發(fā)布時(shí)間：2018-05-09 14:48

  本文選題：類風(fēng)濕關(guān)節(jié)炎 + 食物抗原�。� 參考：《鄭州大學(xué)》2011年碩士論文

【摘要】：類風(fēng)濕關(guān)節(jié)炎(rheumatoid arthritis, RA)是一種以慢性、進(jìn)行性、侵襲性關(guān)節(jié)炎為主要表現(xiàn)的全身性自身免疫疾病。如果不經(jīng)過正規(guī)治療,患者病情會(huì)逐漸發(fā)展,最終導(dǎo)致關(guān)節(jié)畸形、功能喪失。RA具有很高的致殘率,嚴(yán)重影響患者的健康和生活質(zhì)量,同時(shí)也給患者家庭和社會(huì)造成巨大的經(jīng)濟(jì)負(fù)擔(dān)。 眾多研究已表明食物與RA的發(fā)生密切相關(guān)。目前通過皮下注射Ⅱ型膠原蛋白誘導(dǎo)的RA模型是最佳的RA實(shí)驗(yàn)動(dòng)物模型。盡管通過注射的方式能成功有效地誘導(dǎo)大鼠產(chǎn)生類風(fēng)濕關(guān)節(jié)炎,但皮下注射的誘導(dǎo)方式并不符合人體攝食模式,其忽略了胃腸道在RA發(fā)病中的重要性,使RA發(fā)病機(jī)制的研究出現(xiàn)了一個(gè)極為重要的空白點(diǎn)：即食物抗原在人體攝食過程中如何通過腸上皮粘膜屏障進(jìn)入腸道粘膜固有層,引起腸道相關(guān)免疫組織細(xì)胞的一系列免疫應(yīng)答,擾亂腸粘膜免疫組織的固有與適應(yīng)性免疫反應(yīng),并通過分子擬態(tài)和旁觀者效應(yīng)打破免疫耐受,進(jìn)而導(dǎo)致RA發(fā)生的機(jī)理。為了深入RA發(fā)病機(jī)制的研究,建立一種與人RA發(fā)病機(jī)制及病理相同或相似的動(dòng)物模型(口服食物抗原誘導(dǎo)的RA動(dòng)物模型)在RA研究中的作用也就顯得尤為重要。 目的： 建立口服牛Ⅱ型膠原蛋白誘導(dǎo)的RA大鼠模型；確定口服食物抗原與RA的相關(guān)性；并在動(dòng)物模型的基礎(chǔ)上,對(duì)腸道通透性在食物抗原誘導(dǎo)的RA發(fā)病機(jī)理中的作用進(jìn)行初步探索,為臨床RA的預(yù)防和治療提供理論依據(jù)。 方法： 本研究選擇Wistar大鼠作為實(shí)驗(yàn)動(dòng)物。從牛軟骨中提取牛Ⅱ型膠原蛋白,先用阿司匹林破壞大鼠的腸道粘膜屏障功能,然后灌胃給予大鼠較大劑量的牛Ⅱ型膠原蛋白,誘導(dǎo)大鼠類風(fēng)濕關(guān)節(jié)炎的發(fā)生。將4-5周齡雌性Wistar大鼠隨機(jī)分成四組：對(duì)照組(生理鹽水；生理鹽水)、M1組(生理鹽水；生理鹽水+CC14+LPS)、M2組(5mg/kg阿司匹林；牛Ⅱ型膠原蛋白+CC14+LPS)、M3組(10mg/kg阿司匹林；牛Ⅱ型膠原蛋白+CC14+LPS)。在阿司匹林灌胃期間,每七天灌胃給予大鼠一定量的乳果糖和甘露醇,用代謝籠收集大鼠24h尿,高效液相色譜儀(HPLC)檢測(cè)并計(jì)算乳果糖、甘露醇比值(L/M),并將M2組、M3組與對(duì)照組相比,間接評(píng)價(jià)大鼠腸道通透性的變化情況。建模過程中,用游標(biāo)卡尺測(cè)量并記錄大鼠右后腿踝關(guān)節(jié)的直徑,兩周一次,一共5次,并進(jìn)行關(guān)節(jié)指數(shù)(AI)評(píng)定。建模結(jié)束后,取大鼠血清、空腸組織和踝關(guān)節(jié)組織。采用雙抗體夾心ELISA法測(cè)定各組大鼠血清中牛Ⅱ型膠原蛋白特異性抗體水平、細(xì)胞因子TNF-a、IFN-r和IL-4水平；將所取實(shí)驗(yàn)動(dòng)物的空腸和踝關(guān)節(jié)組織用固定液固定后,石蠟包埋切片,蘇木素-伊紅(HE)染色,光學(xué)顯微鏡下觀察其病理形態(tài)結(jié)構(gòu)的變化以及淋巴細(xì)胞浸潤(rùn)滲透的情況；用熒光免疫組織化學(xué)的方法,以小鼠來(lái)源的牛Ⅱ型膠原蛋白單克隆抗體作為一抗,以FITC標(biāo)記的山羊抗小鼠的IgG作為二抗,定位空腸中的牛Ⅱ型膠原蛋白,觀察其是否能夠通過腸粘膜的屏障從而進(jìn)入腸道粘膜固有層；用熒光免疫組織化學(xué)的方法,以小鼠來(lái)源的牛Ⅱ型膠原蛋白單克隆抗體作為一抗,以FITC標(biāo)記的山羊抗小鼠的IgG作為二抗,定位關(guān)節(jié)滑膜上的牛Ⅱ型膠原蛋白,觀察其在關(guān)節(jié)滑膜上的沉積情況。通過以上指標(biāo)對(duì)所建立的類風(fēng)濕關(guān)節(jié)炎大鼠模型進(jìn)行綜合評(píng)價(jià)。 結(jié)果： 在建模結(jié)束后,M2組、M3組大鼠踝關(guān)節(jié)呈現(xiàn)不同程度的腫脹；M2組、M3組大鼠血清中抗牛Ⅱ型膠原蛋白特異性抗體顯著升高(P0.05),血清中TNF-a和IFN-r水平明顯升高(P0.05),并且IL-4水平明顯降低(P0.05)；M2組、M3組大鼠空腸病理學(xué)改變明顯：腸粘膜損傷嚴(yán)重,粘膜下層中性粒細(xì)胞浸潤(rùn)明顯,腸絨毛水腫肥大,腸粘膜上皮細(xì)胞廣泛脫落等；熒光免疫組化顯示未經(jīng)消化的大分子牛Ⅱ型膠原蛋白透過損傷的腸上皮粘膜屏障進(jìn)入腸道粘膜固有層；在M2組、M3組大鼠踝關(guān)節(jié)標(biāo)本組織學(xué)中觀察到滑膜組織增生,其局部骨吸收破壞明顯,軟骨表層膠原纖維松解斷裂、軟骨細(xì)胞變性壞死,軟骨表面凸凹不平等；并通過熒光免疫組化的方法觀察到M2組、M3組大鼠關(guān)節(jié)滑膜上牛Ⅱ型膠原蛋白的沉積。雙糖實(shí)驗(yàn),M2組、M3組大鼠尿中的甘露醇濃度降低(P0.05),乳果糖濃度升高(P0.01),L/M升高(P0.05),說(shuō)明M2組、M3組大鼠的腸道通透性增加。 結(jié)論： 在腸黏膜損傷的條件下,口服牛Ⅱ型膠原蛋白可成功誘導(dǎo)大鼠產(chǎn)生類風(fēng)濕關(guān)節(jié)炎。
[Abstract]:Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic, progressive and invasive arthritis. Without regular treatment, the patient's condition will gradually develop, eventually lead to joint deformity, and the loss of function.RA has a high rate of disability, which seriously affects the health and quality of life of the patients. It also brings enormous financial burden to the family and society.
Many studies have shown that food is closely related to the occurrence of RA. The RA model induced by subcutaneous injection of type II collagen is the best model of the RA experimental animal. Although the injection can effectively induce the rat to produce rheumatoid arthritis, the induction mode of subcutaneous injection does not conform to the model of human feeding, which is ignored. The importance of the gastrointestinal tract in the pathogenesis of RA leads to a very important blank point in the study of the pathogenesis of RA: how food antigens enter the lamina of the intestinal mucosa through the intestinal mucosal barrier during the feeding process of the human body, causing a series of immune responses to the intestinal related immune tissue cells and disturbing the intestinal mucosal immune tissues. Inherent and adaptive immune responses, and break the immune tolerance through molecular mimicry and bystander effect, lead to the mechanism of the occurrence of RA. In order to study the pathogenesis of RA, the establishment of an animal model which is similar or similar to the pathogenesis and pathology of human RA (the RA animal model induced by oral food antigen) is also useful in the study of RA. It is particularly important.
Objective:
The RA rat model induced by oral bovine type II collagen was established, and the correlation between oral food antigen and RA was determined. On the basis of animal model, the role of intestinal permeability in the pathogenesis of RA induced by food antigen was preliminarily explored to provide a theoretical basis for the prevention and treatment of clinical RA.
Method錛，

本文編號(hào)：1866434