【摘要】：背景隨著人們生活水平的提高以及一些綜合因素的作用,糖尿病的發(fā)病率也逐漸上升,并趨向年輕化,與此同時其并發(fā)癥的發(fā)病率也逐年升高。作為糖尿病患者最常見和最嚴(yán)重的并發(fā)癥之一,糖尿病性視網(wǎng)膜病變(diabetic retinopathy,DR)隨著糖尿病病程的延長,如果發(fā)展到視網(wǎng)膜病變的后期即增殖型糖尿病視網(wǎng)膜病變期,將會對視力造成不可逆轉(zhuǎn)的損害。糖尿病性視網(wǎng)膜病變已嚴(yán)重影響了人們的生活質(zhì)量,但目前其發(fā)病機(jī)制并不完全明確,糖尿病性視網(wǎng)膜病變的主要病理改變是由于高血糖引起視網(wǎng)膜毛細(xì)血管滲漏造成視網(wǎng)膜水腫,繼而引起視網(wǎng)膜組織缺血缺氧,最終引發(fā)新生血管的生成。目前糖尿病性視網(wǎng)膜病變中研究較多的是視網(wǎng)膜,研究發(fā)現(xiàn)視網(wǎng)膜水腫、缺血和缺氧等條件均會引起視網(wǎng)膜內(nèi)水通道蛋白4(aquaporin4,AQP4)表達(dá)水平的改變,而近年的研究發(fā)現(xiàn)糖尿病性視網(wǎng)膜病變患者血清中水通道蛋白的水平也發(fā)生改變,但玻璃體內(nèi)水通道蛋白4的表達(dá)水平如何目前尚不明確。目的建立檢測糖尿病性視網(wǎng)膜病變患者玻璃體內(nèi)水通道蛋白4抗原及抗體的酶聯(lián)免疫檢測方法,并用本試驗建立的方法分析糖尿病性視網(wǎng)膜病變患者玻璃體內(nèi)AQP4表達(dá)水平的變化。方法本研究以兔抗人水通道蛋白4抗體為包被抗體,羊抗兔Ig G-HRP為酶標(biāo)抗體,分別建立檢測人眼玻璃體內(nèi)水通道蛋白4抗原抗體的ELISA檢測方法;并對兩種方法的工作條件進(jìn)行優(yōu)化,包括抗體的工作濃度,封閉溫度和時間,孵育溫度和時間,顯色溫度和時間等。隨后對兩種方法的線性、檢測范圍、最低檢測限以及重復(fù)性等性能進(jìn)行評價。最后用這兩種方法分別測定正常人和糖尿病性視網(wǎng)膜病變患者玻璃體內(nèi)水通道蛋白4抗原抗體的水平,統(tǒng)計學(xué)方法分析正常人和糖尿病性視網(wǎng)膜病變患者玻璃體內(nèi)水通道蛋白4抗原抗體表達(dá)水平的差異。結(jié)果初步建立了檢測人眼玻璃體內(nèi)水通道蛋白4抗原的雙抗體夾心ELISA方法和檢測水通道蛋白4抗體的競爭ELISA方法。雙抗體夾心ELISA的工作條件:最佳包被抗體濃度為2.0μg/ml;最佳封閉溫度和時間為37℃封閉1h,夾心一抗最佳工作濃度為1:800,最佳孵育溫度和時間為37℃1h,酶標(biāo)抗體最適濃度為1:3500,最佳顯色條件為時間為37℃顯色15min。標(biāo)準(zhǔn)曲線y=0.0178x+0.1937,決定系數(shù)R2=0.9938,線性范圍為3.125-100ng/ml,最低檢測限為3.125ng/ml,3次批內(nèi)CV%分別為3.80%、4.68%、4.18%,批間CV%為3.01%。競爭法ELISA工作條件:包被抗體和酶標(biāo)抗體的最適濃度分別為1:500和1:5000,最佳封閉溫度和時間為37℃封閉1h,夾心一抗的最佳稀釋度為1:400,實際工作濃度是1:800,最佳孵育條件為37℃孵育1h,最佳顯色條件為37℃顯色15min。標(biāo)準(zhǔn)曲線y=-0.3937lg X+1.4471,決定系數(shù)R2=0.9893,線性范圍為2.5-80ng/ml,最低檢測限為2.5 ng/ml,三批試驗測得的批內(nèi)CV%分別為2.41%,2.60%,1.60%,批間CV%為2.49%。兩種ELISA方法的批內(nèi)變異系數(shù)均小于5%,批間CV%小于10%。正常人與糖尿病性視網(wǎng)膜病變患者玻璃體內(nèi)水通道蛋白4抗原水平比較有顯著性差異(P0.05),而兩組間的抗體水平比較也有統(tǒng)計學(xué)意義(P0.05)。結(jié)論1初步建立了檢測人眼玻璃體內(nèi)AQP4抗原的雙抗體夾心ELISA檢測方法。2初步建立了檢測人眼玻璃體內(nèi)AQP4抗體的競爭法ELISA檢測方法。3糖尿病性視網(wǎng)膜病變患者玻璃體內(nèi)AQP4抗原及抗體的表達(dá)水平均上調(diào)。
[Abstract]:Background With the improvement of people's living standard and the effect of some comprehensive factors, the incidence of diabetes is increasing gradually, and the incidence of complications is increasing year by year. As one of the most common and most serious complications of diabetes mellitus, diabetic retinopathy (DR) increases with the course of diabetes, and will cause irreversible damage to vision if it develops to the late stage of retinopathy, namely proliferative diabetic retinopathy. Diabetic retinopathy has seriously affected people's quality of life, but the pathogenesis of diabetic retinopathy is not completely clear, and the main pathological changes of diabetic retinopathy are retinal edema due to blood capillary leakage caused by hyperglycemia, In turn causes retinal tissue ischemia and hypoxia, resulting in the formation of new blood vessels. At present, there are many studies in diabetic retinopathy, and studies have found that retinal edema, ischemia and hypoxia can cause changes in the expression level of aquaporin 4 (AQP4) in the retina. In recent years, it has been found that the level of water channel protein in serum of diabetic retinopathy is also changed, but the expression level of water channel protein 4 in the vitreous body is unclear at present. Objective To establish an enzyme-linked immunosorbent assay (ELISA) for detecting aquaporin 4 antigen and antibody in vitreous of diabetic retinopathy patients, and to analyze the changes of AQP4 expression level in diabetic retinopathy patients. Methods Using rabbit anti-human aquaporin 4 antibody as coated antibody, goat anti-rabbit Ig G-HRP as enzyme-labeled antibody, we established ELISA detection method for detecting water channel protein 4 antigen antibody in human eye glass body, and optimized the working conditions of two methods, including the working concentration of antibody. Close temperature and time, incubation temperature and time, color temperature and time, etc. The linearity, detection range, minimum detection limit and repeatability of the two methods were evaluated. Finally, the levels of water channel protein 4 antigen in the vitreous body of normal and diabetic retinopathy patients were determined by the two methods. The difference of the expression level of water channel protein 4 antigen was analyzed in normal subjects and diabetic retinopathy patients. Results The double antibody sandwich ELISA for detecting the water channel protein 4 antigen in human eyes was established and the competitive ELISA for detecting the water channel protein 4 antibody was established. The optimal concentration of sandwich antibody was 1: 800, the optimal incubation temperature and time was 37 鈩，

本文編號：2289396