共培养体系中内皮细胞外泌体调控平滑肌细胞表型转化的基因表达谱分析

Gene expression profile analysis of smooth muscle cell phenotypic switching regulated by endothelial cell-derived exosomes in a co-culture system

目的通过建立细胞共培养模型,对其外泌体进行高通量基因测序,初步证实内皮细胞(ECs)外泌体通过差异表达的LncRNA影响平滑肌细胞(SMCs)表型转换。方法原代培养大鼠主动脉ECs,进行形态学观察、免疫荧光鉴定,大鼠主动脉SMCs体外培养扩增,采用Transwell建立两种细胞体外共培养模型,分别为单独培养SMCs组、ECs-SMCs共培养组、ECs-SMCs+GW4869外泌体抑制组。通过Western-Blot比较单独培养SMCs组和ECs-SMCs+GW4B69外泌体抑制组中收缩表型标志蛋白(α-SM actin)、合成表型标志蛋白(Vimentin)的表达,使用外泌体试剂盒分离提取其外泌体,通过透射电镜、纳米粒径分析、Western-Blot进行鉴定。并对外泌体进行高通量基因测序,差异基因表达谱进行生物信息学分析。结果成功分离培养并鉴定出原代主动脉ECs,在Transwell共培养细胞模型中SMCs向收缩表型转化,表现为α-SM actin收缩表型蛋白表达显著升高,Vimentin合成表型标志蛋白逐渐降低。采用试剂盒法成功提取纯化外泌体,鉴定出形态、直径、浓度及标志蛋白CD9、CD63、CD81表达均满意的外泌体,通过对外泌体高通量基因测序及生物信息学分析,筛选出4276个差异表达基因,其中3303个基因呈现上调,973个基因呈现下调。这些差异基因主要分布于细胞核、胞浆、细胞连接结构、囊泡及细胞外基质等区域,在细胞蛋白质的修饰、大分子代谢及氮化合物代谢等多个关键生物学过程中扮演着至关重要的作用。结论采用Transwell法构建ECs-SMCs共培养细胞模型可显著提高SMCs收缩表型蛋白的表达,促进平滑肌细胞向收缩表型转化,其外泌体差异表达基因对SMCs的增殖分化及表型转化产生调控作用。

ObjectiveBy establishing a co-culture model of cells and performing high-throughput gene sequencing on their exosomes,this study aims to preliminarily confirm that the exosomes from endothelial cells(ECs)influence the phenotypic switching of smooth muscle cells(SMCs)through the differential expression of LncRNAs.MethodsPrimary rat aortic ECs were cultured and identified through morphological observation and immunofluorescence.Rat aortic SMCs were cultured and expanded in vitro and a co-culture model of the two cell types was established using a Transwell system.The expressions of contractile phenotype marker protein(α-SM actin)and synthetic phenotype marker protein(Vimentin)was compared between the co-culture and control groups through Western-Blot.Exosomes were isolated and extracted using an exosome isolation kit and identified by transmission electron microscopy,nanoparticle tracking analysis,and Western-Blot.High-throughput gene sequencing of the exosomes was performed,followed by bioinformatic analysis of the differential gene expression profiles.ResultsPrimary aortic ECs were successfully isolated,cultured,and identified.In the Transwell co-culture model,SMCs shifted towards a contractile phenotype,evidenced by a significant increase inα-SM actin expression and a gradual decrease in Vimentin.Exosomes were successfully purified using the kit,with satisfactory morphology,diameter,concentration,and expression of the marker proteins CD9,CD63,and CD81.Through high-throughput gene sequencing and bioinformatic analysis of the exosomes,4,276 differentially expressed genes were identified,of which 3,303 were upregulated and 973 were downregulated.These differential genes were primarily located in the cell nucleus,cytoplasm,cell junctions,vesicles,and extracellular matrix,playing crucial roles in key biological processes such as cellular protein modification,macromolecule metabolism,and nitrogen compound metabolism.ConclusionThe Transwell-based ECs-SMCs co-culture model significantly enhanced the expression ofα-SM actin in SMCs and promoted the transition of SMCs towards a contractile phenotype.The differentially expressed genes in the exosomes regulated the proliferation,differentiation,and phenotypic switching of SMCs.