【摘要】：背景和目的:IgA腎病(IgA nephropathy, IgAN)是目前世界范圍內最常見的原發(fā)性腎小球疾病,是引起終末期腎衰竭的重要原因之一,30%～40%的IgAN患者在診斷該病后20年內發(fā)展成終末期腎病(end stage renal disease,ESRD),給家庭和社會帶來了沉重的負擔。IgAN的確診目前仍依賴于腎活檢,但腎穿刺活檢為有創(chuàng)性檢查,存在一定風險和禁忌證。IgAN起病隱匿,易導致診治不及時,因此早期篩查、診治對于防控,減少ESRD患者數量尤為重要。因此,探尋IgAN的發(fā)病機制和早期無創(chuàng)生物標志物具有重要意義。本研究采用Raybiotech細胞因子抗體芯片技術對IgAN患者和對照組腎臟組織進行檢測;采用高分辨質譜技術對IgAN患者、原發(fā)性膜性腎病(idiopathic membranous nephropathy,IMN)患者和正常人群尿液進行對照檢測分析;篩選IgAN的生物標志物,探討其應用價值,進一步闡明IgAN的發(fā)病機制。方法:(1)入選年齡18～60歲,解放軍總醫(yī)院住院腎活檢確診為IgAN的患者10例,取活檢腎組織進行研究;年齡18～60歲的于解放軍總醫(yī)院泌尿外科行腎癌腎臟切除的患者10例,取腎癌患者癌旁6cm以外的正常腎臟組織作為對照組進行研究。收集上述人群的臨床基本信息及化驗檢查結果。上述人群腎臟組織利用Raybiotech細胞因子抗體芯片進行檢測,篩選差異蛋白質,并對差異蛋白質進行GO(Gene Ontology)分析。結合分析結果,選取了 IL-33利用酶聯(lián)免疫吸附試驗(ELISA)進行驗證。(2)對解放軍總醫(yī)院腎科腎穿刺活檢患者進行篩選,入組20-60歲的48例診斷為IgANⅡ～Ⅲ級的患者和9例診斷為IMNⅠ～Ⅱ期的患者;另入組40例20-60歲尿常規(guī)、血常規(guī)及血生化結果無明顯異常且無基礎疾病的正常人作為健康對照。收集上述人群晨起第1次尿中段尿,通過Lable free LC/MS高分辨質譜技術對29例IgAN患者9例IMN患者和40例健康對照進行尿液蛋白質檢測,篩選差異蛋白質,對差異蛋白質進行GO富集分析;依據蛋白質譜和數據分析的結果,結合IgAN可能的發(fā)病機制,選取48例IgAN患者和40例正常人尿液利用酶聯(lián)免疫吸附試驗(ELISA)對轉鐵蛋白(transferrin,TF)進行驗證。結果:(1)對10例病理診斷為IgANⅡ~Ⅲ級、10例正常腎臟組織進行細胞因子表達譜檢測,共發(fā)現(xiàn)了 25個差異蛋白質,GO分析結果表明:這些蛋白質主要與應激、細胞粘附、蛋白質代謝、信號轉導相關。ELISA驗證結果顯示:IgAN患者腎組織中IL33表達水平顯著高于對照組。(2)利用高分辨質譜技術檢測發(fā)現(xiàn),與正常對照組相比對,在IgAN和IMN疾病組,檢測到53種蛋白質差異表達(p0.05);在IMN組中找到15種顯著差異表達但在IgAN組中無差異表達的蛋白質(p0.05);在IgAN組中找到13種差異表達但在IMN組中無差異表達的蛋白質(p0.05)。IMN組和IgAN組組間,找到了 9種蛋白質差異表達(p0.05)。GO富集發(fā)現(xiàn)這些差異蛋白質主要參與凝血與血栓形成、炎癥反應、補體系統(tǒng)激活、蛋白轉運、鐵代謝、糖及脂質代謝生物過程。差異蛋白C3、EGF、APOD等與以往研究結果相符,ORM1、ORM2可能是診斷IgAN和IMN的生物標志物。ELISA驗證結果顯示:IgAN患者尿液中TF表達水平顯著高于正常對照組,TF具有非常大的診斷價值。結論:IL-33可能是IgAN發(fā)病機制中起重要作用的細胞因子,TF可能是優(yōu)于24h尿蛋白定量診斷IgAN和IMN的早期生物標志物,ORM1、ORM2,尤其ORM2在IMN患者尿液中高表達可能是IMN區(qū)別于IgAN的重要標志物,CETP可能是診斷IgAN的生物標志物。
[Abstract]:Background and Objective: IgA nephropathy (IgAN) is one of the most common primary glomerular diseases in the world, which is one of the most important causes of end-stage renal failure. 30-40% of the IgAN patients developed end-stage renal disease (ESRD) in 20 years after the diagnosis of the disease. The heavy burden on the family and society. The diagnosis of IgAN is still dependent on renal biopsy, but the biopsy of the kidney is invasive and there is a certain risk and contraindication. The diagnosis and treatment of IgAN is very important for prevention and control and reduction of the number of ESRD patients. Therefore, it is of great significance to explore the pathogenesis of IgAN and the early invasive biomarkers. In this study, the renal tissue of the IgAN patients and the control group were tested by the technique of Raybitech's cytokine antibody chip. The detection and analysis of the urine of the patients with IgAN and the primary membranous nephropathy (IMN) and the normal population were analyzed by high-resolution mass spectrometry. The biological markers of the IgAN were selected. The application value of IgAN is discussed, and the pathogenesis of IgAN is further clarified. Methods: (1) 10 patients with IgAN were selected for the diagnosis of renal biopsy from 18 to 60 years old and in the general hospital of the PLA. The normal kidney tissues except 6cm in the carcinoma of the renal cell carcinoma were studied as the control group. Collect the clinical basic information and test results of the above-mentioned population. In the above-mentioned population, the renal tissue was tested using a Raybitech cytokine antibody chip to screen the differential protein, and a GO (Gene Ontology) analysis of the differential protein was performed. In combination with the results of the analysis, an enzyme-linked immunosorbent assay (ELISA) was used for the validation of IL-33. (2) The patients with the renal biopsy of the General Hospital of the PLA General Hospital were selected, 48 of the patients aged 20 to 60 were diagnosed as the patients with the IgAN 鈪，

本文編號：2362360