【作者】 韩琳；

【导师】 余晨；

【作者基本信息】 同济大学 ， 临床医学（专业学位）， 2022， 博士

【摘要】 背景:环境中的铅污染可以引起慢性肾脏疾病,并在世界范围内构成巨大的公共卫生威胁。铅中毒常见的临床表现是肾脏纤维化。我们以往的研究显示:醋酸铅可以通过转化生长因子-β(Transforming Growth Factor-β,TGF-β)/Smad信号通路促进肾脏纤维化的发生,并且能够刺激趋化因子CC chemokine ligand 20(CCL20)基因、信使RNA(m RNA)和蛋白的表达明显升高。目的:在以往研究的基础上,进一步深入研究趋化因子CCL20基因对铅所致的肾脏纤维化的影响,探索调控靶基因CCL20与纤维化相关指标的关系,筛选并验证调控靶基因CCL20的微小核糖核酸(micro RNA或miRNA),并在小鼠铅中毒动物模型上验证miRNA对铅损伤导致的肾脏纤维化的作用。方法:1.体外培养小鼠肾间质成纤维细胞,加入不同浓度醋酸铅(0.5、1、2μmol/L),应用实时定量PCR(Realtime PCR)技术检测CCL20、趋化因子受体(chemokine receptor 6,CCR6)、TGF-β1、纤维连接蛋白(Fibronectin,FN)和I型胶原(Collagen I)的m RNA表达,生化方法检测成纤维细胞羟脯氨酸的含量,酶联免疫吸附测定(ELISA)检测CCL20和TGF-β1的蛋白分泌,采用蛋白质印迹法(Western blot)检测CCL20、CCR6、TGF-β1、Collagen I、FN、smad2/3、磷酸化smad2/3(p-smad2/3)、蛋白激酶B(AKT)和磷酸化蛋白激酶B(p-AKT)蛋白表达。2.1μmol/L的醋酸铅处理小鼠肾间质成纤维细胞,同时用小干扰RNA(small interfering RNA,si RNA)干扰CCL20基因,Realtime PCR检测CCL20、CCR6、TGF-β1、FN和Collagen I的m RNA表达,生化方法检测成纤维细胞羟脯氨酸的含量,ELISA检测CCL20和TGF-β1的蛋白分泌,Western blot检测CCL20、CCR6、TGF-β1、Collagen I、FN、smad2/3、p-smad2/3、AKT和p-AKT蛋白表达,细胞增殖毒性检测(Cell counting kit8,CCK8)实验分别检测0、6、12、24h细胞增殖能力。3.利用miRWalk、Target Scan、miRanda等在线软件预测并筛选出可能调控小鼠CCL20基因分数较高的miRNA,并通过不同浓度醋酸铅(0.5、1、2μmol/L)处理小鼠肾间质成纤维细胞,Real-PCR检测各候选miRNA的表达,并用荧光素酶报告基因分析筛选靶向CCL20基因的miRNA。用1μmol/L的醋酸铅处理小鼠肾间质成纤维细胞,分别加用miR-143-5p模拟物或联合10ng/ml的CCL20重组载体处理,Realtime-PCR检测CCL20、CCR6、TGF-β1、FN和Collagen I的m RNA表达,生化方法检测成纤维细胞羟脯氨酸的含量,ELISA检测CCL20和TGF-β1蛋白分泌,Western blot检测CCL20、CCR6、TGF-β1、Collagen I、FN、smad2/3、p-smad2/3、AKT和p-AKT蛋白表达,用CCK8实验分别检测0、6、12、24h检测细胞增殖能力。4.24只小鼠随机分为A组:对照组,B组:铅损伤组,C组:铅损伤组+miR-143-5p模拟物组,每组8只。使用醋酸铅溶液(10mg/kg)腹腔注射干预4周,C组在醋酸铅干预4周后,尾静脉注射1OD/次/天的miR-143-5p模拟物,连续2周,A组和B组注射生理盐水,处死小鼠后分别用HE染色和MASSON染色观察分析肾脏组织纤维化情况,Realtime-PCR检测CCL20、CCR6、TGF-β1、FN和Collagen I的m RNA表达,ELISA方法检测外周血中CCL20和TGF-β1的表达,肾组织生化法检测羟脯氨酸含量,Western blot检测CCL20、CCR6、TGF-β1、Collagen I、FN、smad2/3、p-smad2/3、AKT和p-AKT蛋白表达。结果:1.小鼠肾间质成纤维细胞在醋酸铅作用下,可以剂量依赖性地增加CCL20和TGF-β1的m RNA水平、蛋白分泌和表达,羟脯氨酸含量也呈剂量依赖性地增加,CCL20的受体CCR6以及细胞外基质Collagen I和FN的m RNA及蛋白表达也随醋酸铅浓度依赖性地增加,信号通路蛋白p-smad2/3和p-AKT的表达水平随着醋酸铅浓度的升高而升高。2.siRNA干扰抑制了醋酸铅诱导的成纤维细胞增殖,能够降低CCL20和TGF-β1的m RNA水平、蛋白分泌和表达,同时也抑制了羟脯氨酸含量的表达、CCR6以及细胞外基质Collagen I和FN的表达,信号通路蛋白p-smad2/3和p-AKT的表达水平也被抑制。3.通过生物信息学分析软件预测了5个评分较高的靶向CCL20的miRNA,其中,在醋酸铅处理成纤维细胞后,miR-143-5p的表达随醋酸铅浓度的增加呈剂量依赖性地降低,通过双荧光素酶报告基因分析,验证了miR-143-5p可以直接靶向结合CCL20来抑制其表达。在功能验证实验中,miR-143-5p可以拮抗醋酸铅诱导的成纤维细胞增殖,降低羟脯氨酸含量、CCL20、CCR6、TGF-β1和细胞外基质的m RNA和蛋白表达,而CCL20过表达后这种作用被抑制。此外,miR-143-5p减弱了醋酸铅暴露的成纤维细胞中Smad/AKT两条通路的激活,而这些影响可以被CCL20的过表达所抵消。4.在动物实验中,尾静脉注射miR-143-5p可以延缓小鼠组织中肾脏纤维化的进展,抑制CCL20和TGF-β1的m RNA水平、蛋白分泌和表达,同时降低羟脯氨酸含量、抑制肾脏组织中CCR6以及细胞外基质Collagen I和FN的表达以及信号通路蛋白p-smad2/3和p-AKT的表达。结论:在本研究中,我们对醋酸铅处理的原代小鼠肾间质成纤维细胞进行了基因表达分析,证实了CCL20基因显著上调。miR-143-5p可以通过干扰CCL20基因表达,减少醋酸铅诱导的肾间质成纤维细胞增殖,降低细胞及组织内TGF-β1和纤维化相关蛋白的表达,延缓肾脏纤维化。因此,目前的研究可能为铅损伤导致的肾脏纤维化提供一个有前景的治疗靶点。更多还原

【Abstract】 Backgroud:Lead pollution in the environment can cause chronic kidney disease and poses a huge public health threat worldwide.The common clinical manifestation of lead poisoning is renal fibrosis.Abnormal proliferation of renal interstitial fibroblasts and accumulation of extracellular matrix are the main pathological causes of renal fibrosis.However,the underlying mechanism of lead induced renal fibrosis remains unclear.Our previous studies have shown that lead acetate can promote renal fibrosis through Transforming Growth Factor-β(TGF-β)/Smad signaling pathway.,and significantly increase the expression of CC chemokine ligand 20 gene,m RNA and protein.Objective:On the basis of previous research,to do further research to investigate the effect of chemokine CCL20 gene on lead induced renal fibrosis,explore the relationship between regulatory target gene CCL20 and fibrosis related factors,screen and verify the miRNA to regulate target gene CCL20,and verify the effect of miRNA on lead induced renal fibrosis in lead poisoning mouse model.Methods:1.Mouse renal interstitial fibroblasts were cultured in vitro and treated with different concentrations of lead acetate(0.5,1,2μmol/L).Realtime PCR was used to detect the m RNA expressions of CCL20,CCR6,TGF-β1,fibronectin(FN)and Collagen I.The content of hydroxyproline in fibroblasts was detected by biochemical methods.The proteins secretions of CCL20 and TGF-β1 were detected by ELISA,and the protein expressions of CCL20,CCR6,TGF-β1,Collagen I,FN,Smad2/3,p-Smad2/3,AKT and p-AKT were detected by Western blot.2.Mouse renal interstitial fibroblasts were treated with 1μmol/L lead acetate,CCL20 gene was interfered by si RNA.The m RNA expressions of CCL20,CCR6,TGF-β1,FN and Collagen I were detected by Realtime PCR,the content of hydroxyproline in fibroblasts was detected by biochemical method,and the protein secretion of CCL20 and TGF-β1 was detected by ELISA.Western blot was used to detect the protein expressions of CCL20,CCR6,TGF-β1,Collagen I,FN,Smad2/3,p-Smad2/3,AKT and p-AKT.Cell proliferation of 0、6、12、24h was detected by CCK8 assay.3.MiRWalk,Target Scan,miRanda and other online software were used to predict and screen out miRNAs that may regulate CCL20 gene with high scores in mice.Mouse renal interstitial fibroblasts were treated with different concentrations of lead acetate(0.5,1,2μmol/L).Real-time PCR was used to detect the expression of candidate miRNAs,and Dual-Luciferase Reporter Assay was used to confirm the miRNA targeting CCL20 gene.Mouse renal interstitial fibroblasts were treated with1μmol/L lead acetate,respectively,plus miR-143-5P mimics or combined with 10ng/m L CCL20 recombinant vector.The m RNA expressions of CCL20,CCR6,TGF-β1,FN and Collagen I were detected by Realtime-PCR,the content of hydroxyproline in fibroblasts was detected by biochemical method,and the protein secretions of CCL20 and TGF-β1 were detected by ELISA.Western blot was used to detect the protein expressions of CCL20,CCR6,TGF-β1,Collagen I,FN,Smad2/3,p-Smad2/3,AKT and p-AKT.Cell proliferation of 0、6、12、24h was detected by CCK8 assay.4.Twenty-four mice were randomly divided into group A: control group,group B:lead injury group,and group C: lead injury group + miR-143-5p mimics group,with 8mice in each group.Lead acetate solution(10mg/kg)was intraperitoneally injected for4 weeks.After 4 weeks of lead acetate intervention,miR-143-5p mimics were injected into the tail vein 1OD/ time/day for 2 consecutive weeks in group C.The mice in group A and group B were injected with normal saline,and the renal tissue fibrosis was observed and analyzed by HE staining and MASSON staining respectively.The m RNA expressions of CCL20,CCR6,TGF-β1,FN and Collagen I were detected by Realtime-PCR.The expressions of CCL20 and TGF-β1 in peripheral blood were detected by ELISA.The content of hydroxyproline in renal tissue was detected by biochemical assay.Western blot was used to detect the protein expressions of CCL20,CCR6,TGF-β1,Collagen I,FN,Smad2/3,p-Smad2/3,AKT and p-AKT.Results:1.The m RNA levels,protein secretion and expression of CCL20 and TGF-β1,as well as the content of hydroxyproline in mouse renal interstitial fibroblasts were dose-dependently increased by lead acetate.The m RNA and protein expressions of CCL20 receptor CCR6,extracellular matrix Collagen I and FN also increased in a dose-dependent manner with lead acetate concentration,and the expression levels of signaling pathway proteins p-Smad2/3 and p-AKT increased with the increased lead acetate concentration.2.SiRNA interference inhibited the proliferation of fibroblasts induced by lead acetate,decreased the m RNA level,protein secretion and expression of CCL20 and TGF-β1,and also inhibited the expression of hydroxyproline content 、CCR6、Collagen I and FN protein.The expression levels of signaling pathway proteins p-Smad2/3 and p-AKT were also inhibited.3.Bioinformatics analysis predicted 5 miRNAs with high scores targeting CCL20.Among them,after the fibroblasts were treated with lead acetate,the expression of miR-143-5p decreased in a dose-dependent manner with the increase of lead acetate concentration.It was verified that miR-143-5p could directly target CCL20 to inhibit its expression through Dual-Luciferase Reporter Assay.In functional validation experiments,miR-143-5p antagonized lead acetate induced fibroblast proliferation,reduced hydroxyproline content、m RNA and protein expression of CCL20,CCR6,TGF-β1 and extracellular matrix,while this inhibition was reversed after overexpression of CCL20.In addition,miR-143-5p attenuates the activation of Smad and AKT pathways in lead acetate exposed fibroblasts,and these effects can be offset by overexpression of CCL20.4.In the animal experiments,tail vein injection of miR-143-5p can delay the progression of renal fibrosis in mouse tissues: inhibited the m RNA levels、protein secretion and expression of CCL20 and TGF-β1,decreased the content of hydroxyproline,inhibited the expressions of CCR6,Collagen I and FN in the kidney tissue,and the signaling pathway proteins p-Smad2/3 and p-AKT.Conclusion:In this study,gene expression analysis of primary mouse renal interstitial fibroblasts treated with lead acetate confirmed significant upregulation of CCL20 gene.Mi R-143-5p can reduce lead acetate induced renal interstitial fibroblast proliferation by interfering the expression of CCL20 gene,and reduce the expression of TGF-β1 and fibrosis-related proteins in cells and tissues,and delay renal fibrosis.Therefore,the present study may provide a promising therapeutic target for renal fibrosis caused by lead injury.更多还原