【作者】 刘容锐；

【导师】 林洪丽；

【作者基本信息】 大连医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:观察TIMP-1短发夹式RNA(TIMP-1shRNA)及来氟米特(leflunomide,LEF)对糖尿病肾病大鼠肾间质纤维化的影响方法:将雄性Wistar大鼠120只随机分为6组:正常对照组(N组)、糖尿病组(DM组)、糖尿病TIMP-1shRNA转染组(DM-RNAi组)、糖尿病HKshRNA转染组(DM-VE组)、糖尿病来氟米特治疗组(DM-LEF组)、糖尿病羧甲基纤维素组(DM-CMC组)。观察各组大鼠肾功能变化;PAS染色观察大鼠肾间质病理形态改变;RT-PCR方法检测大鼠肾组织中TIMP-1、MCP-1、ICAM-1、VCAM-1、TGF-β1、Fractalkine和RANTES mRNA各时间点基因水平的变化;Western Blot方法检测大鼠肾组织中TIMP-1、MMP-2、MMP-9、TGF-β1、MCP-1、ICAM-1、Fractalkine各组蛋白水平的变化,以及免疫组化检测大鼠肾脏FN、TIMP-1、MMP-2、NF-κB、TGF-β1、MCP-1、α-SMA的表达。结果:随时间延长,糖尿病大鼠的肾脏功能逐渐受累,DM组尿蛋白/尿肌酐,血肌酐,血尿素氮以及相对肾重(KW/BW)与N组比较均显著升高(P<0.05),DM-RNAi组,DM-LEF组与DM组比较表达下调(P<0.05)。肾组织病理PAS染色可见DM组肾脏呈进行性肾间质纤维化改变,包括肾小管的萎缩、扩张、炎细胞浸润等;而DM-RNAi组和DM-LEF组肾组织病变明显减轻。基因水平检测TIMP-1在10、12、14、16周表达,随时间延长,与N组比较DM组表达升高(P<0.05),DM-RNAi组和DM-LEF组抑制TIMP-1表达(P<0.05)。RT-PCR检测MCP-1、ICAM-1、VCAM-1、TGF-β1、Fractalkine和RANTES mRNA的表达,可见DM组表达升高(P<0.05),DM-RNAi组和DM-LEF组表达下调(P<0.05)。同时,TIMP-1蛋白表达与基因水平表达一致;DM组MMP-2、MMP-9随时间延长蛋白表达降低(P<0.05),而DM-RNAi组和DM-LEF组表达上调(P<0.05);DM组TGF-β1、MCP-1、ICAM-1、Fractalkine表达升高(P<0.05),DM-RNAi组和DM-LEF组表达下调(P<0.05)。免疫组化示DM组TIMP-1、FN、α-SMA、NF-κB、TGF-β1、MCP-1阳性染色表达面积增多(P<0.05),DM-RNAi组和DM-LEF组明显比DM组减少(P<0.05)。结论:1.TIMP-1在糖尿病肾病大鼠肾脏中的高表达对糖尿病肾病间质纤维化的发生发展起到重要作用。2.TIMP-1shRNA在基因水平阻断TIMP-1表达,促进细胞外基质(extracellular material ,ECM)降解增加改善肾间质纤维化;此外,TIMP-1shRNA可能通过抑制炎症反应改善肾小管间质损伤,对糖尿病大鼠肾脏起到保护作用。3.糖尿病肾病中来氟米特减轻间质炎症反应,抑制炎症因子表达,抑制肌成纤维细胞增殖,从而减轻肾间质纤维化。更多还原

【Abstract】 Objective: To study the effects of TIMP-1 small hairpin RNA (TIMP-1 shRNA) and leflunomide on the renal interstitial fibrosis caused by diabetic nephropathy in rats.Methods: 120 healthy Wister rats were randomly divided into 6 groups: normal control group(N group)、diabetes mellitus(DM group)、TIMP-1 shRNA group(DM-RNAi group)、HKshRNA group(DM-VE group)、leflunomide(DM-LEF group)、Carboxymethyl Cellulose group(DM-CMC group). Rats’ renal function of each group were monitored;The histological changes of renal interstitial tissues were observed by PAS staining. The mRNAs of TIMP-1、MCP-1、ICAM-1、VCAM-1、TGF-β1、Fractalkine and RANTES were reverse transcribed and quantified by RT-PCR .The proteins of TIMP-1、MMP-2、MMP-9、TGF-β1、MCP-1、ICAM-1 and Fractalkine changes were analyzced by Western Blot analysis.The proteins of FN、TIMP-1、MMP-2、NF-κB、TGF-β1、MCP-1 andα-SMA were assessed by immunohistochemistry analysis.Results: After the rat model were established ,with the extension of time, kidney function of the diabetic rats’ suffered gradually,urine protein/ urine creatinine,serum creatinine, blood urea nitrogen,relative kidney weight(KW/BW) in DM group were higher than N group (P<0.05); compared with DM group,the DM-RNAi group and the DM-LEF group were decrease remarkably (P<0.05).Nephridial tissue pathomorphology mani- fested that:In DM group, interstitial fibrosis including tubular astrophy,loss and dilation,inflammatory cell infiltration were evident.These changes were all improved by TIMP-1shRNA and leflunomide treatment.Respectively analysising DN rat kidney TIMP-1mRNA at 10、12、14、16 weeks by RT-PCR,with the extension of time,the DM group’s expression was higher than the N group’s(P<0.05),but the DM-RNAi group and the DM-LEF group were decreased conspicuously than DM group(P<0.05).In the gene level,the expressions of MCP-1、ICAM-1、VCAM-1、TGF-β1、Fractalkine and RANTES mRNA were increased in the DM group,but the DM-RNAi group and the DM-LEF group were decreased.Meanwhile,in the protein level rat TIMP-1 was consonanced with the gene level; compared with N group,MMP-2 and MMP-9 were accrescenced in DM group;yet,the DM-RNAi group and the DM-LEF group descented significantly than DM group(P<0.05) ; either TIMP-1shRNA or leflunomide significantly decreased the levels of TGF-β1、MCP-1、ICAM-1 and Fractalkine,in DM group was inceased dramatically(P<0.05).In nephridial tissue,through the immunohistochemistry,the masc dyeing areas of FN、TIMP-1、MMP-2、NF-κB、TGF-β1、MCP-1、α-SMA enhanced in DM group, compared with the DM group,the DM-RNAi group and the DM-LEF group extracellular matrix diminished (P<0.05).Conclusions:1. TIMP-1 plays an important role in the development of diabetic nephropathy interstitial fibrosis.2. TIMP-1shRNA downregulates the gene expression of TIMP-1,promotes the degradation of extracellular material and improves interstitial fibrosis; in addition,TIMP-1shRNA might inhibit inflammation and improve tubules injury ,therefore protects rat renal function.3. Leflunomide may attenuate interstitial inflammation reaction, suppresse the expressions of inflammatory factors,improve interstitial fibrosis which is probably correlated with myofibroblast proliferation. therefore,it can protect renal function in diabetic nephropathy.更多还原