【作者】 黄琳；

【导师】 林洪丽；

【作者基本信息】 大连医科大学 ， 内科学， 2007， 硕士

【摘要】 目的:糖尿病肾病(Diabetes nephropathy, DN)是糖尿病严重的慢性并发症之一,也是糖尿病患者重要的死亡原因。正常条件下,肾小球细胞外基质(ECM)的合成与降解保持着动态平衡。任何环节的细微变化均可导致肾小球ECM成与降解失衡,出现肾小球基质膜(GBM)的破坏和ECM堆积,长期得不到纠正,即可导致肾小球硬化。DN发病机制复杂,影响因素众多,目前认为凝血是DN发病过程中重要原因之一。众多生长因子、细胞因子启动外源性凝血途径,凝血酶原复合物( Factor Va/Factor Xa/Ca2+和磷脂)激活凝血酶原形成凝血酶,凝血酶催化可溶性纤维蛋白原形成不溶性的纤维蛋白,纤维蛋白单体在肾小球系膜区形成交联纤维(XFB),最后导致细胞外基质(ECM)的积聚。本研究检测Factor V在DN病人血清水平和DN大鼠肾脏组织蛋白水平表达情况及TIMP-1、MMP-9、Ⅳ型胶原、在DN大鼠肾脏组织蛋白水平的表达情况,旨在探讨凝血在DN发病过程中的作用,为DN的诊断治疗寻找新的途径。方法:收集糖尿病肾病病人共60例,按照1999年世界卫生组织(WHO)诊断标准确诊。按Mogensen建议,根据糖尿病患者肾功能和结构病变的演进及临床表现分为Ⅱ-V期;正常对照组:健康正常人10名。取静脉血3ml,采用促凝分离胶,3000r/min离心15 min,取血清-20℃冻存,应用ELISA的方法检测FV血清水平,450nm酶标仪进行吸光度检测。健康雄性Wistar大鼠38只,一次大量腹腔注射streptozotoci(nSTZ)。72小时后测随机血糖>16.6mmol/L,尿糖++-+++,尿量增加150%,为糖尿病肾病大鼠模型的标准进行筛选。分别饲养2、4、6、8周,监测血糖,血糖下降者予以剔除。大鼠腹腔注射柠檬酸-柠檬酸钠缓冲液作为对照组。采用免疫组织化学的方法检测FV、TIMP-1、MMP-9和Ⅳ型胶原蛋白水平的表达。结果:FV在DN病人血清水平是明显升高的,与正常对照组比较P<0.01。FV在2、4、6、8周DN大鼠肾脏组织蛋白水平的表达与对照组比较也有明显升高P<0.05;TIMP-1在对照组与2、4、6、8周DN大鼠肾脏组织中的表达分别为:4.088%±1.041,7.950%±1.418,10.483%±1.107,14.867%±1.034,18.461%±1.98,与正常组比较P<0.01;MMP-9在正常对照组与2、4、6、8周DN大鼠肾脏组织中的表达分别为:9.559%±1.99,23.45%±3.68,16.62%±1.23,13.36%±3.36,7.65%±2.5,正常组与8周比较:P>0.05,2周与对照组比较:P<0.01,6周和8周比较:P≤0.05,6周和4周比较:P>0.05,与TIMP-1作为MMP-9抑制物的结论相一致;Ⅳ型胶原在对照组与2、4、6、8周DN大鼠肾脏组织中的表达分别为:7.917%±0.8,12.474%±0.58,13.5%±0.73,14.77%±1.12,16.12%±1.01,P<0.05,表明在DN发病过程中,有明显的ECM积聚,具有显著统计学意义。结论: DN病人血清FV与正常对照比较有明显升高,并随着病情不断发展而逐渐升高;FV在DN大鼠肾脏组织蛋白水平的阳性表达也是明显升高的;TIMP-1和Ⅳ型胶原在DN大鼠肾脏组织与对照比较有明显表达,而MMP-9随着病情的发展其阳性表达是下降的。更多还原

【Abstract】 Objective: Diabetic nephropathy(DN) is one of serious chronic diabetic complications, is aso the main death cause of diabetic patients. The synthesis and degradation of glomerular extracellular matrix(ECM) maintained a dynamic balance . Any of the slight changes can lead to imbalance of synthesis and degradation of the ECM, which result in a damage of glomerular matrix membrane(GBM) and the accumulation of ECM. If this situation can not be treat properly for a long time, glomerulosclerosis will develope. Pathogenesis of DN is complex and related to many factors. Currently, coagulation is considered as one of the reasons in the development of DN. Many cytokines activat extrinsic coagulation pathway. Prothrombin complex, which is Factor V a/Factor X a/Ca2+ and phospholipid, converts prothrombin to thrombin. Then thrombin turn into fibrinogen to fibrin. Fibrin monomer converts to cross-linked fiber(XFB) in glomerular mesangial. Finally, ECM accumulate in Glomerular mesangial cells. Therefore, in this study, we examined the expression of FV in DN patients and kidney of DN rats. At the same time,we also examined expression of TIMP-1、MMP-9、PAI-1、typeⅣcollagen and P-selection in kidney of DN rats. The objective is to discuss the role of coagulation in DN and find a new way of diagnosis and treatment for DN.Methods: Sixty patients with Diabetic nephropathy were included, These petients were diagnosed according to the diagnosis standards issued by World Health Organization (WHO) in 1999. By Mogensen, the clinical manifestations of the patients ranged from II-V stage based on renal function and structure changes of the diabetic patients. The control group:ten healthy controls. 3ml of intravenous blood were taken, using procoagu-lant separation plastic, centrifuged at 3000r/min for 10 minutes, obtained blood serum sample and stored it at -20°C. Measuring FV serum levels by ELISA and measuring absorbance at 450nm with spectrophotometer.One hundred and fifty male wistar rats were administrated streptozotocin (STZ) at high dose by intraperitoneal injection. Seventy two hours later, random serum glucose >16.6mmol/L, urine glucose ++-+++, urine output increased by 150%, so that the rats are prepared for selection of diabetic nephropathy model. Then the rats were grouped into 5 groups and were fed up to 2,4,6,8 weeks respectively. During this experiment, Serum glucose was monitored and rats with low glucose would be excluded.Control group: Rats were administrated citric acid-itric acid sodium citrate buffer by intraperitoneal injection. According to Clinical manifestations, renal function and pathology change, classified intoⅡ-V stages. The expression of Factor V、TIMP-1, MMP-9, PAI-1, P-selectin and type IV collagen protein in kidney of DN were measured with immunohistochemical method.Results: FVblood serum is significantly higher in DN patients than control group, P<0.01.The expression of FVincreased significantly compared with control group in kidney of DN rats, P<0.05; The expression of TIMP-1 in the control group and DN rats with II-V stage are 4.088%±1.041,7.950%±1.418,10.483%±1.107,14.867%±1.034,18.461%±1.98(P<0.01).The expression of MMP-9 in the control group and DN rats with II-V stage are 9.559%±1.99,23.45%±3.68,16.62%±1.23,13.36%±3.36,7.65%±2.5. Control group compared with Vstage, P>0.05. Control group compared withⅡstage: P<0.01.Ⅳstage compared with Vstage:P≤0.05.Ⅳstage compared withⅢstage: P>0.05. these results are consistent with the inclusion that TIMP-1 is the inhibitor of MMP-9. The expression of typeⅣcollagen in the control group and DN rats II-V stage are 7.917%±0.8,12.474%±0.58,13.5%±0.73,14.77%±1.12,16.12%±1.01,P<0.05. These results show that ECM accumulated significantly in the development of DN and the results is statistically significant.Conclusion: Present data suggest that the expression of FV increased significantly in DN patients and it is increasing with the development of DN. Similarly, the expression of FV also increased in and DN rats. The resultsare similar in patients and rats. Compared with the control group, the expression of TIMP-1, MMP-9, PAI-1, P-selectin and type IV collagen in DN rats increased significantly.howerer, with the development of DN, the expression of MMP-9 declined. So we concluded that MMP-9 was regulated by TIMP-1.更多还原