【作者】 余荣杰；

【导师】 吴雄飞；

【作者基本信息】 第三军医大学 ， 外科学， 2006， 博士

【摘要】 肾组织炎性细胞浸润是多种慢性肾病(Chronic kidney disease,CKD)慢性化进程中的常见表现,在介导肾小管间质慢性炎症和肾纤维化的发生发展中具有重要作用。在病理状态下外周血炎症介质表达及活性增加,同时肾脏固有细胞激活并表达各种趋化因子,募集单核-巨噬细胞、多形核白细胞和T细胞等炎症细胞向肾组织浸润,与肾固有细胞相互作用,表达一系列细胞因子/生长因子、粘附分子和细胞外基质(extracellular matrix, ECM)成分,从而直接或间接放大肾间质损伤,同时细胞因子和趋化因子的协同作用可以启动更多的炎症反应途径,形成瀑布效应,导致肾间质不可逆损伤。但是关于炎症细胞向炎症部位募集、迁徙机制,除公认的炎症趋化因子作用外,是否存在其它因素影响炎细胞迁徙浸润的生物学行为,如炎症细胞迁徙移行路径中结构及微环境变化等,目前尚不清楚。炎症细胞趋向炎症反应区必须穿越由细胞外基质和基底膜组成的屏障。该屏障主要由结构蛋白和糖氨聚糖两种成分构成,后者主要成分是硫酸乙酰肝素蛋白多糖(heparan sulfate proteoglycan,HSPGs)。HSPGs主要由一个蛋白核心和数个与之共价连接的线性硫酸乙酰肝素(heparan sulfate,HS)侧链组成。起初认为结构蛋白的破坏是细胞侵袭、转移的决定性步骤。事实上糖氨聚糖作为ECM和基底膜的另一主要成分,其降解也是细胞侵袭游走所必须的。类肝素酶(heparanase,Hpa)是近年发现的一种以HSPGs为底物、裂解HSPGs核心分子HS的内切糖苷酶,Hpa在炎症及肿瘤细胞的侵袭、转移中具有重要作用。在炎症和免疫反应中,淋巴细胞、单核巨噬细胞等受刺激后可立即释放Hpa,裂解HSPG,突破血管壁基底膜屏障和内皮的完整性,有利于循环中的免疫活性细胞外渗,迁徙移行到炎症区域而发生炎症反应。炎症反应的发动是机体防御性反应,但病理情况下如炎症趋化及炎症反应不去除,持续的修复过度,则导致炎症反应区域不可逆纤维化。鉴于近年关于Hpa在肿瘤细胞转移以及免疫活性细胞向炎症区迁徙中作用的认识,我们推想在进展性CKD中,肾组织炎性细胞的浸润以及此后的慢性进行性肾纤维化进程中可能也涉及Hpa的异常表达,激活的T淋巴细胞等炎性细胞在肾脏局部发挥Hpa活性,促进肾组织炎细胞浸润,激活肾脏固有细胞,释放HS结合因子、释放促纤维化细胞因子和生长因子等,从而触发肾脏纤维化进程。所以本课题旨在观察CKD中Hpa与肾组织炎细胞浸润的相关性;利用阿霉素肾病模型持续大量蛋白尿、肾间质进行性炎症性损伤、纤维化的特点,模拟人类CKD,观察Hpa在阿霉素肾病中的表达,并探讨Hpa抑制剂的抗炎症治疗作用,为防治CKD及延缓其纤维化进程提供新思路。主要实验内容:1、第一部分采用RT-PCR及免疫组化方法观察进展期慢性肾炎患者及健康肾移植供者外周血淋巴细胞及肾组织Hpa表达,同时观察肾组织炎细胞浸润情况;2、第二部分通过细胞培养,RT-PCR方法观察Hpa抑制剂海带多糖对培养的CKD患者外周血淋巴细胞Hpa表达的影响;3、第三部分建立阿霉素肾病模型,采用RT-PCR及免疫组化方法进一步观察肾病大鼠外周血淋巴细胞及肾组织Hpa表达及其与肾组织炎细胞浸润的相关性,检测临床指标观察海带多糖对阿霉素肾病的治疗作用,液闪计数观察海带多糖对阿霉素肾病大鼠淋巴细胞增殖活性的影响,以及免疫组化检测肾组织中重要的趋化因子、粘附分子以及致纤维化因子的表达变化。主要实验结果:1、CKD患者组外周血淋巴细胞Hpa mRNA表达较健康对照组增高;正常肾组织Hpa仅低表达,而慢性肾病患者肾组织中Hpa表达均不同程度增高,主要表达在肾小管间质炎细胞浸润部位,与炎细胞浸润程度具有相关性。2、培养CKD患者外周血淋巴细胞中Hpa mRNA表达仍高于正常对照,海带多糖可减弱Hpa表达。3、阿霉素肾病大鼠外周血淋巴细胞及肾组织Hpa表达均较正常大鼠增高,与肾组织炎细胞浸润程度的加重具有相似的时相性。4、海带多糖治疗组大鼠尿蛋白、血肌酐、血尿素氮等临床指标均较肾病对照组减轻;肾组织炎细胞浸润程度及纤维化程度较肾病对照组减轻。5、海带多糖治疗组大鼠外周血淋巴细胞Hpa mRNA及肾组织Hpa表达均低于肾病对照组。6、海带多糖治疗组大鼠淋巴细胞增殖活性较肾病对照组减弱。7、海带多糖治疗组肾组织IL-6、IL-8、ICAM-1及TGF-β表达较肾病对照组减弱。全文主要结论:1、Hpa在CKD中存在高表达,肾组织炎性细胞浸润与Hpa高表达具有相关性。2、海带多糖对CKD淋巴细胞Hpa表达具有抑制作用。3、海带多糖对阿霉素肾病具有治疗作用,其机制可能通过抑制Hpa、抑制淋巴细胞增殖而减轻肾间质炎症反应,从而减轻肾组织重要的趋化因子、粘附分子及致纤维化因子表达,延缓CKD纤维化。更多还原

【Abstract】 As a common symptome in the development of various Chronic kidney diseases(CKD), renal inflammatory cell infiltration plays an important role in mediation of the occurrence and development of both chronic renal tubulointerstitial inflammation and renal fibrosis. Pathologically , there is an increase of expression and activity of inflammation mediators in peripheral blood, meanwhile, intrinsic renal cells are activated , express various chemotatic factors, and conduct inflammatory cells such as mononuclear-macrophage, polymorphonuclear leucocytes and T cells to nephridial tissue ,which interact with intrinsic renal cells ,then express a series of cytokines and growth factors, adhesion molecules and extracellular matrix (ECM), so renal interstitial injury is directly or indirectly magnified, meanwhile, synergistic effect of cytokines and chemotatic factors will lead to more paths of inflammatory reactions to form a water fall effect. This cascade effect results in irreversible injury of renal interstitium. However, with regard to the mechanism of recruitment and migration of inflammatory cell to inflammatory site , it is still unclear whether there are other factors contributing to the biological behavior of migration and infiltration of inflammatory cells beside the effects of known inflammatory chemotatic factors up to now.To migrate to inflammatory reaction site, inflammatory cells have to pass through the barrier composed of extracellular matrix and basement membrane .This barrier mainly contains structural protein and glycosaminoglycan, the main component of glycosaminoglycan is heparan sulfate proteoglycan (HSPGs). HSPGs are glycoproteins mainly consisting of a protein core and side chains of several linear heparan sulfate (HS) covalent linking to the protein core. At the beginning, destruction of structural protein is regarded as the decisive step for cell invasion and migration. But actually, as a component of ECM and basement membrane, the degradation of glycosaminoglycan is essensial for cell invasion.Heparanase(Hpa)is an endoglycosidase which takes HSPGs as substrate,then splits core molecule HS of HSPGs . Hpa plays an important role in inflammation, invasion and migration of tumor cells. In inflammation and immune reaction , once on stimulation , lymphocytes and mononuclear-macrophages immediately release Hpa and split HSPG, so integrity of vessel wall basement membrane is destroyed ,which enables the extravasation of immune competent cell and they migrate to the inflammation site. The body’s defensive reaction leads to inflammatory reaction, but pathologically, if inflammatory chemotaxis and inflammatory reaction haven’t been stopped, persistent excessive repair will contribute to irreversible fibrosis of the inflammatory site.According to recent knowledge of the role that Hpa plays in metachoresis of immunocompetence cells and migration to inflammatory site of immune competent cells, we guess that in progressive CKD, renal inflammatory cell infiltration and successive chronic progressive renal fibrosis may have relationship with abnormal expression of Hpa, inflammatory cells such as activated T lymphocytes secrete Hpa, and Hpa performs its activity in regional kidney, which promotes inflammatory cell infiltration into nephridial tissue, activates intrinsic renal cells, releases HS binding factors, pro-fibrotic cytokines and growth factors, which triggers renal fibrosis proceeding.Therefore, our study aimed at investigation of the relationship between Hpa and renal inflammatory cell infiltration. As adriamycin nephrosis model has a character of continuous quantitative proteinuria, progressive inflammatory injury and fibrosis of renal interstitium, so similarly, we observed Hpa expression in adriamycin nephrosis and discussed the anti-inflammatory effect of Hpa inhibitors to explore new way to prevent CKD and delay its fibrosis.Main contents of the experiment1. In the first part of our study, RT-PCR and immunochemical method were used to investigate the Hpa expression in the nephridial tissue and peripheral blood lymphocyte of patients with progressive chronic nephritis and healthy donor for kidney transplant. Meanwhile, we observed the inflammatory cell infiltration of the nephridial tissue.2. In the second part, after cell culture, we used RT-PCR to observe the effect of Hpa inhibitor laminaran on Hpa expression in cultured peripheral blood lymphocytes from CKD patients.3. In the third part, we first established an adriamycin nephrosis model, then investigated the Hpa expression in murine peripheral blood lymphocytes and nephridial tissue, and their relationship with nephridial tissue inflammatory cell with RT-PCR and immunochemical method, tested clinical indexes to see the therapeutical effect of laminaran on adriamycin nephrosis model, observed the influence of laminaran on proliferation of lymphocytes in adriamycin nephrosis rat with liquid scintillation method, and measured the expression variance of chemokines, adhesion molecules and pro-fibrotic cytokines that were crucial to nephridial tissue with immunochemical method.Main result1. We use RT-PCR and immunohistochemistry to investigate the Hpa expression of peripheral blood lymphocytes and nephridial tissues in CKD patients and healthy donors of kidney for transplant, as well as the infiltration of inflammatory cells2. Hpa mRNA expression is still higher in cultured peripheral blood lymphocytes than normal control group. Laminaran can decrease Hpa expression.3. The Hpa expressions, in peripheral blood lymphocytes and nephridial tissue from adriamycin nephrosis rat, both are higher than normal rat, which has a similar phase characteristic to the degree of aggravation of inflammatory cells infiltration in nephridial tissue.4. Compared with control group with renal disease, clinical indexes including Urine protein, serum creatinine, blood urea nitrogen, etc, show that the disease is relieved in laminaran treated group rat, extent of inflammatory cell infiltration and fibrosis in nephridial tissue are palliated.5. Both Hpa mRNA expression in peripheral blood lymphocytes and nephridial tissue of laminaran treated group rats are lower than these of control group with renal disease.6. Lymphocytes proliferation activitiesof laminaran treated group rats are lower than these of control group with renal disease.7. Expression of IL-6、IL-8、ICAM-1 and TGF-βin nephridial tissue of laminaran treated group is lower than that of control group with renal disease.Main Conclusion1.There is high Hpa expression in CKD, infiltration extent of inflammatory cells in nephridial tissue closely relates with high expression of Hpa.2. Laminaran can inhibit the expression of Hpa in CKD Lymphocytes. 3. Laminaran has curative effect on adriamycin nephrosis, probably by inhibiting Hpa and Lymphocytes proliferation to palliate inflammatory reaction in renal interstitium, which palliates the expression of important chemotactic factors, adhesion molecules, profibrotic cytokines in nephridial tissue, and delays CKD fibrosis.更多还原