共聚焦激光显微内镜在体评估NSAIDs相关胃黏膜损伤及其药物干预效果

发布时间：2018-05-30 21:37

  本文选题：共聚焦激光显微内镜(CLE) + NSAIDs相关胃黏膜损伤　； 参考：《山东大学》2014年硕士论文

【摘要】：研究背景 非甾体类抗炎药(NSAIDs)因其抗炎、抗风湿、解热、镇痛作用广泛用于临床,然而NSAIDs广泛应用已逐渐成为消化道黏膜损伤的主要致病因素之一。NSAIDs可通过抑制环氧化酶(COX)从而导致前列腺素E2(PGE2)合成减少以及其他不依赖于COX的机制引起消化道黏膜的损伤,因此NSIADs相关胃黏膜损伤已成为目前迫切需要解决的临床难题,明确发病机制对其预防与治疗至关重要,然而目前尚缺乏对NSAIDs相关胃黏膜损伤黏膜屏障功能障碍的研究。 雷贝拉唑与替普瑞酮分别是临床常用的质子泵抑制剂与胃肠黏膜保护剂,两者被广泛应用于包括NSAIDs相关胃黏膜损伤在内的消化道黏膜损伤性病变的治疗与预防当中。目前研究已表明,两者可通过提高黏膜COX与PGE2水平,增加黏膜表面黏液的合成等多种途径对NSAIDs相关胃黏膜损伤起保护作用。但两药对NSAIDs相关胃黏膜损伤的保护机制尚缺乏深入研究,尤其两药物在NSAIDs相关胃黏膜损伤中改善胃黏膜屏障功能的作用机制仍需进一步证实及完善。 共聚焦激光显微内镜(confocal laser endomicroscopy, CLE)可以在活体实时大面积评价组织的形态学改变,同时可以观察细胞坏死与脱落过程,通过荧光素渗漏情况判断细胞活性及通透性,从而判断组织损伤程度,使得我们对消化道黏膜损伤及通透性的评价有了更准确,更全面及客观的评价方法。共聚焦激光显微内镜在NSAIDs相关胃黏膜损伤中的应用价值尚缺乏验证。 目的 本研究旨在通过应用共聚焦激光显微内镜在体观察NSAIDs相关胃黏膜损伤中黏膜通透性的改变以评价黏膜屏障功能障碍。同时结合在体与体外实验方法,深入探索NSAIDs相关胃黏膜损伤的形态学与分子机制,并在此方法下探索替普瑞酮和雷贝拉唑对急性胃黏膜损伤中黏膜屏障功能的保护作用。 方法 1动物模型制备：成年雄性Wistar大鼠96只随机分为8组：正常组、模型组、替普瑞酮预防组、雷贝拉唑预防组、空白治疗组、替普瑞酮治疗组、雷贝拉唑治疗组、联合治疗组进行动物模型制各。 2胃黏膜肉眼损伤评估：按照Guth标准在10倍放大镜下评估各组胃黏膜损伤情况并计算溃疡指数。 3Optiscan FIVE1共聚焦激光显微内镜评估：应用FIVE1共聚焦显微内镜系统在体对各组胃黏膜形态学进行观察评估,建立NSAIDs相关胃黏膜损伤共聚焦显微内镜评分系统。 4常规HE染色评估各组胃黏膜组织学改变并进行评估,建立与共聚焦显微内镜相对应的NSAIDs相关胃病组织学评分方法。同时免疫组化方法检测各组大鼠胃黏膜上皮紧密连接蛋白occludin与ZO-1的表达水平。 5扫描电子显微镜下观察胃黏膜表面超微结构变化,建立与共聚焦显微内镜相对应的NSAIDs相关胃病扫描电镜评分方法。 6酶联免疫吸附试验检测各组大鼠血清TNF-α的表达。 7Western blot方法检测胃黏膜上皮细胞NF-κB与caspase-3的表达。 8统计学分析：采用SPSS13.0软件进行统计分析,数据以均数±标准差(mean±SD)表示,多样本之间比较检验方差齐性,方差齐性时采用多样本方差分析,不齐时用非参数秩和检验,均以双侧P0.05为有统计学意义。 结果： 1根据胃黏膜损伤严重程度的不同,建立了4级NSAIDs相关胃黏膜损伤共聚焦显微内镜评分系统。 2共聚焦显微内镜黏膜损伤评分与组织学评分及肉眼下黏膜损伤评分具有良好的一致性。NSAIDs相关胃黏膜损伤模型组黏膜损伤程度明显重于正常对照组,其共聚焦内镜评分、组织学评分及扫描电镜评分均明显高于正常对照组,而替普瑞酮与雷贝拉唑预防组与治疗组评分分别较模型组与空白治疗组降低。 3NSAIDs相关胃黏膜损伤模型组TNF-α水平明显高于正常对照组,而替普瑞酮与雷贝拉唑预防组及治疗组TNF-α的表达较对照组相比明显降低。 4NSAIDs相关胃黏膜损伤模型组胃黏膜上皮细胞caspase-3和NK-κB水平明显高于正常对照组,替普瑞酮与雷贝拉唑预防组及治疗后,胃黏膜上皮caspase-3和NK-κB水平较模型组及治疗对照组明显降低。 5NSAIDs胃黏膜损伤后,紧密连接蛋白ZO-1与occludin表达明显减少,同时存在分布不均,而替普瑞酮与雷贝拉唑的应用后,可见occludin与ZO-1表达增多。 结论： 1CLE可以客观、实时、准确地评价NSAIDS相关胃黏膜损伤中黏膜上皮细胞损伤及黏膜通透性的改变。 2NSAIDs可通过上调TNF-α增加,继而经caspase-3和NK-κB途径引起胃黏膜上皮细胞紧密连接蛋白的表达异常,从而导致细胞坏死及黏膜通透性增加。 3替普瑞酮与雷贝拉唑可通过抑制TNF-α、 caspase-3和NK-κB的表达上调,同时促进紧密连接蛋白的表达从而维持黏膜屏障功能的完整性,成为NSAIDs相关胃黏膜损伤性疾病有效的预防与治疗药物。
[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锛，

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