【作者】 胡晓晶；

【导师】 柳方娥；

【作者基本信息】 山东大学 ， 儿科学， 2010， 硕士

【摘要】 目的通过制备脂多糖(LPS)致大鼠肾小球系膜细胞(GMCs)损伤模型,研究二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)对炎症状态下GMCs表达基质金属蛋白酶(MMPs)及其抑制剂金属蛋白酶组织抑制因子(TIMPs)、过氧化物酶体增殖物激活受体γ(PPARγ)、单核细胞趋化蛋白-1(MCP-1)、转化生长因子-β1(TGF-β1)的影响,探讨EPA、DHA对损伤的GMCs可能的保护机制。方法与结果1.实时定量PCR方法检测EPA、DHA对LPS刺激GMCs MMP-2/TIMP-2 mRNA的表达体外培养大鼠GMCs,取第4代细胞计数后按1×105细胞／孔浓度接种于6孔培养板,实验分六组：正常对照组；LPS刺激组(LPS终浓度为10μg·mL-1)EPA(终浓度10μmol·L-1、100μmol·L-1)组；DHA(终浓度10μmol·L-1、100μmol·L-1)组。培养48h后,实时定量PCR方法检测MMP-2、TIMP-2mRNA表达。结果显示：LPS刺激能够使GMCs表达MMP-2 mRNA及MMP-2/TIMP-2比值明显降低、TIMP-2 mRNA明显增高(P<0.01)。EPA、DHA显示出了对MMP-2、TIMP-2 mRNA表达的抑制作用及对MMP-2/TIMP-2比值的促进作用。2.实时定量PCR方法检测EPA、DHA对LPS刺激GMCs MMP-9/TIMP-1 mRNA的表达取第4代细胞计数后按1×105细胞／孔浓度接种于6孔培养板,实验分组同上。培养48h后,实时定量PCR方法检测MMP-9、TIMP-1mRNA表达。结果显示：LPS刺激能够使GMCs表达MMP-9 mRNA及MMP-9/TIMP-1比值明显降低、TIMP-1 mRNA明显增高(P<0.01)。EPA、DHA显示出了对MMP-9、TIMP-1mRNA表达的抑制作用及对MMP-9/TIMP-1比值的促进作用。3.实时定量PCR方法检测EPA、DHA对LPS刺激GMCs PPARy mRNA的表达细胞计数后按1×105细胞／孔浓度接种于6孔培养板,实验分组同上。分别培养24h、48h、72h后,实时定量PCR方法检测PPARy mRNA表达。结果显示：LPS刺激GMCs后,细胞表达PPARy mRNA明显减少(P<0.01)。EPA、DHA能促进GMCs PPARy mRNA的表达。4.实时定量PCR方法检测EPA、DHA对LPS刺激GMCs MCP-1、TGF-β1 mRNA的表达细胞计数后按1×105细胞／孔浓度接种于6孔培养板,实验分组同上。分别培养24h、48h、72h后,实时定量PCR方法检测MCP-1、TGF-β1 mRNA表达。结果显示：LPS刺激GMCs后,细胞表达MCP-1、TGF-β1 mRNA明显增加(P<0.01)。EPA、DHA能抑制GMCs MCP-1、TGF-β1 mRNA的表达。5.免疫细胞化学方法检测EPA、DHA对LPS刺激GMCs PPARγ、TGF-β1蛋白的表达细胞计数后按1×104细胞／孔浓度接种于24孔培养板,实验分组同上。培养48h后,免疫细胞化学方法检测PPARγ、TGF-β1蛋白。结果显示：LPS刺激GMCs后,细胞分泌PPARy蛋白明显减少、TGF-β1蛋白明显增加(P<0.05)。正常组细胞的胞浆区染色低,LPS组的细胞则可见明显的黄染。EPA、DHA显示出了对PPARγ蛋白分泌的促进作用及对TGF-β1蛋白分泌的抑制作用。结论1 EPA、DHA能抑制LPS刺激的GMCs表达MMP-2、MMP-9、TIMP-1、TIMP-2mRNA,提高MMP-2/TIMP-2、MMP-9/TIMP-1比值。2 EPA、DHA能促进LPS刺激的GMCs PPARy mRNA及蛋白的表达。3 EPA、DHA能抑制LPS刺激的GMCs MCP-1 mRNA的表达。4 EPA、DHA能抑制LPS刺激的GMCs TGF-β1 mRNA及蛋白的表达。更多还原

【Abstract】 ObjectiveTo investigate the effects of eicosapentaenoic acid(EPA) and docosahexaenoic acid(DHA) on matrix metalloproteinases(MMPs)、tissue inhibitors of metalloprotein-ases(TIMPs)、peroxisome proliferator-activated receptor gamma(PPARy)、monocyte chemoattractant protein-1(MCP-1)、transforming growth factor beta 1(TGF-β1) on rat glomerular mesangial cells(GMCs) stimulated by lipopolysaccharide(LPS), and explore the possible protective mechanism of EPA and DHA to impaired GMCs.Methods and Results1. Effect of EPA and DHA on the expression of MMP-2/TIMP-2 mRNA on GMCs stimulated by LPS by real-time PCRGMCs were cultured in vitro. Count the 4th generation cell at a 1×105per well to the kind of 6-well plates, and then divided into six groups:control group; LPS group(with LPS final concentration of 10μg-mL-1); EPA group(with EPA final concentration of 10μmol·L-1 and 100μmol·L-1); DHA group(with DHA final concentration of 10μmol·L-1 and 100μmol·L-1). The expression of MMP-2 and TIMP-2 mRNA of GMCs was measured by real-time PCR after incubated 48h.The result showed that the level of MMP-2 mRNA and the ratio of MMP-2/TIMP-2 were significantly attenuated, TIMP-2 mRNA significantly increased when stimulated by LPS(P＜0.01). EPA and DHA could decrease the level of MMP-2 and TIMP-2 mRNA, increase the ratio of MMP-2/TIMP-2. 2. Effect of EPA and DHA on the expression of MMP-9/TIMP-1 mRNA on GMCs stimulated by LPS by real-time PCRCount the 4th generation cell at a 1×105per well to the kind of 6-well plates, and then divided into the same six groups. The expression of MMP-9 and TIMP-1 mRNA of GMCs was measured by real-time PCR after incubated 48h.The result showed that the level of MMP-9 mRNA and the ratio of MMP-9/TIMP-1 were significantly attenuated, TIMP-1 mRNA significantly increased when stimulated by LPS(P＜0.01). EPA and DHA could decrease the level of MMP-9 and TIMP-1 mRNA, increase the ratio of MMP-9/TIMP-1.3. Effect of EPA and DHA on the expression of PPARy mRNA on GMCs stimulated by LPS by real-time PCRGMCs were cultured for 24h、48h、72h, respectively. The expression of PPARy mRNA of GMCs was measured by real-time PCR. The result showed that the level of PPARγmRNA was significantly attenuated when stimulated by LPS(P<0.01). EPA and DHA could increase the level of PPARy mRNA.4. Effect of EPA and DHA on the expression of MCP-1、TGF-β1 mRNA on GMCs stimulated by LPS by real-time PCRGMCs were cultured for 24h、48h、72h, respectively. The expression of MCP-1、TGF-β1 mRNA of GMCs was measured by real-time PCR. The result showed that the level of MCP-1、TGF-β1 mRNA was significantly increased when stimulated by LPS(P＜0.01). EPA and DHA could decrease the level of MCP-1、TGF-β1 mRNA.5. Effect of EPA and DHA on the level of PPARγ、TGF-β1 protein on GMCs stimulated by LPS by immunocytochemistryCount the cell at a 1×104per well to the kind of 24-well plates, and then divided into the same six groups. The expression of PPARγ、TGF-β1 protein of GMCs was detected by immunocytochemistry after incubated 48h. The result showed that the level of PPARy protein was significantly attenuated and TGF-β1 protein significantly increased when stimulated by LPS(P<0.05). EPA and DHA could increase the level of PPARy mRNA and decrease the level of TGF-β1 mRNA. Conclusion1. EPA and DHA can decrease the level of MMP-2、MMP-9、TIMP-1、TIMP-2 mRNA of GMCs stimulated by LPS, increase the ratio of MMP-2/TIMP-2、MMP-9/TIMP-1.2. EPA and DHA can increase the level of PPARy mRNA and protein of GMCs stimulated by LPS.3. EPA and DHA can decrease the level of MCP-1 mRNA of GMCs stimulated by LPS.4. EPA and DHA can decrease the level of TGF-β1 mRNA and protein of GMCs stimulated by LPS.更多还原