肾间质纤维化的分子机制探讨以及秋水仙碱防治作用的研究

【作者】 黄文彦；

【导师】 陈荣华；

【作者基本信息】 南京医科大学 ， 儿科学， 2003， 博士

【摘要】 肾间质纤维化(Renal Interstitial Fibrosis，RIF)是各种肾脏疾病进展为终未期肾衰的共同途径和结局。是由于各种原因引起的肾小管间质炎性细胞浸润、间质细胞(尤其成纤维细胞)增生并产生各种炎症介质，从而使细胞外基质(Extracellular Matrix，ECM)过度沉积于肾小管间质，最终导致纤维组织过度增生并使正常组织结构毁坏。间质纤维化的程度不仅与肾脏疾病慢性进展速度和病人的预后密切相关，而且其较肾小球与肾脏功能的关系更为密切。尽管目前对RIF有了一定的认识，但有关RIF的发生机制，尤其分子机制目前尚不完全清楚。所以努力探讨RIF发病机制并积极寻找抗肾脏纤维化药物一直是肾脏病研究者们的热门话题。 特异性微管解聚剂秋水仙碱成功地用于治疗痛风已有几百年历史。最近资料表明，其不仅为一种抗炎症药物，也具有明显抗纤维化作用。但秋水仙碱对RIF的确切防治效果尚不清楚，也更缺乏系统的研究观察。 所以，为了进一步探讨RIF的发病机制以及秋水仙碱对RIF的防治作用和机制，我们进行了下列五部分研究。 第一部分：秋水仙碱对肾间质纤维化防治作用的形态学观察 目的：观察秋水仙碱对肾间质纤维化的防治作用及其效果。 方法：(1)动物分组：56只SD雄性大鼠随机分成3组：对照组即假手术组(16只)；模型组(20只)和治疗组(20只)。治疗组在单侧输尿管结扎(Unilateral Ureteral Obstruction，UUO)基础上予秋水仙碱100.0μg／kg·d腹腔注射(1次／天)每周5天，模型组在UUO基础上予生理盐水代替秋水仙碱腹腔注射每周5天。分别于实验第3天、第7天、第14天和第21天分批处死大鼠，对照组每次处死4只，模型组和治疗组每次各处死5只。大鼠处死后留取结扎侧肾组织进行形态学和免疫组织化学研究。(2)指标观察：肾组织经HE、PAS、Masson’s三色染色观察各实验组肾小管损伤、肾小 南京医科人学博十学位论义管萎缩，肾间质炎性细胞浸润，间质纤维化程度。同时利用兔疫组织化学技术观察肾＊、管上皮细胞、间质细胞增殖细胞核抗原oCNA）以及间质胶原 Ill（COL Ill）表达。结果：口 ）UUO后出现了广泛肾小管损伤，秋水仙碱对肾小管损伤具有一定的保护作用。秋水仙碱具有促进肾小管上皮细胞再生、修复能力，以及抑制间质细胞增生作用／m模型组肾间质炎性细胞浸润随病程进展进行性加重；秋水仙碱治疗后其炎性细胞浸润明显减少。＊X疗组间质纤维化指数、间质 COL Ill阳性表达指数均显著低于同期模型组。结论：＊）首次通过UUO肾间质纤维化动物模型发现秋水仙碱具有明显抗纤维化作用。m秋水仙碱预防肾间质纤维化是通过减少和抑制炎症细胞浸润，促进肾小管上皮细胞再生等作用而实现的。第二部分：肾间质纤维化以及秋水仙碱抗纤维化的分子机制研究目的：口）寻找肾间质纤维化以及秋水仙碱抗纤维化相关基因。仪）阐明炎症因子和趋化因子在＊UO纤维化肾组织中的动态表达规律以及秋水仙碱对其影响。方法：（仔用基因芯片枝术观察纤维化大鼠肾组织、秋水仙碱治疗组－肾组织基因表达谱改变，并通过生物信息学处理寻找纤维化相关基因和秋水仙碱抗纤维化基因。仅）分别采用逆转录多聚酶链式反应（RTPCR）和免疫组织化学技术观察UU O不同时期肾组织转化生长因子川厂＊卜pl）、白细胞介素一16（IL一仰、M管i田胞粘附分子一l（Vascular Cell Adhesion Molec。fief，VCAM－l）、i田胞间粘附分子 （Intercellula Adhesion Molecule，ICP。M－l）mRNA，TGF乃、11。八 蛋白动态表达规律；以及秋水仙碱对其表达的影 。向。结果：门）＊＊O后以及秋水仙碱治疗后均发生了广泛基因表达谱改变；差 异表达基固涉及免疫作胞粘附、DNA转录、蛋白修饰、信号转导、细胞分裂分化、能量代谢、物质转运、细胞骨架等不同功能的基因群。筛选出重要的纤维｛匕相关基队 TGF卡、IL刁、血管内皮生长因子（VE（iFX 2 南京L科人学阶1，牛仆论义ICAM－l、Prohibitin（PHB）、胰岛素样结合蛋白 7（Insuin卜ke Growth FactorBinding Protein 7，IGFBP7）、PTKZ、核转录因子 EZFS、基质金属蛋白酶15（MMP 5）、RacZ、生长激素抑制素支体 3（Somatostatin receptor 3，SSTR3）、丝氨酸／苏氨酸激酶15（Serine／TIre。nine Kin。sc 15，STK15）等。秋水仙碱可能王要通过调节 TGF抑、VCAM1、CFLAR、MMP、ROC口等基因表达及其功能而起到抗纤维化效果。mUUO纤维化大鼠肾组织TGF卡、ILl m删A和蛋白表达明显上调并随纤维化程度加重而进行性上升，秋水仙碱对前者具有显著抑制效果，对后者具有明显促进作用。UUO纤维化时 VCAMl 表达明显上调并持续高表达，而 ICAM刁在RIF早期表达明显上调并迅速下降至正常水平。秋水仙碱对ICAM刁、VCAM刁 表达均具有显著抑制效果。结论：厂）初步筛选出重要的＊?更多还原

【Abstract】 Renal Interstitial Fibrosis (RIF), a major consequence and common pathway of all kidney diseases associated with progression to end-stage renal failure, is the result of proliferation of fibroblasts within the interstitium, infiltration of the interstitium by monocytes or inflamed cells, and the excess production of matrix within the interstitium. The severity and degree of RIF is strongly correlation between renal functional loss and the prognosis of renal patients, recent studies have shown that histological grading of the fibrosis of the tubulointerstitium is more closely correlated with the loss of renal function than the histological grading of glomerulosclerosis. The mechanisms of RIF, especially the molecular pathogenesis of which, were little understood, So the recent interests of nephrologists have focused on elucidating pathogenesis of RIF and how to ameliorate the courses of progressive renal diseases.As a specific microtubule disrupter, colchicine has been succeeding used for centuries in treatment of gout. Recently its anti-inflammatory and antifibrotic properties of colchicine have been employed for an increasing number of suggested and approved indications of anti-fibrosis effects (including primary biliary cirrhosis, idiopathic pulmonary fibrosis, liver fibrosis). However, the effects of colchicine on the RIF were unclear.In order to research the mechanisms of RIF, and to understand the roles of treatment of RIF by colchicine, the present study consists five parts.Part I: Morphological research on treatment of RIF in rats with Unilateral ureteral obstruction (UUO) by colchicineObjective: The purpose of this study was to observe the effects of colchicine on RIF in UUO rats.Methods: 26 male Sprague-Dawley rats were divided into three groups: sham-operated control group (16 rats), colchicine-treated group (20 rats): UUO + colchicine 100ug/kg.d I.P for 5 days per week, model group (group of unilateral ureteral obstruction, 20 rats): UUO+ saline 100ug/kg.d I.P for 5 days per week. The rats of three groups were sacrificed at 3, 7, 14, 21 days. The renal tissues were stained by hematoxylin and eosin (HE), periodic acid-Schiff reaction (PAS), Masson’s trichrome in general morphological research; and expression of PCNA and COL III were also evaluated immunohistochemically in this study.Results: (1) the extensive and progressive of renal tubular lesions were found in morphologically in UUO in rats, the changes of tubular damages were ameliorated after treatment with colchicine. Colchicine could be beneficial in regenerations of renal tubular epithelial cells, and inhibit the inflammatory cells infiltration in tubulointerstitium. (2) The numbers of infiltrated cells in interstitium were significantly loss in colchicine-treatment groups than those of model groups. (3) Colchicine could decrease the deposition of extracellular matrix (including collagen type III) in renal tubulointerstitium space.Conclusion: Colchicine had beneficial effects on interstitial fibrosis in rats with UUO Sprague-Dawley.Part II: The study on molecular mechanisms of renal interstitial fibrosis and its influences of that with treatment of colchicineObjective: To Screen the genes involved in RIF and investigate the effect of colchicine on the fibrosis and to elucidate the expression profile of cytokines and chemokines in UUO rats.Methods: (1) The gene expression pattern of renal tissues from both UUO group and those from colchicine-treated group was detected by cDNA microarray and the resulting information was analyzed with bioinformatics. (2)The expression pattern of TGF-β1, IL-1β, VCAM-1, and ICAM-1 were confirmed by RT-PCR and immunohistochemistry.Results: (1) Differently expressed gene pattern consists of the genes encode the Immune/cell adhesiveness, transcription of DNA, post-transcriptional modification of protein, signal transduction, cell division and differentiation, energy metabolism, substance transportation, as well as cytoskeleton associated gene nes更多还原