【作者】 聂春岩；

【导师】 陈莉明； 常宝成； 梁东春；

【作者基本信息】 天津医科大学 ， 内科学， 2010， 硕士

【摘要】 目的糖尿病肾病(diabetic nephropathy, DN)是糖尿病重要的微血管并发症之一,目前20%-30%的糖尿病患者合并肾病,已成为糖尿病主要致死原因。DN的病理学特征包括肾小球系膜区增宽和肾小球基底膜弥漫性增厚,这种变化的组织学基础即是细胞外基质(ECM)积聚。正常情况下,ECM产生和降解的动态平衡处于机体的精确调控下。基质金属蛋白酶(MMPs)及其组织抑制剂(TIMPs)是ECM降解的主要调节因素。高糖通过何种途径调节肾脏中MMPs和TIMPs的表达在国内外的研究中尚不明确。丝裂原活化蛋白激酶(MAPK)信号通路作为直接调节转录因子活性和基因表达的信息传递系统与MMPs及TIMPs表达的关系值得深入探讨。MMPs主要降解底物是ECM的重要成分Ⅳ型胶原,但p38MAPK信号通路在糖尿病肾病发生中的作用与Ⅳ型胶原的关系尚无定论。本研究通过观察高糖刺激下SD大鼠肾小球系膜细胞p38MAPK、MMP-2/TIMP-2、Ⅳ型胶原mRAN及蛋白表达的变化和MMP-2/TIMP-2比值的改变,以及使用特异性抑制剂SB203580干预后它们的改变,明确糖尿病肾病时MMP-2/TIMP-2及Ⅳ型胶原的表达变化,探讨p38MAPK信号通路是否参与高糖诱导的肾小球系膜细胞MMP-2/TIMP-2及Ⅳ型胶原的表达变化的调节。方法将培养的SD大鼠肾小球系膜细胞分为6组：(1)正常对照组：5.6mmol/L的葡萄糖；(2)高糖A组：10 mmol/L的葡萄糖；(3)高糖B组：20 mmol/L的葡萄糖；(4)高糖C组：30 mmol/L的葡萄糖；(5)SB203580组：30 mmol/L的葡萄糖+10μmol/L SB203580; (6)甘露醇组：5.6mmol/L的葡萄糖+24.4mmol/L的甘露醇。待细胞生长状况良好,细胞生长至90%左右密度后,同步化24小时,分别用以上各因素处理细胞并继续培养,于培养48小时后收集细胞,提取总mRNA,逆转录成cDNA,采用实时荧光定量PCR法检测各组肾小球系膜细胞p38MAPK、MMP-2、TIMP-2及Ⅳ型胶原mRNA表达；提取总蛋白,采用Western-blot法检测MMP-2、TIMP-2及Ⅳ型胶原的蛋白表达。结果(1)正常条件下系膜细胞p38MAPK、MMP-2、TIMP-2及Ⅳ型胶原在mRNA水平上均有表达,MMP-2、TIMP-2及Ⅳ型胶原在蛋白水平上均有表达；(2)高糖组和甘露醇组p38MAPKmRNA的表达均高于正常组,但高糖组p38MAPKmRNA的表达增加更明显,且当葡萄糖浓度由10mmol/L增加到20mmol/L及30mmol/L时,p38MAPKmRNA的表达也逐渐增加；(3)高糖组和甘露醇组TIMP-2 mRNA和蛋白的表达较正常组增高,而MMP-2 mRNA和蛋白的表达较正常组下降,MMP-2/TIMP-2比值较正常组下降,高糖组的这些改变较相同渗透压的甘露醇组更为明显,且随着葡萄糖浓度的增加,这些变化也越来越明显；(4)高糖组和甘露醇组Ⅳ型胶原mRNA和蛋白的表达较正常组增高,高糖组的这些改变较相同渗透压的甘露醇组更为明显,Ⅳ型胶原mRNA表达随着葡萄糖浓度增加而增加,但Ⅳ型胶原的蛋白表达并无此规律；(5)与同浓度葡萄糖组相比,p38MAPK抑制剂SB203580组系膜细胞MMP-2mRNA与蛋白表达和MMP-2/TIMP-2比值增加,TIMP-2及Ⅳ型胶原mRNA与蛋白表达下降,p38MAPK mRNA表达无明显变化。结论(1)高糖诱导SD大鼠肾小球系膜细胞p38MAPK和TIMP-2 mRNA和蛋白表达升高,而使系膜细胞MMP-2 mRNA和蛋白表达降低,MMP-2/TIMP-2比值下降,且高糖引起的这些变化呈浓度依赖性；(2)高糖诱导SD大鼠肾小球系膜细胞Ⅳ型胶原mRNA和蛋白表达升高,且Ⅳ型胶原的mRNA的表达呈葡萄糖浓度依赖性,而Ⅳ型胶原的蛋白表达并非呈葡萄糖浓度依赖性；(3)甘露醇增加系膜细胞p38MAPK和TIMP-2及Ⅳ型胶原mRNA和蛋白表达,减少MMP-2mRNA与蛋白表达。高糖诱导的系膜细胞p38MAPKmRNA表达的增多可能部分是由于高渗透压引起的；(4) p38MAPK抑制剂SB203580能逆转高糖诱导的MMP-2、TIMP-2及Ⅳ型胶原mRNA和蛋白表达,却不能改变高糖诱导的p38MAPKmRNA表达的增加；(5)在糖尿病肾病中,p38MAPK的表达水平及活性与MMP-2、TIMP-2及Ⅳ型胶原mRNA和蛋白表达有密切关系。p38MAPK信号通路与MMP-2/TIMP-2在糖尿病肾病时细胞外Ⅳ型胶原的聚集过程发挥了重要作用,对p38MAPK信号通路与MMP-2、TIMP-2的深入研究将为今后糖尿病肾病的预防和治疗及新药研发提供思路。更多还原

【Abstract】 Objective Diabetic nephropathy (DN) is an important microvascular complication in diabetes, about 20%-30% of diabetic patients complicate with kidney disease. Diabetic nephropathy has become a major cause of death in diabetic patients. The pathological features of DN include glomerular mesangial broadening and diffuse thickening of glomerular basement membrane. The histological basis for this change is extracellular matrix (ECM) accumulation. Under normal circumstances, ECM production and degradation in body is under precise control. Matrix metalloproteinases (MMPs) and tissue inhibitor metalloproteinases (TIMPs) is the main regulator of ECM degradation. How high glucose regulating the expression of MMPs and TIMPs is not clear. The relationship between the expression of MMPs/ TIMPs and the p38MAPK should be explored. How p38MAPK signal pathway regulating the expression of collagen IV in diabetic nephropathy is inconclusive. In this study, we investigated the effect of high glucose and SB203580, inhibitor of p38MAPK, on the expression of p38MAPK and MMP-2/TIMP-2 in cultured SD rat glomerular mesangial cell and discussed the mechanism of p38MAPK signal transduction pathways in DN as well as the relation between p38MAPK and MMP-2/TIMP-2.Methods The cultured SD rat mesangial cells were divided into six groups, normal glucose group(5.6mmol/L), high glucose group(including different concentration gluclose:10 mmol/L,20mmol/L,30 mmol/L), SB203580 group(30 mmol/L glucose plus 10μmol/L SB203580) and mannitol group(5.6mmol/L glucose+24.4mmol/L mannitol). After the cells were incubated in different conditions for 48 hours, total RNA was isolated with Trizol reagent, the expression of p38MAPK, MMP-2 and TIMP-2 mRNA were examined by Real time Quantitative PCR. Total protein was isolated with RIPA, the expression of MMP-2/TIMP-2 and collagenⅣprotein were examined by western blot.Results (1) p38MAPK, MMP-2 and TIMP-2 mRNA express in normal glucose group; (2) The expression of p38MAPK mRNA is increased in high glucose group and mannitol group than that in normal glucose group, but the expression of p38MAPK mRNA in high glucose groups is much higher than mannitol group, which is in a concentration-dependent manner; (3) Compared with normal glucose group, the expression of TIMP-2 mRNA and protein is increased, but MMP-2 mRNA and protein and MMP-2/TIMP-2 mRNA and protein ratio in high glucose groups and in mannitol group are decreased; these changes in high glucose groups are more significantly than that in mannitol group, which are dependent of glucose concentration; (4) Compared with normal glucose group, the expression of collagen IVmRNA and protein is increased, these changes in high glucose groups are more significantly than that in mannitol group, the expression of collagenⅣmRNA are dependent of glucose concentration, but the expression of collagenⅣprotein are independent of glucose concentration,(5) The mRNA and protein expression of MMP-2 and MMP-2/TIMP-2 mRNA and protein ratio are higher in SB203580 group, and TIMP-2 mRNA and protein is lower than that in high glucose group (30 mmol/L), whereas there is no significant difference between high glucose group and SB203580 group in the expression of p38MAPK mRNAConclusion (1) High glucose can increase significantly the mRNA and protein expression of p38MAPK and TIMP-2, and decrease the mRNA and protein expression of MMP-2, and also decrease MMP-2/TIMP-2 mRNA and protein ratio in cultured mesangial cells, and these changes are dependent of glucose concentration; (2) The expression of collagen IV mRNA are dependent of glucose concentration, but the expression of collagen IV protein are independent of glucose concentration; (3) Mannitol increase the expression of p38MAPK mRNA in mesangial cells, high osmitic pressure at least partly participates in increasing of p38MAPK mRNA induced by high glucose; (4) The inhibitor of p38MAPK can reverse the changes of MMP-2, TIMP-2 mRNA、protein and MMP-2/TIMP-2 mRNA and protein ratio induced by high glucose, but can not inhibit the increased expression of p38MAPK mRNA induced by high glucose; (5) P38MAPK is related to the mRNA and protein expression of MMP-2、TIMP-2 and collagen IV in diabetic nephropathy.更多还原