【作者】 陈磊；

【导师】 邵怡；

【作者基本信息】 上海交通大学 ， 外科学（泌尿外科）， 2017， 硕士

【摘要】 目的:1.研究RLX对梗阻性肾病肾脏巨噬细胞极化以及肾纤维化的影响。2.研究RLX对梗阻性肾病后TLR4信号通路的影响。3.验证RLX通过TLR4信号通路调控巨噬细胞极化改善梗阻性肾病肾纤维化。方法:1.以单侧输尿管梗阻小鼠为梗阻性肾病模型,运用渗透压泵泵入RLX,观察给药后肾纤维化程度以及肾组织巨噬细胞极化情况;使用RAW264.7巨噬细胞系诱导形成M1型巨噬细胞和M2型巨噬细胞,研究RLX干预后巨噬细胞极化情况变化。2.以单侧输尿管梗阻小鼠为梗阻性肾病模型,运用渗透压泵泵入RLX,观察给药后TLR4信号通路变化情况;使用RAW264.7巨噬细胞系诱导形成M1型巨噬细胞和M2型巨噬细胞,研究RLX干预后巨噬细胞TLR4信号通路情况变化。3.使用RAW264.7巨噬细胞系诱导形成M1型巨噬细胞和M2型巨噬细胞,研究RLX和TLR4信号通路抑制剂干预后巨噬细胞极化情况变化。结果:1.UUO组较Sham组肾脏病理损害变化增加,并出现单核细胞渗出,并且肾脏胶原蛋白和Fibronectin的表达显著增高。UUO+relaxin组较UUO组肾脏病理损害变化减轻,单核细胞渗出情况减轻,胶原蛋白的表达显著降低。运用western blot的方法检测Fibronectin的表达情况,结果显示UUO+relaxin组较UUO组肾脏Fibronectin表达降低。UUO+relaxin组M1巨噬细胞标志物的表达情况较UUO组显著下降,而UUO+relaxin组M2巨噬细胞标志物的表达较UUO组显著上调。M0、M1和M2巨噬细胞在RLX处理后,其M1巨噬细胞标志物(iNOS,TNF-α,CCL-3,IL-23)的基因表达量下降而M2巨噬细胞标志物(arginase,CX3CR1,IL-4,Arg-1,IL-10,Ym1)的基因表达量升高。2.UUO组较Sham组TLR4信号通路蛋白TLR4、Myd88、p65、p-p65蛋白表达显著升高。UUO+relaxin组较UUO组TLR4信号通路蛋白TLR4、Myd88、p65、p-p65蛋白表达显著下降。M0、M1和M2巨噬细胞在RLX处理后TLR4信号通路相关蛋白TLR4、Myd88、p65、p-p65蛋白表达以及基因表达显著下降。3.M1巨噬细胞TAK-242+Relaxin组较Relaxin组M1巨噬细胞标志物iNOS蛋白表达升高,M2巨噬细胞TAK-242+relaxin组较Relaxin组M2巨噬细胞标志物iNOS蛋白表达下降。TAK-242+Relaxin组与TAK-242组比较,除了M2巨噬细胞中,M1巨噬细胞标志物IL-23和M2巨噬细胞标志物IL-10基因表达有差异,其余M1、M2巨噬细胞标志物iNOS、TNF-α,CCL-3和M2巨噬细胞标志物MRC、IL-4、arginase、iNOS、Ym1基因表达均无差异。结论:1.RLX能够改善梗阻性肾病肾纤维化;RLX能够促进梗阻性肾病肾脏巨噬细胞形成M2型极化;体外实验中RLX能够促进巨噬细胞形成M2型极化。2.RLX能够抑制UUO后小鼠肾脏TLR4信号通路,体外实验中RLX能够抑制巨噬细胞TLR4信号通路。3.T LR4信号通路抑制剂能够阻断RLX对巨噬细胞极化的调控更多还原

【Abstract】 Purpose:1.To study the effects of Relaxin on renal macrophage polarization and renal fibrosis in obstructive nephropathy.2.To study on the effect of Relaxin on TLR4 signal pathway after obstructive nephropathy.3.To verify that RLX abrogates renal interstitial fibrosis by regulating macrophage polarization via inhibition of Toll-like receptor 4 signaling.Materials and method:1.Mice with unilateral ureteral obstruction in obstructive nephropathy model,observed renal fibrosis and renal tissue macrophage polarization after RLX treatment;RAW264.7 macrophage cell line induced into M1 macrophages and M2 macrophages,observed macrophage polarization changes after Relaxin treatment.2.Mice with unilateral ureteral obstruction in obstructive nephropathy model,observed the change of TLR4 signaling after RLX treatment;RAW264.7 macrophage cell line induced into M1 macrophages and M2 macrophages,observed the change of TLR4 signaling after Relaxin treatment.3.RAW264.7 macrophage cell line induced into M1 macrophages and M2 macrophages,observed macrophage polarization changes after Relaxin and TAK-242 treatment.Results:1.H&E and Sirius red staining showed that marked interstitial inflammation and fibrosis occurred 5 days after UUO and that relaxin can reduce cell infiltration and interstitial fibrosis.Compared with the kidneys of mice pretreated with relaxin,the kidneys of day 5 UUO-injured mice had higher fibronectin levels.Five days following UUO,gene expression analysis of macrophages from the kidneys revealed increased both M1 and M2 markers.Relaxin pretreatment caused significant increases in the gene expression levels of most renal macrophage M2 markers and decrease M1 markers.the expression levels of most of the M1 marker genes decreased while M2 markers increased when macrophages were treated with relaxin.2.TLR4,Myd88,p65 and pp65 expression levels were upregulated at day 5 following UUO,while relaxin significantly decreased their mRNA and protein expression levels.TLR4,Myd88,p65 and pp65 genes and proteins were expressed at low levels after relaxin treatment in the different macrophage phenotypes compared with those treated with vehicle3.The proteins and genes expression profiles of M1 markers and M2 markers in macrophage were not different between those treated with TAK-242 and those treated with TAK-242 and relaxin.Conclusions:Relaxin can downregulate the Toll-like receptor(TLR)4 signaling,shift macrophage polarization toward the M2 phenotype and ameliorate renal fibrosis at the early stages of UUO.In vitro experiments confirmed that relaxin can downregulate the TLR4 signaling and induce the M2 macrophage transition.Furthermore,the macrophage phenotype transition actions of relaxin were significantly blocked by TAK-242,a TLR4 antagonist,in vitro.更多还原