VEGF protein was detected in intestinal tissue by ELISA.
Caco2 intestinal epithelial cell monolayer model 2. in vitro culture, application of hypoxia mimetic cobalt chloride (1mM) stimulation of intestinal epithelial cells 24 hours, observation of sodium valproate (2mM) on VEGF, HIF-1 alpha, the expression of MLCK and ZO-1 protein expression in HIF-1; alpha siRNA specifically inhibited by transfection of HIF-1 alpha. HIF-1 alpha on VEGF, MLCK and ZO-1, and analyze the specific mechanism of sodium valproate protect intestinal epithelial barrier function.
Result
1. scald + saline group rats 2 hours after injury of intestinal barrier permeability, intestinal barrier damage score Chiu 's, HIF-1 alpha, VEGF expression and MCLK protein were significantly increased, respectively 2.61 times, sham + saline group 7.76 times, 3.88 times, 1.94 times and 1.71 times (P < 0.05); the expression of Ac-H3K9 and ZO-1 protein were significantly reduced, only 0.73 times the scald + saline group and 0.65 times (P < 0.05); the change in 6 hours after injury is more obvious, 4.80 times, scald + saline group the indicators were sham + saline group 17.33 times, 4.42 times, 2.43 times, 2.80 times, 0.48 times and 0.43 times (P < 0.05).
2. VPA treated rats after burn 2 hours of intestinal mucosal protein level of HIF-1 alpha began to decrease, while Ac-H3K9 increased, the changes in 6 hours after injury was more significant (P < 0.05). Sodium valproate in the treatment of rats can cut 6 hours after injury of intestinal mucosal VEGF and protein levels of MLCK at the same time, the inhibition of ZO-1 degradation, reduce intestinal barrier permeability and reduce the damage of the intestinal mucosa (P < 0.05).
3. cobalt chloride after 24h stimulation of intestinal epithelial cells HIF-1 alpha protein levels of VEGF and MLCK increased significantly, while the level of ZO-1 protein decreased significantly, respectively 5.84 times, 9.97 times of the normal control group, 3.49 times and 0.42 times (P < 0.05); sodium valproate treatment can significantly reduce the intestinal epithelial cells HIF-1 a, the expression of VEGF and MLCK protein also increased the protein level of ZO-1 (P < 0.05).
4., using siRNA interference technology to specifically inhibit HIF-1 alpha can significantly reduce VEGF and MLCK protein expression and ZO-1 protein degradation in intestinal epithelial cells after CO chloride stimulation (P is less than 0.05).
conclusion
Intestinal mucosa histone acetylation and the level of ZO-1 decreased, and cause the impairment of intestinal barrier function; sodium valproate can improve the histone acetylation and the level of ZO-1, protect the intestinal epithelial barrier function, the inhibitory effect and its mechanism of sodium valproate on HIF-1 alpha and its downstream target genes VEGF and MLCK.

【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R644

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本文編號(hào)：1568081