分泌型IgA在IgA肾病发病中的作用及机制研究

发布时间：2018-05-29 01:45

本文选题：Ig + A肾病　；参考：《郑州大学》2015年博士论文

【摘要】：研究背景： Ig A肾病(Ig A nephropathy,Ig AN)是我国乃至全球最常见的原发性肾小球肾炎,其特征是免疫荧光下见到以Ig A为主的免疫复合物在肾小球系膜区和/或肾小球毛细血管襻沉积。据2014年最新资料显示,全球Ig AN的发病率为2.5人/10万人/每年,约20%-50%的患者20年可进展为终末期肾脏病。但是,Ig AN的发病机制仍然不十分清楚。因Ig AN患者常在上呼吸道或肠道等粘膜感染后发生肉眼血尿和/或蛋白尿,提示粘膜免疫与Ig AN的发病机制密切相关。分泌型Ig A(secretory Ig A,SIg A)是参与粘膜免疫最重要的抗体,近年来的研究发现,SIg A在Ig AN的发病中可能也起着一定的致病作用。Ig AN的病理类型及临床表现具有多样性,是否因为有SIg A的参与?SIg A可以沉积在Ig AN患者的系膜区,那么它对系膜细胞具有损伤作用吗?SIg A发挥这些致病损伤作用的机制是什么呢?迄今为止,还未见相关报道。Micro RNAs(mi RNAs)是真核生物中一类具有内源性调控功能的非编码RNA,它通过与靶基因信使RNA(messenger RNA,m RNA)的3’端非编码区(untranslated region,UTR)结合,发挥抑制翻译的作用。研究提示,mi RNAs在肾脏病中也起了重要的作用。那么SIg A是否通过作用mi RNAs来发挥在Ig AN中的致病性呢?第一部分Ig A肾病患者分泌型Ig A在肾组织的沉积以及同临床病理之间的关系目的：检测Ig AN患者肾组织中SIg A沉积的比例;分析SIg A与临床及肾脏病理之间的关系。方法：选取263名原发性Ig AN患者,应用激光共聚焦显微镜观察肾组织SIg A的沉积,并比较有SIg A沉积和无沉积患者的临床及肾脏病理各项指标的不同。结果： 1.263名Ig AN患者中87名可见到系膜区SIg A的沉积,约占29.28%;2.同系膜区无SIg A沉积的病人相比较,有SIg A沉积的病人具有明显的感染史(P=0.016)及血尿(P=0.024)发生率,临床指标上具有明显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肾脏小管间质损伤(P=0.030)和更低的T评分(牛津分型,P=0.026)。结论：粘膜免疫同Ig AN密切相关,SIg A参与了Ig AN的发病,它在肾组织的沉积与某些的临床肾脏病理指标相联系。第二部分分泌型Ig A在Ig AN患者肾组织激活的补体通路研究目的：检测Ig AN患者肾组织中SIg A激活的补体通路来研究SIg A的肾损伤作用,以及比较肾组织经不同补体途径激活的Ig AN患者之间的临床病理特征。方法：选取87名原发性IgAN患者,应用激光共聚焦显微镜观察肾组织SIgA以及各补体通路相关蛋白的沉积,探讨SIg A与各补体通路激活的关系,并比较不同补体激活通路的患者临床及肾病理特点之间的差别。结果： 1.87名IgAN患者中有22名可见到系膜区SIgA沉积,其中经旁路途径激活的占77.27%,在65名无SIg A沉积的患者中经凝集素途径激活的占72.30%,两组比较无明显差别(P=0.648);2.同肾组织经凝集素途径激活的患者相比较,经旁路途径激活的64人(73.6%)在临床指标方面具有更低的肾小球滤过率(P=0.043),在肾病理方面肾小球硬化比例(P=0.035)、小管间质损伤程度(P=0.030)、C3的免疫荧光强度(P=0.012)以及牛津评分的S(P=0.006)和T评分(P=0.039)均损伤更严重。结论：在Ig AN中SIg A具有致病性,可能通过激活旁路途径和凝集素途径造成肾损伤;经旁路途径激活的Ig AN患者部分临床和肾病理特征比经凝集素途径激活的患者严重。第三部分分泌型Ig A对系膜细胞的生物学效应研究目的：研究SIgA对人系膜细胞的生物学效应,探讨SIgA在IgAN中的致病作用。方法：运用亲和层析柱提纯Ig AN患者及正常人唾液中的SIg A,应用亲和层析柱和分子筛提纯Ig AN患者及正常人血中的多聚Ig A(polymeric Ig A,p Ig A);将提纯的SIg A及p Ig A分别刺激人系膜细胞,检测细胞的增殖及各种促炎促纤维化因子(IL-6、IL-8、MCP-1、TGF-β1和纤维粘连蛋白)在细胞中m RNA及蛋白水平的表达情况,并相互比较。结果： 1.SIg A和p Ig A均能使人系膜细胞明显增殖(P0.05),表达IL-6、IL-8、MCP-1、TGF-β1和纤维粘连蛋白明显增多(m RNA和蛋白水平,P0.05),并且患者来源的SIg A和p Ig A对细胞的上述生物学效应均明显强于正常人来源的刺激作用(P0.05);2.经Ig AN患者唾液纯化的SIg A对系膜细胞的增殖作用及刺激其分泌IL-6、IL-8和纤维粘连蛋白的作用要明显弱于经Ig AN患者血清纯化的p Ig A作用(P0.05)。结论：在Ig AN中SIg A有致病作用,SIg A具有刺激系膜细胞增殖和释放促炎促纤维化细胞因子的生物学效应,并且SIg A与p Ig A对系膜细胞的生物学效应是不完全相同的。第四部分与分泌型Ig A致病性有关的micro RNAs筛选及验证目的：筛选与SIg A致病性有关的mi RNAs,研究SIg A在Ig AN中产生致病性的分子机制。方法：运用Agilent human mi RNAs芯片,检测经Ig AN患者及正常人SIg A刺激的系膜细胞中的mi RNAs,并分析筛选其中差异性表达的mi RNAs,应用实时荧光定量PCR分别进行验证。结果： 1.通过mi RNAs芯片筛选了17个经Ig AN患者及正常人的SIg A刺激后细胞内差异表达的mi RNAs,其中上调5个、下调12个;2.经实时荧光定量PCR验证芯片筛选结果中的mi RNAs,趋势均一致,其中16个表达差异显著(P0.05),1个差异性不显著(P0.05)。结论： SIgA在IgAN发挥致病性的机制中,mi RNAs参与其中。第五部分mi R-16-2-3p和mi R-100-3p调控IL-6和IL-8的分子机制目的：预测和验证了miR-16-2-3p和mi R-100-3p的靶基因,进一步探讨SIgA在Ig AN中产生致病性的分子机制。方法：通过生物信息学方法对mi RNAs芯片筛选结果中的mi R-16-2-3p和mi R-100-3p进行靶基因预测,建立融合靶基因3‘UTR的荧光素酶报告基因载体,通过双报告荧光素酶基因检测方法确定mi R-16-2-3p和mi R-100-3p的靶基因;应用实时荧光定量PCR和Western blot研究mi R-16-2-3p和mi R-100-3p对其靶基因的内源性调控作用。结果： 1.成功构建了能够过表达mi R-16-2-3p和mi R-100-3p的报告基因载体;2.荧光素酶双报告基因检测显示,mi R-16-2-3p和mi R-100-3p的复制物可以分别与报告基因载体的IL-6和IL-8 3’UTR野生序列结合,使载体的荧光素酶活性降低,而IL-6和IL-8 3’UTR突变序列不能产生这样的作用(P0.05);3.Mi R-16-2-3p过表达可以抑制SIg A刺激系膜细胞中分泌IL-6蛋白过多的情况,但不影响IL-6 m RNA的水平(P0.05),同样mi R-100-3p过表达可以抑制其分泌IL-8蛋白过多的情况,但不能影响IL-8 m RNA的水平(P0.05)。结论： 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通过作用mi R-16-2-3p和mi R-100-3p来分别调控系膜细胞产生炎症因子IL-6和IL-8,是SIg A在Ig AN中产生致病性的分子机制之一。全文结论 SIg A参与了Ig AN的发病,它在肾组织的沉积同一定的临床肾脏病理指标相联系;SIg A在Ig AN中具有致病作用,表现在有可能激活旁路途径和凝集素途径造成肾损伤,刺激系膜细胞增殖并释放促炎促纤维化细胞因子;SIg A通过作用mi R-16-2-3p和mi R-100-3p来分别调控系膜细胞产生炎症因子IL-6和IL-8,可能是SIg A在Ig AN中发挥致病作用的机制之一。
[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.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R692.31

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