本文選題：Ig + A腎病��； 參考：《鄭州大學》2015年博士論文

【摘要】：研究背景： Ig A腎病(Ig A nephropathy,Ig AN)是我國乃至全球最常見的原發(fā)性腎小球腎炎,其特征是免疫熒光下見到以Ig A為主的免疫復合物在腎小球系膜區(qū)和/或腎小球毛細血管襻沉積。據(jù)2014年最新資料顯示,全球Ig AN的發(fā)病率為2.5人/10萬人/每年,約20%-50%的患者20年可進展為終末期腎臟病。但是,Ig AN的發(fā)病機制仍然不十分清楚。因Ig AN患者常在上呼吸道或腸道等粘膜感染后發(fā)生肉眼血尿和/或蛋白尿,提示粘膜免疫與Ig AN的發(fā)病機制密切相關(guān)。分泌型Ig A(secretory Ig A,SIg A)是參與粘膜免疫最重要的抗體,近年來的研究發(fā)現(xiàn),SIg A在Ig AN的發(fā)病中可能也起著一定的致病作用。Ig AN的病理類型及臨床表現(xiàn)具有多樣性,是否因為有SIg A的參與?SIg A可以沉積在Ig AN患者的系膜區(qū),那么它對系膜細胞具有損傷作用嗎?SIg A發(fā)揮這些致病損傷作用的機制是什么呢?迄今為止,還未見相關(guān)報道。Micro RNAs(mi RNAs)是真核生物中一類具有內(nèi)源性調(diào)控功能的非編碼RNA,它通過與靶基因信使RNA(messenger RNA,m RNA)的3’端非編碼區(qū)(untranslated region,UTR)結(jié)合,發(fā)揮抑制翻譯的作用。研究提示,mi RNAs在腎臟病中也起了重要的作用。那么SIg A是否通過作用mi RNAs來發(fā)揮在Ig AN中的致病性呢?第一部分Ig A腎病患者分泌型Ig A在腎組織的沉積以及同臨床病理之間的關(guān)系目的： 檢測Ig AN患者腎組織中SIg A沉積的比例;分析SIg A與臨床及腎臟病理之間的關(guān)系。方法：選取263名原發(fā)性Ig AN患者,應用激光共聚焦顯微鏡觀察腎組織SIg A的沉積,并比較有SIg A沉積和無沉積患者的臨床及腎臟病理各項指標的不同。結(jié)果： 1.263名Ig AN患者中87名可見到系膜區(qū)SIg A的沉積,約占29.28%;2.同系膜區(qū)無SIg A沉積的病人相比較,有SIg A沉積的病人具有明顯的感染史(P=0.016)及血尿(P=0.024)發(fā)生率,臨床指標上具有明顯低水平的血清胱抑素C(P=0.027)和β2微球蛋白(P=0.018),病理特征上具有更微的腎臟小管間質(zhì)損傷(P=0.030)和更低的T評分(牛津分型,P=0.026)。結(jié)論： 粘膜免疫同Ig AN密切相關(guān),SIg A參與了Ig AN的發(fā)病,它在腎組織的沉積與某些的臨床腎臟病理指標相聯(lián)系。第二部分分泌型Ig A在Ig AN患者腎組織激活的補體通路研究目的： 檢測Ig AN患者腎組織中SIg A激活的補體通路來研究SIg A的腎損傷作用,以及比較腎組織經(jīng)不同補體途徑激活的Ig AN患者之間的臨床病理特征。方法： 選取87名原發(fā)性IgAN患者,應用激光共聚焦顯微鏡觀察腎組織SIgA以及各補體通路相關(guān)蛋白的沉積,探討SIg A與各補體通路激活的關(guān)系,并比較不同補體激活通路的患者臨床及腎病理特點之間的差別。結(jié)果： 1.87名IgAN患者中有22名可見到系膜區(qū)SIgA沉積,其中經(jīng)旁路途徑激活的占77.27%,在65名無SIg A沉積的患者中經(jīng)凝集素途徑激活的占72.30%,兩組比較無明顯差別(P=0.648);2.同腎組織經(jīng)凝集素途徑激活的患者相比較,經(jīng)旁路途徑激活的64人(73.6%)在臨床指標方面具有更低的腎小球濾過率(P=0.043),在腎病理方面腎小球硬化比例(P=0.035)、小管間質(zhì)損傷程度(P=0.030)、C3的免疫熒光強度(P=0.012)以及牛津評分的S(P=0.006)和T評分(P=0.039)均損傷更嚴重。結(jié)論： 在Ig AN中SIg A具有致病性,可能通過激活旁路途徑和凝集素途徑造成腎損傷;經(jīng)旁路途徑激活的Ig AN患者部分臨床和腎病理特征比經(jīng)凝集素途徑激活的患者嚴重。第三部分分泌型Ig A對系膜細胞的生物學效應研究目的： 研究SIgA對人系膜細胞的生物學效應,探討SIgA在IgAN中的致病作用。方法： 運用親和層析柱提純Ig AN患者及正常人唾液中的SIg A,應用親和層析柱和分子篩提純Ig AN患者及正常人血中的多聚Ig A(polymeric Ig A,p Ig A);將提純的SIg A及p Ig A分別刺激人系膜細胞,檢測細胞的增殖及各種促炎促纖維化因子(IL-6、IL-8、MCP-1、TGF-β1和纖維粘連蛋白)在細胞中m RNA及蛋白水平的表達情況,并相互比較。結(jié)果： 1.SIg A和p Ig A均能使人系膜細胞明顯增殖(P0.05),表達IL-6、IL-8、MCP-1、TGF-β1和纖維粘連蛋白明顯增多(m RNA和蛋白水平,P0.05),并且患者來源的SIg A和p Ig A對細胞的上述生物學效應均明顯強于正常人來源的刺激作用(P0.05);2.經(jīng)Ig AN患者唾液純化的SIg A對系膜細胞的增殖作用及刺激其分泌IL-6、IL-8和纖維粘連蛋白的作用要明顯弱于經(jīng)Ig AN患者血清純化的p Ig A作用(P0.05)。結(jié)論：在Ig AN中SIg A有致病作用,SIg A具有刺激系膜細胞增殖和釋放促炎促纖維化細胞因子的生物學效應,并且SIg A與p Ig A對系膜細胞的生物學效應是不完全相同的。第四部分與分泌型Ig A致病性有關(guān)的micro RNAs篩選及驗證目的： 篩選與SIg A致病性有關(guān)的mi RNAs,研究SIg A在Ig AN中產(chǎn)生致病性的分子機制。方法： 運用Agilent human mi RNAs芯片,檢測經(jīng)Ig AN患者及正常人SIg A刺激的系膜細胞中的mi RNAs,并分析篩選其中差異性表達的mi RNAs,應用實時熒光定量PCR分別進行驗證。結(jié)果： 1.通過mi RNAs芯片篩選了17個經(jīng)Ig AN患者及正常人的SIg A刺激后細胞內(nèi)差異表達的mi RNAs,其中上調(diào)5個、下調(diào)12個;2.經(jīng)實時熒光定量PCR驗證芯片篩選結(jié)果中的mi RNAs,趨勢均一致,其中16個表達差異顯著(P0.05),1個差異性不顯著(P0.05)。結(jié)論： SIgA在IgAN發(fā)揮致病性的機制中,mi RNAs參與其中。第五部分mi R-16-2-3p和mi R-100-3p調(diào)控IL-6和IL-8的分子機制目的： 預測和驗證了miR-16-2-3p和mi R-100-3p的靶基因,進一步探討SIgA在Ig AN中產(chǎn)生致病性的分子機制。方法：通過生物信息學方法對mi RNAs芯片篩選結(jié)果中的mi R-16-2-3p和mi R-100-3p進行靶基因預測,建立融合靶基因3‘UTR的熒光素酶報告基因載體,通過雙報告熒光素酶基因檢測方法確定mi R-16-2-3p和mi R-100-3p的靶基因;應用實時熒光定量PCR和Western blot研究mi R-16-2-3p和mi R-100-3p對其靶基因的內(nèi)源性調(diào)控作用。結(jié)果： 1.成功構(gòu)建了能夠過表達mi R-16-2-3p和mi R-100-3p的報告基因載體;2.熒光素酶雙報告基因檢測顯示,mi R-16-2-3p和mi R-100-3p的復制物可以分別與報告基因載體的IL-6和IL-8 3’UTR野生序列結(jié)合,使載體的熒光素酶活性降低,而IL-6和IL-8 3’UTR突變序列不能產(chǎn)生這樣的作用(P0.05);3.Mi R-16-2-3p過表達可以抑制SIg A刺激系膜細胞中分泌IL-6蛋白過多的情況,但不影響IL-6 m RNA的水平(P0.05),同樣mi R-100-3p過表達可以抑制其分泌IL-8蛋白過多的情況,但不能影響IL-8 m RNA的水平(P0.05)。結(jié)論： Mi R-16-2-3p的靶基因是IL-6,mi R-100-3p的靶基因是IL-8;SIg A通過作用mi R-16-2-3p和mi R-100-3p來分別調(diào)控系膜細胞產(chǎn)生炎癥因子IL-6和IL-8,是SIg A在Ig AN中產(chǎn)生致病性的分子機制之一。全文結(jié)論 SIg A參與了Ig AN的發(fā)病,它在腎組織的沉積同一定的臨床腎臟病理指標相聯(lián)系;SIg A在Ig AN中具有致病作用,表現(xiàn)在有可能激活旁路途徑和凝集素途徑造成腎損傷,刺激系膜細胞增殖并釋放促炎促纖維化細胞因子;SIg A通過作用mi R-16-2-3p和mi R-100-3p來分別調(diào)控系膜細胞產(chǎn)生炎癥因子IL-6和IL-8,可能是SIg A在Ig AN中發(fā)揮致病作用的機制之一。
[Abstract]:Background: Ig A nephropathy (Ig A nephropathy, Ig AN) is the most common primary glomerulonephritis in China and in the world. It is characterized by the deposition of Ig A based immune complexes in the glomerular mesangial region and / or glomerular capillary loops under immunofluorescence. According to the latest data in 2014, the incidence of Ig AN in the global Ig is 2.5 people. People / each year, about 20%-50% patients can progress to end-stage renal disease in 20 years. However, the pathogenesis of Ig AN is still not very clear. Because Ig AN patients often occur naked hematuria and / or proteinuria after infection of the upper respiratory tract or intestinal mucosa, suggesting that mucosal immunity is closely related to the pathogenesis of Ig AN. Secretory Ig A (secretory Ig A) It is the most important antibody involved in mucosal immunization. Recent studies have found that SIg A may also play a certain pathogenicity in the pathogenesis of Ig AN in the pathological type and clinical manifestations of.Ig AN. Is there the participation of SIg A? SIg A can be deposited in the mesangial region of Ig AN patients, and is it damaging to mesangial cells? What is the mechanism of Ig A playing these pathogenetic effects? So far, no related reports have been reported that.Micro RNAs (MI RNAs) is a non coded RNA with endogenous regulatory functions in eukaryotes, which combines with the 3 'end non coding region of the target gene messenger RNA (messenger RNA, m RNA) and exerts inhibition. The study suggests that MI RNAs plays an important role in renal disease. Then, does SIg A play the pathogenicity in Ig AN by acting mi RNAs? Analysis of the relationship between SIg A and clinical and renal pathology. Methods: 263 patients with primary Ig AN were selected and the deposition of SIg A in renal tissue was observed by laser confocal microscopy, and the clinical and renal pathological indexes of SIg A and non deposition patients were compared. Fruit: 87 of the 1.263 Ig AN patients could see the mesangial region. The deposition of SIg A accounted for about 29.28%; 2. compared with patients without SIg A deposition in the homologous membrane region, the patients with SIg A deposition had obvious infection history (P=0.016) and hematuria (P=0.024), and the clinical indicators had obvious low levels of serum cystatin C (P=0.027) and beta 2 microglobulin (P=0.018), and the pathological features had a slightly smaller tubulointerstitium. Damage (P=0.030) and lower T score (Oxford classification, P=0.026). Conclusion: mucosal immunity is closely related to Ig AN, SIg A participates in the pathogenesis of Ig AN, and its deposition in the renal tissue is associated with some clinical renal pathological indexes. Second part secretory Ig A in the renal tissue activated complement pathway of the Ig patients: detection The role of SIg A activated complement pathway in renal tissue to study the renal damage of SIg A and to compare the clinicopathological features of Ig AN patients activated by different complement pathways in renal tissue. Methods: 87 primary IgAN patients were selected to observe the deposition of SIgA in renal tissue and the proteins related to complement pathway by laser confocal microscopy. The relationship between SIg A and the activation of complement pathway was investigated and the difference between the clinical and renal pathological features of the patients with different complement activation pathways was compared. Results: 22 of the 1.87 IgAN patients could see the SIgA deposition in the mesangial region, among which 77.27% were activated by the bypass pathway, and 7 of the 65 patients without SIg A were activated by the agglutinin pathway. 2.30%, there was no significant difference in the two groups (P=0.648); 2. the 64 (73.6%) activated by the bypass pathway (73.6%) had lower glomerular filtration rate (P=0.043), renal pathological glomerular sclerosis ratio (P=0.035), tubulointerstitial damage (P=0.030), and C3 immunofluorescence Light intensity (P=0.012) and the S (P=0.006) and T score (P=0.039) of the Oxford score were more severe. Conclusion: SIg A in Ig AN is pathogenicity and may cause renal injury by activating the bypass pathway and lectin pathway; some of the clinical and renal pathological features of Ig AN patients activated by the bypass pathway are more severe than those activated by the lectin pathway. The biological effects of third partial secretory Ig A on mesangial cells: To study the biological effects of SIgA on human mesangial cells and to explore the pathogenicity of SIgA in IgAN. Methods: purification of SIg A in Ig AN patients and normal human saliva by affinity chromatography column, and the purification of Ig AN patients and normal by affinity chromatography column and molecular sieve. Ig A (polymeric Ig A, P Ig A) in human blood, and the purified SIg A and P Ig, respectively, to stimulate human mesangial cells and to detect the proliferation of cells and the expression of various proinflammatory fibrotic factors in cells. The proliferation of human mesangial cells (P0.05), IL-6, IL-8, MCP-1, TGF- beta 1 and fibronectin were significantly increased (m RNA and protein level, P0.05), and the biological effects of SIg A and P Ig from the patients were significantly stronger than those of normal human sources. 2. The proliferation and stimulation of the secretion of IL-6, IL-8 and fibronectin should be significantly weaker than the P Ig A action (P0.05) purified by Ig AN patients. Conclusion: SIg A in Ig AN has the pathogenicity, which has the biological effect of stimulating mesangial cell proliferation and releasing proinflammatory cytokines. The biological effects of the cells are not exactly the same. Fourth the screening and verification of micro RNAs related to the pathogenicity of the secretory Ig A is to screen the MI RNAs related to the pathogenicity of SIg A and to study the molecular mechanism of the pathogenesis of SIg A in Ig AN. The MI RNAs in the mesangial cells stimulated by A was analyzed and the differential expression of MI RNAs was screened, and the real-time fluorescent quantitative PCR was used to verify the results. Results: 1. through mi RNAs chip, 17 cells were screened by Mi RNAs chip and the differential expression in the cells after SIg A stimulation, up regulation 5, down 12; 2. by real time fluorescence determination. The trend of MI RNAs in the screening results of PCR was the same, and the 16 differences were significant (P0.05) and 1 differences were not significant (P0.05). Conclusion: SIgA is involved in the pathogenesis of IgAN, MI RNAs participates in it. Fifth part mi R-16-2-3p and Mi regulates and regulates the molecular mechanisms. The target genes of 6-2-3p and MI R-100-3p further explore the molecular mechanism of the pathogenicity of SIgA in Ig AN. Method: target gene prediction of MI R-16-2-3p and MI R-100-3p in the screening results of MI RNAs chips by bioinformatics method, and to establish a fusion target gene 3 'fluorescein reporter gene carrier, through double reporting fluorescein. The target genes of MI R-16-2-3p and MI R-100-3p were determined by enzyme gene detection. The endogenous regulatory effects of MI R-16-2-3p and MI R-100-3p on the target genes were studied by real time fluorescent quantitative PCR and Western blot. Results: 1. the reporter gene vector was successfully constructed and the 2. luciferase double reporter gene was successfully constructed. The results showed that the replication of MI R-16-2-3p and MI R-100-3p could be combined with the IL-6 and IL-8 3 'UTR wild sequence of the reporter gene vector, and the luciferase activity of the carrier was reduced, while IL-6 and IL-8 3' UTR mutation sequence could not produce such action. L-6 protein is too much, but it does not affect the level of IL-6 m RNA (P0.05), and the overexpression of MI R-100-3p can inhibit its secretion of IL-8 protein, but it can not affect the IL-8 m RNA level. The formation of inflammatory factors IL-6 and IL-8 respectively in mesangial cells is one of the molecular mechanisms of the pathogenesis of SIg A in Ig AN. Conclusion SIg A is involved in the pathogenesis of Ig AN, and its deposition in the renal tissue is associated with a certain clinical renal pathological index; SIg A has a pathogenic role in the pathogenesis of the bypass pathway and the possibility of activating the bypass pathway and coagulation. The collection pathway causes renal injury, stimulates the proliferation of mesangial cells and releases proinflammatory cytokines, and SIg A regulates the inflammatory factors IL-6 and IL-8 by Mi R-16-2-3p and MI R-100-3p, which may be one of the mechanisms of SIg A in Ig AN.
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R692.31

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8 周華波;房慧伶;賈永起;王曉麗;吳欣欣;李紅蕾;;傳染性支氣管炎弱毒苗黏膜免疫途徑對雛雞上呼吸道及哈德氏腺SIgA細胞分布的影響[A];中國畜牧獸醫(yī)學會動物解剖學及組織胚胎學分會第十四次學術(shù)研討會論文集[C];2006年

9 汪常偉;李凡成;;綜合療法對常年性變態(tài)反應性鼻炎患者SIgA、IgE水平的影響[A];世界中聯(lián)耳鼻喉口腔科專業(yè)委員會第五屆學術(shù)年會、中華中醫(yī)藥學會耳鼻喉科分會第十九屆學術(shù)交流會暨貴州省中西醫(yī)結(jié)合學會耳鼻咽喉分會第二次學術(shù)交流會論文匯編[C];2013年

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8 羅樹立;盲升結(jié)腸可控膀胱術(shù)后尿sIgA含量研究[D];延邊大學;2007年

9 王艷;唾液sIgA納米免疫生物傳感器的實驗研究[D];重慶醫(yī)科大學;2011年

10 朱鈺;IL-21，地塞米松，，苯甲酸雌二醇對小鼠SIgA形成的影響[D];重慶醫(yī)科大學;2012年

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