【作者】 赵辉；

【导师】 焦波；

【作者基本信息】 山东大学 ， 药理学， 2011， 硕士

【摘要】 肾脏疾病如IgA肾病(IgA nephropathy, IgAN)、肾小球肾炎等是常见的原发性肾小球疾病,部分患者可能进展为终末期肾病,危害人们的健康。转化生长因子β1 (transforming growth factor beta 1, TGF-β1)、整合素连接激酶(integrin-linked kinase, ILK)、β1整合素(β1 integrin)及过氧化物酶体增贿物激活受体γ(peroxisome proliferators-activated receptor gamma, PPARγ)等与肾脏病的进展有着十分密切的关系。同时,细胞外基质(extracellular matrix, ECM)代谢失衡,合成增多或降解减少导致的ECM蛋白组分过度积聚是肾小球硬化的主要病理特征。鱼油由深海鱼类中提取,含丰富的n-3多不饱和脂肪酸(n-3 polyunsaturated fatty acid, n-3 PUFA),其主要活性成分是二十碳五烯酸(eicosapentaenoic acid, EPA)和二十二碳六烯酸(docosahexaenoic acid, DHA)。鱼油除具有抗血栓、舒血管、调血脂、抗肿瘤等经典作用外,还具有一定的肾脏保护作用,延缓肾脏疾病的进展。本实验通过脂多糖(lipopolysaccharide, LPS)致大鼠肾小球系膜细胞(glomerular mesangial cells, GMCs)损伤模型观察EPA和DHA对GMCs MMPs、PPARγ、ILK、β1整合素、TGF-β1表达的影响,探讨其对损伤的GMCs可能的保护机制,为临床应用提供理论依据。方法取对数生长期GMCs,常规培养24 h后,换无血清培养24 h,使细胞同步于G0期。分为：正常对照组；LPS(模型组,LPS10.0mg/L)组；EPA (10μmol /L、100μmol/L)组；DHA (10μmol/L、100μmol/L)组,其中用药组均用含LPS培养基培养。各组分别培养24 h、48 h后,进行指标测定。1采用ELISA法观察EPA和DHA对LPS刺激GMCs TGF-β1分泌的影响；2采用SDS-PAGE gelatin zymopraphy法分析EPA和DHA对LPS刺激GMCs MMP-2、9活性的影响；3采用Western Blot法观察EPA和DHA对LPS刺激GMCs PPARy和ILK蛋白表达的影响；4采用RT-PCR法观察EPA和DHA对LPS刺激GMCs PPARy、ILK、β1整合素及TGF-β1 mRNA表达的影响；结果1 EPA、DHA对LPS刺激GMCs TGF-β1的影响LPS刺激下,GMCs TGF-β1合成分泌和mRNA的表达较正常对照组增加EPA和DHA可使LPS诱导的GMCs TGF-β1合成分泌和mRNA的表达下降。2 EPA、DHA对LPS刺激GMCs MMP-2,9活性的影响LPS刺激下,GMCs MMP-2,9合成分泌较正常对照组减少；EPA和DHA使LPS诱导的GMCs MMP-2,9合成分泌增加3 EPA、DHA对LPS刺激GMCs PPARy表达的影响LPS可使GMCs PPARy mRNA和蛋白表达下降；EPA和DHA可增加LPS诱导的GMCs PPARy mRNA和蛋白的表达。4 EPA、DHA对LPS刺激GMCs ILK表达的影响LPS可增加GMCs ILK mRNA和蛋白的表达；EPA和DHA可使LPS诱导的GMCs ILK mRNA和蛋白表达下降。5 EPA、DHA对LPS刺激GMCsβ1整合素mRNA表达的影响LPS可增加GMCsβ1整合素mRNA的表达；EPA和DHA可使LPS诱导的GMCsβ1整合素mRNA表达下降。结论1 EPA和DHA可以降低LPS诱导的GMCs TGF-β1蛋白和基因的表达。2 EPA和DHA能够促进LPS诱导的GMCs分泌MMP-2,9。3 EPA和DHA能够促进LPS诱导的GMCs PPARy蛋白和基因的表达。4 EPA和DHA可以降低LPS诱导的GMCs (31整合素和ILK蛋白和基因的表达。更多还原

【Abstract】 BackgroundRenal diseases such as IgA nephropathy, glomerular nephritis is a common primary glomerulonephritis, some patients may develop into end stage renal disease which endangers heath. Factors such as transforming growth factor beta 1 (TGF-β1), integrin linked kinase (ILK),β1 integrin and peroxisome proliferator-activated receptor y (PPARy) have a very close relationship with renal diseases. Meanwhile, the accumulation of extracellular matrix (ECM) protein components caused by metabolic imbalance like increased synthesis or decreased degradation is the main pathological features of glomerulosclerosis.Fish oil is extracted from deep-sea fish, and is rich in n-3 polyunsaturated fatty acids (n-3 PUFA). Its main active ingredients are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Besides the anti-thrombotic effect, vasodilation, blood lipoid adjustment and anti-tumor effect, fish oil has a renal protective effect. In this study, we investigated the effects of EPA and DHA on the expression of MMPs, PPARy, ILK,β1 integrin and TGF-β1 in glomerular mesangial Cells (GMCs) stimulated by LPS (LPS) and to explore its possible protection mechanisms.Methods The mesangial cells in logarithmic growth phase were used. After serum-free cultured for 24 h, the cells were synchronized in the Go period, and then divided into six groups:the control group; LPS group (model group, LPS 10.0 mg/L); LPS plus EPA (10μmol/L、100μmol/L) group; LPS plus DHA (10μmol/L、100μmol/L) group. All the cells were cultured in 37℃and 5% CO2 for 24 h or 48 h.1 The TGF-β1 secretion was measured by enzyme linked immunosorbent assay (ELISA);2 The MMP-2,9 concentration in supernatant was measured by gelatin zymography;3 The expression of PPARγand ILK was determined by Western Blotting;4 The mRNA expression of PPARγ, ILK,β1 integrin and TGF-β1 were determined by RT-PCR;Results1 Effect of EPA and DHA on the expression of TGF-β1 in GMCs stimulated by LPSLPS could promote TGF-β1 secretion and mRNA expression; EPA and DHA could inhibit the GMCs TGF-β1 secretion and mRNA expression stimulated by LPS.2 Effects of EPA and DHA on the Secretion of MMP-2,9 in GMCs stimulated by LPSLPS could inhibit MMP-2,9 secretion; EPA and DHA could promote the GMCs MMP-2,9 secretion stimulated by LPS.3 Effects of EPA and DHA on the PPARγexpression in GMCs stimulated by LPSLPS could inhibit PPARγprotein and mRNA expression; EPA and DHA could promote the GMCs PPARγprotein and mRNA expression stimulated by LPS.4 Effects of EPA and DHA on the ILK expression in GMCs stimulated by LPSLPS could promote ILK protein and mRNA expression; EPA and DHA could inhibit the GMCs ILK protein and mRNA expression stimulated by LPS.5 Effects of EPA and DHA on the mRNA expression ofβ1 integrin in GMCs stimulated by LPSLPS could promoteβ1 integrin mRNA expression; EPA and DHA could inhibit the GMCsβ1 integrin mRNA expression stimulated by LPS.Conclusion1 EPA and DHA could promote the GMCs MMP-2,9 secretion stimulated by LPS.2 EPA and DHA could inhibit the GMCs TGF-β1 secretion and mRNA expression stimulated by LPS.3 EPA and DHA could promote GMCs PPARy protein and mRNA expression stimulated by LPS.4 EPA and DHA could inhibit the GMCsβ1 integrin and ILK protein and mRNA expression stimulated by LPS.更多还原