發(fā)布時(shí)間：2018-05-30 21:37

  本文選題：共聚焦激光顯微內(nèi)鏡(CLE) + NSAIDs相關(guān)胃黏膜損傷�。� 參考：《山東大學(xué)》2014年碩士論文

【摘要】：研究背景 非甾體類(lèi)抗炎藥(NSAIDs)因其抗炎、抗風(fēng)濕、解熱、鎮(zhèn)痛作用廣泛用于臨床,然而NSAIDs廣泛應(yīng)用已逐漸成為消化道黏膜損傷的主要致病因素之一。NSAIDs可通過(guò)抑制環(huán)氧化酶(COX)從而導(dǎo)致前列腺素E2(PGE2)合成減少以及其他不依賴(lài)于COX的機(jī)制引起消化道黏膜的損傷,因此NSIADs相關(guān)胃黏膜損傷已成為目前迫切需要解決的臨床難題,明確發(fā)病機(jī)制對(duì)其預(yù)防與治療至關(guān)重要,然而目前尚缺乏對(duì)NSAIDs相關(guān)胃黏膜損傷黏膜屏障功能障礙的研究。 雷貝拉唑與替普瑞酮分別是臨床常用的質(zhì)子泵抑制劑與胃腸黏膜保護(hù)劑,兩者被廣泛應(yīng)用于包括NSAIDs相關(guān)胃黏膜損傷在內(nèi)的消化道黏膜損傷性病變的治療與預(yù)防當(dāng)中。目前研究已表明,兩者可通過(guò)提高黏膜COX與PGE2水平,增加黏膜表面黏液的合成等多種途徑對(duì)NSAIDs相關(guān)胃黏膜損傷起保護(hù)作用。但兩藥對(duì)NSAIDs相關(guān)胃黏膜損傷的保護(hù)機(jī)制尚缺乏深入研究,尤其兩藥物在NSAIDs相關(guān)胃黏膜損傷中改善胃黏膜屏障功能的作用機(jī)制仍需進(jìn)一步證實(shí)及完善。 共聚焦激光顯微內(nèi)鏡(confocal laser endomicroscopy, CLE)可以在活體實(shí)時(shí)大面積評(píng)價(jià)組織的形態(tài)學(xué)改變,同時(shí)可以觀(guān)察細(xì)胞壞死與脫落過(guò)程,通過(guò)熒光素滲漏情況判斷細(xì)胞活性及通透性,從而判斷組織損傷程度,使得我們對(duì)消化道黏膜損傷及通透性的評(píng)價(jià)有了更準(zhǔn)確,更全面及客觀(guān)的評(píng)價(jià)方法。共聚焦激光顯微內(nèi)鏡在NSAIDs相關(guān)胃黏膜損傷中的應(yīng)用價(jià)值尚缺乏驗(yàn)證。 目的 本研究旨在通過(guò)應(yīng)用共聚焦激光顯微內(nèi)鏡在體觀(guān)察NSAIDs相關(guān)胃黏膜損傷中黏膜通透性的改變以評(píng)價(jià)黏膜屏障功能障礙。同時(shí)結(jié)合在體與體外實(shí)驗(yàn)方法,深入探索NSAIDs相關(guān)胃黏膜損傷的形態(tài)學(xué)與分子機(jī)制,并在此方法下探索替普瑞酮和雷貝拉唑?qū)�急性胃黏膜損傷中黏膜屏障功能的保護(hù)作用。 方法 1動(dòng)物模型制備：成年雄性Wistar大鼠96只隨機(jī)分為8組：正常組、模型組、替普瑞酮預(yù)防組、雷貝拉唑預(yù)防組、空白治療組、替普瑞酮治療組、雷貝拉唑治療組、聯(lián)合治療組進(jìn)行動(dòng)物模型制各。 2胃黏膜肉眼損傷評(píng)估：按照Guth標(biāo)準(zhǔn)在10倍放大鏡下評(píng)估各組胃黏膜損傷情況并計(jì)算潰瘍指數(shù)。 3Optiscan FIVE1共聚焦激光顯微內(nèi)鏡評(píng)估：應(yīng)用FIVE1共聚焦顯微內(nèi)鏡系統(tǒng)在體對(duì)各組胃黏膜形態(tài)學(xué)進(jìn)行觀(guān)察評(píng)估,建立NSAIDs相關(guān)胃黏膜損傷共聚焦顯微內(nèi)鏡評(píng)分系統(tǒng)。 4常規(guī)HE染色評(píng)估各組胃黏膜組織學(xué)改變并進(jìn)行評(píng)估,建立與共聚焦顯微內(nèi)鏡相對(duì)應(yīng)的NSAIDs相關(guān)胃病組織學(xué)評(píng)分方法。同時(shí)免疫組化方法檢測(cè)各組大鼠胃黏膜上皮緊密連接蛋白o(hù)ccludin與ZO-1的表達(dá)水平。 5掃描電子顯微鏡下觀(guān)察胃黏膜表面超微結(jié)構(gòu)變化,建立與共聚焦顯微內(nèi)鏡相對(duì)應(yīng)的NSAIDs相關(guān)胃病掃描電鏡評(píng)分方法。 6酶聯(lián)免疫吸附試驗(yàn)檢測(cè)各組大鼠血清TNF-α的表達(dá)。 7Western blot方法檢測(cè)胃黏膜上皮細(xì)胞NF-κB與caspase-3的表達(dá)。 8統(tǒng)計(jì)學(xué)分析：采用SPSS13.0軟件進(jìn)行統(tǒng)計(jì)分析,數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示,多樣本之間比較檢驗(yàn)方差齊性,方差齊性時(shí)采用多樣本方差分析,不齊時(shí)用非參數(shù)秩和檢驗(yàn),均以雙側(cè)P0.05為有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1根據(jù)胃黏膜損傷嚴(yán)重程度的不同,建立了4級(jí)NSAIDs相關(guān)胃黏膜損傷共聚焦顯微內(nèi)鏡評(píng)分系統(tǒng)。 2共聚焦顯微內(nèi)鏡黏膜損傷評(píng)分與組織學(xué)評(píng)分及肉眼下黏膜損傷評(píng)分具有良好的一致性。NSAIDs相關(guān)胃黏膜損傷模型組黏膜損傷程度明顯重于正常對(duì)照組,其共聚焦內(nèi)鏡評(píng)分、組織學(xué)評(píng)分及掃描電鏡評(píng)分均明顯高于正常對(duì)照組,而替普瑞酮與雷貝拉唑預(yù)防組與治療組評(píng)分分別較模型組與空白治療組降低。 3NSAIDs相關(guān)胃黏膜損傷模型組TNF-α水平明顯高于正常對(duì)照組,而替普瑞酮與雷貝拉唑預(yù)防組及治療組TNF-α的表達(dá)較對(duì)照組相比明顯降低。 4NSAIDs相關(guān)胃黏膜損傷模型組胃黏膜上皮細(xì)胞caspase-3和NK-κB水平明顯高于正常對(duì)照組,替普瑞酮與雷貝拉唑預(yù)防組及治療后,胃黏膜上皮caspase-3和NK-κB水平較模型組及治療對(duì)照組明顯降低。 5NSAIDs胃黏膜損傷后,緊密連接蛋白ZO-1與occludin表達(dá)明顯減少,同時(shí)存在分布不均,而替普瑞酮與雷貝拉唑的應(yīng)用后,可見(jiàn)occludin與ZO-1表達(dá)增多。 結(jié)論： 1CLE可以客觀(guān)、實(shí)時(shí)、準(zhǔn)確地評(píng)價(jià)NSAIDS相關(guān)胃黏膜損傷中黏膜上皮細(xì)胞損傷及黏膜通透性的改變。 2NSAIDs可通過(guò)上調(diào)TNF-α增加,繼而經(jīng)caspase-3和NK-κB途徑引起胃黏膜上皮細(xì)胞緊密連接蛋白的表達(dá)異常,從而導(dǎo)致細(xì)胞壞死及黏膜通透性增加。 3替普瑞酮與雷貝拉唑可通過(guò)抑制TNF-α、 caspase-3和NK-κB的表達(dá)上調(diào),同時(shí)促進(jìn)緊密連接蛋白的表達(dá)從而維持黏膜屏障功能的完整性,成為NSAIDs相關(guān)胃黏膜損傷性疾病有效的預(yù)防與治療藥物。
[Abstract]:Research background
Non steroidal anti-inflammatory drugs (NSAIDs) are widely used in clinical because of their anti-inflammatory, anti rheumatic, antipyretic and analgesic effects. However, the widespread use of NSAIDs has gradually become one of the main pathogenic factors for the damage of digestive tract mucosa,.NSAIDs can reduce the synthesis of prostatic E2 (PGE2) by inhibiting cyclooxygenase (COX) and other mechanisms that do not depend on COX. The mucosal lesion of the digestive tract is damaged, so the NSIADs related gastric mucosal injury has become an urgent problem to be solved. The clear pathogenesis is very important for the prevention and treatment of the gastric mucosa. However, there is still a lack of research on the dysfunction of the mucosal barrier of NSAIDs related gastric mucosa injury.
Rabeprazole and teprirone are commonly used proton pump inhibitors and gastrointestinal mucosal protectant, respectively. Both are widely used in the treatment and prevention of digestive tract mucosal damage, including NSAIDs related gastric mucosal damage. The present study has shown that both of them can increase the level of mucosal COX and PGE2 and increase the surface of mucous membrane. Many ways of mucus synthesis have protective effects on NSAIDs related gastric mucosal damage. However, the protective mechanism of two drugs for NSAIDs related gastric mucosal damage is still lacking in depth. Especially, the mechanism of improving the function of gastric mucosal barrier in NSAIDs related gastric mucosa injury still needs to be confirmed and improved by two drugs.
Confocal laser microendoscopy (confocal laser endomicroscopy, CLE) can evaluate the morphological changes in the tissue in real time in large area, and observe the cell necrosis and exfoliation process at the same time. The cell activity and permeability can be judged by fluorescein leakage, so as to judge the damage degree of the group and make the damage to the digestive tract mucosa and the damage of the digestive tract. The evaluation of permeability has a more accurate, more comprehensive and objective evaluation method. The value of confocal laser endoscopy in NSAIDs related gastric mucosal injury is still lacking.
objective
The purpose of this study is to evaluate the mucosal barrier dysfunction by observing the changes of mucosal permeability in NSAIDs related gastric mucosal injury by using confocal laser microendoscopy in vivo and to explore the morphological and sub mechanism of NSAIDs related gastric mucosal injury in vivo and in vitro, and to explore teprunone under this method. Protective effects of Lei Bella azole on mucosal barrier function in acute gastric mucosal injury.
Method
1 animal model preparation: 96 adult male Wistar rats were randomly divided into 8 groups: normal group, model group, tipreone prevention group, riprazole prevention group, blank treatment group, tipreone treatment group, riprazone treatment group and combined treatment group for animal model system.
2 gross injury assessment of gastric mucosa: according to the Guth standard, the gastric mucosal lesions were assessed under 10 magnifying lenses and the ulcer index was calculated.
3Optiscan FIVE1 confocal laser endoscopy evaluation: the morphology of gastric mucosa in each group was observed and evaluated by FIVE1 confocal microendoscopic system in vivo, and the confocal endoscopic scoring system of NSAIDs related gastric mucosal lesion was established.
4 routine HE staining was used to evaluate the histological changes of gastric mucosa in each group and to evaluate the histology of NSAIDs related gastropathy, which was corresponding to confocal microendoscopy. The expression of occludin and ZO-1 of gastric mucosa epithelium in each group was measured by immunohistochemistry.
5 scanning electron microscope (SEM) was used to observe the ultrastructural changes of the gastric mucosal surface. A scanning electron microscopic scoring system for NSAIDs related gastropathy corresponding to confocal microendoscopy was established.
6 enzyme linked immunosorbent assay (ELISA) was used to detect the expression of serum TNF- alpha in rats of each group.
7Western blot was used to detect the expression of NF- kappa B and Caspase-3 in gastric epithelial cells.
8 statistical analysis: the statistical analysis was carried out by SPSS13.0 software. The data were expressed in the mean number of standard deviation (mean + SD), and the variance was homogeneous. The multiple sample variance analysis was used in the homogeneity of variance. The non parametric rank sum test was used when the inhomogeneous was inhomogeneous, both of which were statistically significant with the bilateral P0.05.
Result錛，

本文編號(hào)：1956916