【作者】 方成；

【导师】 罗凡砚；

【作者基本信息】 中南大学 ， 心脏大血管外科， 2023， 硕士

【摘要】 背景:随着全球人口的老龄化和慢性疾病的生存率增加,心房颤动的发病率和患病率正在上升,心房颤动已成为当今最常见的心律失常类型之一。心肌纤维化是房颤的潜在关键因素和标志,也是导致房颤患者心脏重构的主要原因之一。心肌纤维化是由于心脏成纤维细胞激活,过度增殖,分化为肌成纤维细胞,导致心肌间质中细胞外基质蛋白分泌和沉积增加,从而在局部形成纤维化病变。纤维化的存在会干扰正常心脏电生理信号的传导,诱发心房颤动或者增加心房颤动的易感性。网膜素(Omentin-1)作为一种由心外膜脂肪组织分泌的新型脂肪因子,可以抑制血管平滑肌细胞的增殖、迁移和ECM产生。有研究发现,血清omentin-1水平与心房颤动发展和心房结构重构呈负相关。钙黏蛋白-11(CDH11)是钙依赖性的粘附蛋白,被认为是纤维化的介质,在各种纤维化疾病中上调,通过调节细胞收缩性和局灶粘连发挥生物学作用。但目前对于omentin-1抗纤维化的机制以及能否调控CDH11尚不明确。本文旨在研究Omentin-1拮抗CDH11诱导的心脏成纤维细胞激活的调控作用及潜在机制。方法:本研究首先使用数据集比较房颤与窦律样本中纤维化相关基因(α-SMA和COL1)以及CDH11的表达差异,然后通过组织免疫荧光定量分析入院诊断为房颤或窦律患者的心肌样本中纤维化标志物α-SMA以及CDH11表达差异。随后,在TGF-β1诱导纤维化激活的小鼠心脏成纤维细胞(MCFs)模型中进行纤维化相关基因和CDH11表达差异验证。接下来在MCFs细胞中转染CDH11过表达质粒,采用Western blot、CCK8实验和Transwell实验分别检测其纤维化相关基因表达水平、CDH11上调情况、增殖和迁移能力。然后,对正常和纤维化激活状态的MCFs细胞加入si RNA敲降CDH11的表达,进行了Western blot、CCK8实验和Transwell实验分别检测其纤维化相关基因和CDH11表达水平、增殖以及迁移能力,进一步探讨CDH11在促纤维化中的作用。接着,研究对纤维化激活的MCFs细胞加入了omentin-1重组蛋白处理,并进行了Western blot和天狼星红染色实验检测纤维化相关基因和CDH11表达水平。随后,在过表达CDH11的MCFs细胞中加入omentin-1处理,并进行了Western blot、天狼星红染色、CCK8实验和Transwell实验分别检测其纤维化相关基因和CDH11表达水平、胶原分泌、增殖以及迁移能力,探讨CDH11的促纤维化作用是否可以被omentin-1抑制。最后,采用转录组测序对空载组、CDH11过表达组和CDH11过表达+omentin-1处理组进行分析,初步探究了omentin-1与CDH11对心肌纤维化潜在的调节通路机制。结果:通过分析GEO数据集中房颤和窦律转录组数据,显示房颤组心肌组织中纤维化相关基因(α-SMA和COL1)以及CDH11表达明显高于窦律组。免疫荧光提示纤维化标志物α-SMA和CDH11在房颤患者的心肌组织样本中上调。Western blot实验显示在使用TGF-β1进行纤维化激活的MCFs细胞中,纤维化相关基因明显上调,CDH11的表达也明显升高。在过表达CDH11后,Western blot实验显示MCFs细胞纤维化相关基因表达增高,Transwell和CCK8实验显示过表达组细胞迁移和增殖能力增强。在正常状态和纤维化激活的MCFs中敲降CDH11后,Western blot显示正常和纤维化激活的MCFs细胞纤维化水平均降低,Transwell和CCK8实验显示敲降CDH11降低了两组细胞迁移和增殖能力。对纤维化激活的MCFs细胞进行omentin-1处理后,Western blot和天狼星红染色提示MCFs细胞纤维化水平降低。在转染CDH11过表达质粒的MCFs细胞中加入omentin-1,Western blot实验显示omentin-1可以降低CDH11转染组α-SMA和COL1的表达,并且也降低了CDH11的表达,细胞培基天狼星红染色显示omentin-1减少转染组细胞胶原分泌,Transwell和CCK8实验显示omentin-1降低转染组细胞迁移和增殖能力。对空载组、转染CDH11组和转染CDH11组+omentin-1处理组进行转录组测序和Western blot实验,发现PI3K/Akt信号通路被富集和磷酸化激活。结论:CDH11促进MCFs细胞的纤维化进程,脂肪因子omentin-1可能作为上游调控因子,通过降低心脏成纤维细胞的CDH11表达来发挥对纤维化的抑制作用。PI3K/Akt信号通路可能参与omentin-1的抗纤维化作用。图表参考文献的数量:图16张,表11个,参考文献78篇更多还原

【Abstract】 Background: With the aging of the global population and increased survival from chronic diseases,the incidence and prevalence of atrial fibrillation are increasing,and atrial fibrillation has become one of the most common types of arrhythmia today.Myocardial fibrosis is considered to be a potential key factor and indicator of atrial fibrillation,and one of the main factors leading to cardiac remodeling in atrial fibrillation patients.Myocardial fibrosis is caused by the activation,excessive proliferation and differentiation of cardiac fibroblasts into myofibroblasts,which leads to the increased secretion and deposition of extracellular matrix proteins in the myocardial interstitium resulting in local formation of fibrotic lesions.The presence of fibrosis can interfere with the conduction of normal cardiac electrophysiological signals and induce or increase the susceptibility to atrial fibrillation.Omentin-1,as a novel adipokine secreted by epicardial adipose tissue,can inhibit the proliferation,migration and ECM production of vascular smooth muscle cells.Some studies have found that serum omentin-1 levels are negatively correlated with the development of atrial fibrillation and atrial structural remodeling.CDH11,a calcium-dependent adhesion protein,is considered to be a mediator of fibrosis and is upregulated in various fibrotic diseases,playing a biological role by regulating cell contractility and focal adhesion.However,the anti-fibrosis mechanism of omentin-1 and whether it can regulate CDH11 are still unclear.The aim of this study is to investigate the regulatory effect and potential mechanism of CDH11 and omentin-1 on myocardial fibrosis.Methods: In this study,we first used the data set to compare the expression differences of fibrosis-related genes(α-SMA and COL1)and CDH11 in AF and sinus rhythm samples,and then used tissue immunofluorescence to quantitatively analyze the expression differences of fibrosis markers α-SMA and CDH11 in myocardial samples from patients with AF or sinus rhythm.Subsequently,the differential expression of fibrosis-related genes and CDH11 was verified in mouse cardiac fibroblasts(MCFs)model induced by TGF-β1.Next,CDH11 overexpression plasmid was transfected into MCFs cells,and Western blot,CCK8 assay and Transwell assay were used to detect the expression levels of fibrosis-related genes,the up-regulation of CDH11,proliferation and migration ability of MCFS cells,respectively.Then,si RNA was added to knock down the expression of CDH11 in normal and fibrosisactivated MCFs,and Western blot,CCK8 assay and Transwell assay were used to detect the expression levels of fibrosis-related genes and CDH11,proliferation and migration ability of MCFS,respectively.The role of CDH11 in promoting fibrosis was further investigated.Then,the fibrosisactivated MCFs were treated with omentin-1 recombinant protein,and the expression levels of fibrosis-related genes and CDH11 were detected by Western blot and Sirius red staining.Subsequently,the CDH11-overexpressing MCFs were treated with omentin-1,and the expression levels of fibrosis-related genes and CDH11,collagen secretion,proliferation and migration abilities were detected by Western blot,Sirius red staining,CCK8 and Transwell assays,respectively.To investigate whether the pro-fibrotic effect of CDH11 can be inhibited by omentin-1.Finally,transcriptome sequencing was used to analyze the empty vector group,CDH11 overexpression group,and CDH11 overexpression+omentin-1 treatment group,and the potential regulatory pathway mechanism of omentin-1 and CDH11 on myocardial fibrosis was preliminarily explored.Results: By analyzing the transcriptome data of AF and sinus rhythm in GEO database,the expression of fibrosis related genes α-SMA,COL1 and CDH11 in myocardial tissue of AF group was significantly higher than that of sinus rhythm group.Immunofluorescence showed that fibrosis markers α-SMA and CDH11 were up-regulated in myocardial tissue samples from patients with AF.Western blot showed that fibrosisrelated genes and CDH11 expression were significantly up-regulated in fibrosis-activated MCFs by TGF-β1.After overexpression of CDH11,Western blot showed that the expression of fibrosis-related genes in MCFs cells was increased.Transwell and CCK8 experiments showed that the migration and proliferation ability of MCFS cells in the overexpression group was enhanced.After knockdown of CDH11 in normal and fibrosis-activated MCFs,Western blot showed that the fibrosis level of normal and fibrosis-activated MCFs was reduced.Transwell and CCK8 assays showed that knockdown of CDH11 reduced the migration and proliferation of both groups of MCFS.After omentin-1treatment of fibrosis-activated MCFs,Western blot and Sirius red staining suggested that the level of fibrosis in MCFs cells was reduced.Western blot analysis showed that omentin-1 decreased the expression of α-SMA and COL1 in CDh11-transfected MCFs cells,and also decreased the expression of CDH11.Sirius red staining showed that omentin-1 reduced the secretion of collagen in the transfected cells.Transwell and CCK8 assays showed that omentin-1 reduced the migration and proliferation of the transfected cells.Transcriptome sequencing of the empty vector group,CDH11 transfected group and CDH11 +omentin-1 transfected group showed that PI3K/Akt signaling pathway was enriched and activated by phosphorylation.Conclusion: CDH11 promotes the progression of MCFs fibrosis.omentin-1 may act as an upstream regulator to inhibit fibrosis by reducing the expression of CDH11 in cardiac fibroblasts.PI3K/Akt signaling pathway may be involved in the anti-fibrosis effect of omentin-1.更多还原