IL-17及相关趋化因子在阿霉素肾病小鼠和足细胞中的作用机制研究

发布时间：2018-07-07 21:33

本文选题：阿霉素 + 肾病　；参考：《山东大学》2016年博士论文

【摘要】：研究背景及研究目的：原发性肾病综合征(Primary Nephrotic syndrome, PNS)是最常见的儿童肾脏疾病之一,其临床症状是肾病范围的蛋白尿、低白蛋白血症、水肿和高胆固醇血症。PNS的发病机制现仍未知,而且它是一种多元素性疾病。PNS按肾脏病理分为微小病变肾病综合征(minimal change nephrotic syndrome,MCNS),局灶节段性肾小球硬化(focal segmental glomerulosclerosis,FSGS),系膜增生性肾小球肾炎(mesangial proliferative glomerulonephritis,MsPGN),和膜性增生性肾小球肾炎(membranoproliferative glomerulonephritis,MNGN)等。PNS主要病理类型是MCNS(77.1%)和FSGS(7.9%)。患儿大都对激素敏感,但少数患儿对激素依赖或抵抗,最后发展为终末期肾病。因此阐明PNS的发病机制,针对其发病过程进行药物干预,可以为肾病患儿开辟新的诊疗途径。白介素17(Interleukin-17,IL-17),是一种促炎的细胞因子,由活化的CD4+T细胞亚群(常称为Th17)产生。IL-17家族由6种家庭成员构成,它们分别是IL-17(又称IL-17A), IL-17B, IL-17C, IL-17D, IL-17 E(又称IL-25),和IL-17F。IL-17受体家族由5种成员组成,它们分别是IL-17RA、IL-17RB、 IL-17RC、IL-17RD和IL-17RE。近年来国内外对IL-17在肾脏疾病方面的研究集中在狼疮性肾炎、糖尿病肾病、肾小球肾炎、肾损伤和肾移植排斥损伤。有关IL-17在肾病综合征的研究报道很少。我们知道T细胞参与PNS的发病机制,而Th17分泌的细胞因子是否参与PNS的免疫机制还不明确。有研究者提出,IL-17很可能是T细胞活化和炎症之间的一座桥梁。Wang等发现Th17/IL-17通过下调podocalyxin表达水平,和诱导足细胞凋亡,从而促进PNS的发病。Liu等报道MCNS潜在的发病机制是Th17/Treg不平衡。新近少数研究资料表明IL-17在MCNS发病机制中起到主要的作用。新近资料显示,利妥昔单抗可以通过抑制IL-17,影响B细胞的消耗,或者改变足细胞的细胞骨架,干预MCNS的发病机制。IL-17A产生的主要途径是肾脏Th17细胞上的趋化因子受体CCR6与CCL20反应。IL-17信号是通过其与受体相结合来发挥作用。在炎症反应时,IL-17A可以募集中性粒细胞和巨噬细胞,从而产生多肽类蛋白,细胞因子和趋化因子(CXCL1, CXCL2,和CXCL5)。CXCL1、CXCL2能够促进中性粒细胞的趋化。IL-8也可以促进感染部位中性粒细胞的聚集,最后清除炎症病灶。CXC趋化因子配体9(chemokine ligand9,CXCL9)和CXCL10能调节Thl。在胞内病原体、肿瘤抗原以及干扰素-γ(interferon-γ,IFN-γ)和IL-12的诱导下,Th0向Thl分化；Thl分泌IFN-γ、IL-2等；Th2分泌IL-4。因此,我们可以认为IL-17相关的趋化因子是CCR6、CCL20,中性粒细胞相关的趋化因子是CXCL1、CXCL2、IL-8, Th1相关的趋化因子和细胞因子是CXCL9、CXCL10、IL-12、IFN-γ。目前,国内外关于IL-17及相关趋化因子在PNS中的研究尚无报道。PNS的主要损伤是肾脏丢失蛋白质,PNS范围的蛋白尿是由于肾小球滤过膜通透性的损害,和肾小管重吸收障碍所致。肾小球滤过屏障是由三层组成：内皮细胞层,肾小球基底膜(glomerular basement membrane, GBM),和足细胞足突层。。肾小球疾病的特点大都是裂孔隔膜(slit diaphragm,SD)分子结构的改变,和足突结构重组(融合和消失)。足突消失和蛋白尿的最主要原因是GBM或足细胞一GBM之间相互作用的异常,SD区域损害,以及肌动蛋白细胞骨架和相关蛋白的改变。足细胞结构的改变引发足细胞损伤,从而导致足突的丢失。足细胞缺乏是肾小球硬化的开始。丝裂原活化激酶(mitogen-activated protein kinase,MAPK)是细胞功能活动的应激通路,对于细胞的生长、发育、分裂、凋亡等起到重要作用。哺乳细胞的MAPK通路有四种：细胞内信号调节酶(extracellular signal-regulated kinase,Erk)-1和-2,c-Jun N末端酶(c-Jun N-terminal kinase,JNK), p38MAPK和细胞内单个调节酶-5(extracellular signal-regulated kinase,-5,Erk5/BMK1)。研究已证实,MAPK通路在急慢性炎症中起到重要的作用。P38MAPK在抗肾小球基底膜肾炎中的表达增加,并且影响炎症细胞的聚集和肾小管的损伤。本研究通过阿霉素(Adriamycin, ADR)诱导建立的小鼠肾病综合征模型(微小病变型),观察IL-17在肾脏组织和外周血水平的变化,以及IL-17相关趋化因子CCR6、CCL20,中性粒细胞相关趋化因子CXCL1、CXCL2、IL-8,Thl相关趋化因子CXCL9、CXCL10、IL-12、IFN-γ,三组因子在肾组织中的表达变化。同时对模型小鼠应用中和IL-17抗体进行干预,观察其炎症水平和肾脏功能情况的变化。从而探讨IL-17在PNS中的促炎机制。此外,本研究亦通过体外培养小鼠肾足细胞,研究IL-17在肾足细胞炎症中的分子作用机制,以及与MAPK信号路径的相关性。进一步探讨IL-17及其相关趋化因子在儿童PNS发病及发展中的作用,为临床上进一步防治儿童PNS提供新的理论依据。研究方法：1.动物实验：随机将Balb/c小鼠分为三组,正常对照组,模型组(阿霉素肾病),IL-17中和抗体组,每组共10只小鼠。给予小鼠尾静脉注射阿霉素诱导阿霉素肾病模型(微小病变型),1周后其中1组模型小鼠给予腹腔注射IL-17中和抗体,2周后将全部小鼠处死,留取尿液、血液和肾组织。24小时尿蛋白定量采取邻苯三酚红比色法测试,ELISA检测血清IL-17、Cys C、 Kim-1、NGAL的浓度。应用HE染色、免疫组化DAB染色制作肾组织病理切片,观察肾脏组织中IL-17的表达。IL-17相关趋化因子CCR6、CCL20的mRNA表达,中性粒细胞相关趋化因子CXCL1、CXCL2、IL-8 的 mRNA表达,Thl相关趋化因子CXCL9、CXCL10、IL-12、IFN-γ的mRNA表达,均使用荧光定量PCR进行检测。2.肾足细胞培养：小鼠肾足细胞加入含10%胚胎小牛血清的1640倍的培养液,和10U/ml γ干扰素后,然后放入33℃孵育箱,细胞传代培养；然后转入无干扰素、37℃培养孵箱,使细胞分化2周后。足细胞种植于平皿,在无血清的清洁培养液中培养24小时,应用重组的小鼠IL-17蛋白100ng/ml,刺激小鼠肾足细胞共同孵养24小时。随后应用Erk、p38抑制剂10umol/l分别与IL-17刺激的足细胞孵育24小时后；收集细胞,Western Blot检测：小鼠肾足细胞中p38、Erk及p-p38、p-Erk的蛋白水平。在加用p38和Erk抑制剂后,中性粒细胞相关趋化因子IL-8、Th1相关趋化因子IL-12的mRNA表达,采取荧光定量PCR测定。结果：1.光镜的观察结果同对照组比较,模型组有球囊部分粘连,系膜细胞增生,基质增多,肾小管腔部分出现蛋白管型,肾间质炎症细胞浸润；中和IL-17抗体组比模型组轻。2.电镜的观察结果模型组与对照组比较,肾小球形态结构轻微异常,足突普遍融合。IL-17中和抗体组病变较轻。3.各组小鼠肾组织IL-17蛋白表达DAB染色显示,IL-17的阳性细胞多为胞质染色阳性,正常肾组织中有少量IL-17的表达,主要集中在肾小管,而肾小球中无表达；模型组中,肾小管上IL-17表达显著,它在肾小球中有少量表达；IL-17中和抗体组较模型组轻。同对照组比较,阿霉素肾病组、IL-17中和抗体组细胞的阳性着色度明显增强,P0.05。模型组比IL-17中和抗体组细胞阳性着色度明显增高,P0.05。4.各组小鼠肾组织IL-17相关趋化因子CCR6、CCL20的mRNA表达与正常对照组对比,模型组、IL-17中和抗体组CCR6、CCL20的mRNA表达均升高,P0.05。模型组的CCR6、CCL20mRNA表达比IL-17中和抗体组增强,P0.05。5.各组小鼠肾组织中性粒细胞相关趋化因子CXCL1、CXCL2、IL-8的mRNA表达与正常对照组比较,模型组、IL-17中和抗体组CXCL1、CXCL2、IL-8的mRNA表达均升高,P0.05。模型组比IL-17中和抗体组CXCL1、CXCL2、 IL-8的mRNA表达升高,P0.05。6.各组小鼠肾组织Thl相关趋化因子CXCL9、CXCL10、IL-12、IFN-γ的mRNA表达与正常对照组比较,模型组、IL-17中和抗体组CXCL9、CXCL10、IL-12、 IFN-γ的表达下降,P0.05。模型组比IL-17中和抗体组CXCL9、CXCL10、 IL-12、IFN-γ的表达降低,P0.05。7.各组小鼠血清IL-17水平的变化与正常对照组比较,模型组、IL-17中和抗体组的IL-17水平升高,P0.05。模型组比IL-17中和抗体组的IL-17水平升高,P0.05。8.各组小鼠肾功能指标血清KIM-1、NGAL、Csy C水平的变化与正常对照组比较,模型组、IL-17中和抗体组的KIM-1、NGAL、Csy C血清水平均升高,P0.05。模型组比IL-17中和抗体组的KIM-1、NGAL、 Csy C水平升高,P0.05。9.小鼠肾足细胞中p38、Erk及p-p38、p-Erk的蛋白水平与正常组比较,IL-17刺激组Ep-Erk、p38、p-p38的蛋白表达水平升高,其中p-p38的蛋白水平升高最明显。10.应用p38抑制剂和Erk抑制剂后,中性粒细胞相关趋化因子IL-8 mRNA表达与正常组对比,IL-17刺激组、p38抑制剂组、Erk抑制剂组的IL-8 mRNA表达均明显升高,P0.05。与IL-17刺激组比较,p38抑制剂组、Erk抑制剂组IL-8mRNA表达均下降,P0.05。p38抑制剂组比Erk抑制剂组的IL-8 mRNA表达明显升高,P0.05。11.应用p38抑制剂和Erk抑制剂后,Thl相关趋化因子IL-12 mRNA表达与正常组对比,IL-17刺激组IL-12 mRNA表达明显下降,p38抑制剂组、Erk抑制剂组均明显升高,P0.05。与IL-17刺激组比较,p38抑制剂组、Erk抑制剂组IL-12 mRNA表达均明显升高,P0.05。p38抑制剂组比Erk抑制剂组IL-12 mRNA明显升高,P0.05。结论：1.在阿霉素诱导的小鼠肾病模型中,肾组织IL-17表达异常增加,可以使中性粒细胞募集,参与ADR小鼠的炎症病理过程,少量的IL-17对正常肾脏功能的维持有一定作用。2.在阿霉素诱导的小鼠肾病模型中,血清IL-17的水平明显增高,提示IL-17促进ADR小鼠的炎症过程,而且它还能促使Thl细胞分化降低,大量的IL-17可以导致ADR小鼠严重的肾功能损害。3.小鼠腹腔注射中和IL-17抗体能够降低ADR小鼠血清IL-17的水平,减轻中性粒细胞募集,中和IL-17抗体抑制Thl细胞分化降低,肾功能获得改善。4.IL-17可能通过MAPK信号通路对小鼠肾足细胞炎症因子进行调控。5.IL-17能够激活p38及Erk路径信号,此过程中p38路径信号是主要的。6.IL-17促进中性粒细胞聚集,产生炎症反应,并且改变了天然CD4+T细胞向Th1/Th2分化的平衡,诱导其向Th2分化,抑制其向Thl分化。
[Abstract]:Research background and purpose: Primary Nephrotic syndrome (PNS) is one of the most common renal diseases in children. The clinical symptoms are proteinuria, hypoalbuminemia, edema, and hypercholesterolemia, the pathogenesis of.PNS in nephrotic range is still unknown, and it is a multielement disease.PNS by the kidney. The pathology was divided into minimal change nephrotic syndrome (MCNS), focal segmental glomerulosclerosis (focal segmental glomerulosclerosis, FSGS), mesangial proliferative glomerulonephritis (mesangial proliferative glomerulonephritis,), and membranous proliferative glomerulonephritis. Ephritis, MNGN) and other major pathological types of.PNS are MCNS (77.1%) and FSGS (7.9%). Most of the children are sensitive to hormone, but a few children are dependent on or resisted by hormone, and finally develop to end-stage renal disease. Therefore, the pathogenesis of PNS and the drug intervention in the pathogenesis of the disease can open up a new way of diagnosis and treatment for children with kidney disease. IL 17 (Inte). Rleukin-17, IL-17) is a pro-inflammatory cytokine, produced by the activated CD4+T cell subgroup (often called Th17). The.IL-17 family is composed of 6 family members. They are IL-17 (also known as IL-17A), IL-17B, IL-17C, IL-17D, IL-17 E (also known as Th17). -17RC, IL-17RD, and IL-17RE. have focused on the study of IL-17 for renal disease in recent years in lupus nephritis, diabetic nephropathy, glomerulonephritis, renal injury and renal transplantation rejection. There are few reports about IL-17 in nephrotic syndrome. We know that T cells are involved in the pathogenesis of PNS, and Th17 secreted cytokines are The immune mechanism involved in PNS is not clear. Some researchers suggest that IL-17 is likely to be a bridge between T cell activation and inflammation, such as a bridge.Wang, and so on, that the pathogenesis of MCNS latent by Th17/IL-17 by downregulating the podocalyxin expression level, and inducing the apoptosis of the podocyte, and thus promoting the pathogenesis of PNS, is Th17/Treg imbalances. Several research data show that IL-17 plays a major role in the pathogenesis of MCNS. Recent data show that rituximab can inhibit IL-17, affect the consumption of B cells, or change the cytoskeleton of the podocytes. The main way to interfere with the pathogenesis of MCNS,.IL-17A, is the chemokine receptor CCR6 and CCL20 on the renal Th17 cells. The.IL-17 signal is combined with the receptor. In the inflammatory response, IL-17A can raise the concentration of granulocytes and macrophages to produce polypeptide proteins, cytokines and chemokines (CXCL1, CXCL2, and CXCL5).CXCL1, CXCL2 can promote the chemotaxis.IL-8 of neutrophils and can promote neutrophil in the infected site. .CXC chemokine ligand 9 (chemokine ligand9, CXCL9) and CXCL10 can regulate Thl. in the intracellular pathogen, tumor antigen and interferon - gamma (interferon- gamma, IFN- gamma) and IL-12, and Th0 toward Thl; Thl secretes gamma, and so on. CCR6, CCL20, neutrophil related chemokines are CXCL1, CXCL2, IL-8, and Th1 related chemokines and cytokines are CXCL9, CXCL10, IL-12, IFN- gamma. Currently, there is no report on IL-17 and related chemokine in PNS. The damage of glomerular filtration membrane permeability and renal tubular reabsorption disorders. The glomerular filtration barrier consists of three layers: the endothelial cell layer, the glomerular basement membrane (glomerular basement membrane, GBM), and the podocyte Poddar layer.. the characteristics of the glomerular diseases are the changes in the molecular structure of the slit diaphragm (SD), and the changes in the molecular structure of the slit diaphragm. The most important reason for the disappearance of podocyte structure (fusion and disappearance). The most important reason for the disappearance of podocyte and proteinuria is the abnormality of the interaction between GBM or GBM, the damage of the SD region, and the changes of the actin cytoskeleton and related proteins. The changes of the podocyte structure cause the foot cell damage and the loss of the poddate. The deficiency of the podocyte is a small kidney. The beginning of the hardening of the ball. Mitogen-activated protein kinase (MAPK) is the stress pathway of cell function activity. It plays an important role in cell growth, development, division and apoptosis. There are four kinds of MAPK pathway in mammalian cells: intracellular signal regulating enzyme (extracellular signal-regulated kinase, Erk) -1 and -2, c-Jun C-Jun N-terminal kinase (JNK), p38MAPK and intracellular single regulatory enzyme -5 (extracellular signal-regulated kinase, -5, Erk5/BMK1). Studies have shown that the MAPK pathway plays an important role in acute and chronic inflammation and increases the expression in anti glomerular basilar glomerulonephritis and affects the aggregation of inflammatory cells and renal tubules. In this study, the mouse nephrotic syndrome model (small lesion type) induced by Adriamycin (ADR) was used to observe the changes in the level of IL-17 in the renal tissue and peripheral blood, as well as the IL-17 related chemokine CCR6, CCL20, and neutrophil related chemokine related chemokine, CXCL1, CXCL2, IL-8, Thl related chemokine CXCL9, CXCL10, Thl. Gamma, three groups of factors in the renal tissue expression changes. Meanwhile, the model mice were used to neutralize the IL-17 antibody and observe the changes in the inflammatory level and renal function. Thus the proinflammatory mechanism of IL-17 in PNS was explored. In addition, this study also studied the molecules of renal foot cells in vitro by culture in mice, and studied the molecules of IL-17 in the inflammation of the renal poddine. The mechanism of action and the correlation with the MAPK signal pathway. Further explore the role of IL-17 and its related chemokines in the pathogenesis and development of children's PNS, and provide a new theoretical basis for the further prevention and treatment of children's PNS. 1. animal experiments: randomly divided Balb/c mice into three groups, normal control group, model group (adriamycin) Nephrosis), IL-17 neutralization antibody group, 10 mice in each group. Adriamycin induced adriamycin induced nephropathy model was induced by adriamycin in the tail vein of mice. After 1 weeks, 1 models of mice were injected with IL-17 neutralization antibody. After 2 weeks, all mice were killed and urine was left, and.24 hour urine protein in blood and kidney tissues was quantified by o-phenolol. The concentration of IL-17, Cys C, Kim-1, NGAL in serum was measured by ELISA. HE staining, immunohistochemical DAB staining was used to make pathological sections of renal tissue, and the expression of.IL-17 related chemokine in renal tissue was observed. The mRNA expression of CXCL9, CXCL10, IL-12, IFN- gamma,.2. renal podocyte culture was detected by fluorescence quantitative PCR: mice kidney foot cells added 1640 times the culture solution containing 10% embryo calf serum, and 10U/ml gamma interferon, and then incubated at 33 centigrade and cultured for generation, then transferred to no interferon and incubated at 37 centigrade to make cells After 2 weeks of differentiation, the Poddar was cultivated in a Petri dish and cultured for 24 hours in a serum-free cleaning medium. The recombinant mouse IL-17 protein 100ng/ml was used to stimulate the mouse kidney foot cells to incubate for 24 hours. Then, Erk, p38 inhibitor 10umol/l was incubated with IL-17 stimulated podar respectively for 24 hours, and cells were collected and Western Blot detected: small The protein level of p38, Erk and p-p38, p-Erk in rat renal podropoda. After adding p38 and Erk inhibitors, the expression of neutrophil related chemokine IL-8, Th1 related chemokine IL-12 mRNA expression and fluorescence quantitative PCR determination. Results: the results of the 1. light microscopy were compared with the control group, the model group had partial adhesion of balloon, mesangial cell proliferation and matrix. In addition, the renal tubular cavity partially appeared protein tube type and renal interstitial inflammatory cell infiltration, and the observation result model group of neutralizing IL-17 antibody group compared with the model group was compared with the control group, the glomerular morphology and structure were slightly abnormal, and the podocyte process was generally fused with the.IL-17 neutralization antibody group and the IL-17 protein expression of the renal tissue of the kidney tissues of the mice with light.3. was stained with DAB. The positive cells of IL-17 were mostly cytoplasmic staining positive, and a small amount of IL-17 expression in normal renal tissue, mainly concentrated in the renal tubules, but no expression in the glomeruli; in the model group, the expression of IL-17 on the renal tubules was significant, and it had a small amount of expression in the glomeruli; the IL-17 neutralization antibody group was lighter than the model group. Compared with the control group, the adriamycin nephrotic group, IL- The positive coloring degree of cells in the 17 neutralization antibody group was obviously enhanced and the positive coloring degree of the P0.05. model group was significantly higher than that of the IL-17 neutralization antibody group. The IL-17 related chemokines in the renal tissue of P0.05.4. mice were CCR6, the mRNA expression of CCL20 was compared with the normal control group. The CCR6 in the model group, the IL-17 and the antibody groups increased, the mRNA expression of CCL20 was increased, P0.05. modules were increased. The expression of CCR6 and CCL20mRNA in the group was stronger than that of the IL-17 neutralization antibody group. The expression of mRNA expression of neutrophils related chemokine in the renal tissue of each group of P0.05.5. was compared with that of the normal control group. The expression of CXCL1, CXCL2, and IL-8 was higher in the model group and in the IL-17 neutralization antibody group. The expression of mRNA increased, and the expression of mRNA expression of Thl related chemokines, CXCL10, IL-12, IFN- gamma in the kidney tissues of P0.05.6. mice was compared with the normal control group. The expression of CXCL9, CXCL10, IL-12, and gamma decreased in the model group and in the IL-17 neutralization antibody group, and the expression of the model group was lower than that of the neutralization antibody group. The level of serum IL-17 in mice was compared with that in the normal control group. The level of IL-17 in the model group and the IL-17 neutralization antibody group increased and the IL-17 level of the P0.05. model group was higher than that of the IL-17 neutralization antibody group. The changes of the serum KIM-1, NGAL, Csy C in the P0.05.8. groups were compared with those of the normal control group, the model group, the IL-17 neutralization antibody group and the normal control group. The level of serum M-1, NGAL, and Csy C increased, and the level of KIM-1, NGAL, Csy C in the P0.05. model group was higher than that of the IL-17 neutralizing antibody group. The expression of neutrophil related chemokine IL-8 mRNA was compared with that of normal group after p38 inhibitor and Erk inhibitor. The IL-8 mRNA expression in IL-17 stimulation group, p38 inhibitor group and Erk inhibitor group increased obviously. P0.05. and IL-17 stimulation group were compared with p38 inhibitor group and inhibitor group. The expression of IL-8 mRNA in the agent group was significantly higher. The expression of Thl related chemokine IL-12 mRNA expression was compared with the normal group after P0.05.11. application of p38 inhibitor and Erk inhibitor. The IL-12 mRNA expression in the IL-17 stimulation group decreased obviously, and the p38 inhibitor group and the inhibitor group all increased obviously. The expression of A was significantly increased, and the P0.05.p38 inhibitor group was significantly higher than that of the Erk inhibitor group IL-12 mRNA. P0.05. conclusion: 1. in the adriamycin induced mouse nephropathy model, the expression of IL-17 in the renal tissue is abnormal, which can raise the neutrophils and participate in the pathological process of inflammation in ADR mice. A small amount of IL-17 can maintain the normal function of the kidney. In the mouse nephropathy model induced by adriamycin, the level of serum IL-17 increased significantly, suggesting that IL-17 promotes the inflammatory process in ADR mice, and it can also promote the decrease of the differentiation of Thl cells. A large number of IL-17 can cause serious impairment of renal function in ADR mice, and the peritoneal injection of.3. mice and IL-17 antibody can reduce the IL-17 of ADR mice in.3. mice. Level, alleviating neutrophils recruitment, neutralizing IL-17 antibody and inhibiting Thl cell differentiation, and renal function improvement,.4.IL-17 may regulate the inflammatory factors of renal podocyte in mice by MAPK signaling pathway and.5.IL-17 can activate p38 and Erk pathway signals. In this process, p38 path signal is the main.6.IL-17 promoting neutrophil aggregation. It produces inflammatory response and changes the balance of natural CD4+T cell differentiation to Th1/Th2, induces differentiation into Th2 and inhibits Thl.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R726.9

【相似文献】