【摘要】 目的 探究性别决定区Y相关高迁移率族盒蛋白9(SOX9)与Smad家族成员4(Smad4)在静息态角膜基质细胞(KCs)、角膜成纤维细胞(CFs)及角膜肌成纤维细胞(CMFs)中的表达差异及其相互作用，为角膜纤维化疾病的机制解析与靶向治疗提供实验依据。方法 采用生物信息学方法分析GEO数据库中GSE287915数据集，筛选差异表达基因并聚焦SOX9;通过实时荧光定量反转录聚合酶链反应(qRT-PCR)验证SOX9在小鼠KCs(m-KCs)、小鼠CFs(m-CFs)及小鼠CMFs(m-CMFs)中的表达。利用基因转录调控数据库(JASPAR)预测SOX9与Smad4的潜在结合位点，qRT-PCR检测Smad4在三类细胞中的表达水平，最后通过免疫共沉淀(Co-IP)验证SOX9与Smad4蛋白的相互作用。结果 生物信息学分析显示，与KCs比，SOX9在CFs及CMFs中显著高表达(均为P<0.05);qRT-PCR检测结果显示，Smad4、SOX9表达量均呈m-KCs组<m-CFs组<m-CMFs组的趋势，三组间差异均有统计学意义(均为P<0.001)。JASPAR数据库预测显示，SOX9与Smad4启动子区域存在潜在结合位点。Co-IP结果显示，以SOX9、Smad4抗体分别为捕获抗体时，m-CMFs组Smad4与SOX9结合量均高于m-CFs组及m-KCs组(均为P<0.05),m-KCs组未检测到明显结合。结论 SOX9与Smad4在体外培养的m-KCs、m-CFs、m-CMFs中的表达随细胞活化及纤维化程度加重而升高，且二者在体外诱导的纤维化表型m-CFs、m-CMFs中存在特异性相互作用。二者的表达上调及其蛋白相互作用可能参与体外角膜基质细胞的纤维化活化进程，为角膜纤维化疾病的机制研究提供实验参考。更多还原

【Abstract】 Objective To investigate the differential expression and interaction of SRY-box transcription factor 9(SOX9) and Smad family member 4(Smad4) in resting-state keratocytes(KCs),corneal fibroblastic cells(CFs),and corneal myofibroblasts(CMFs),and to provide experimental evidence for the mechanism and targeted therapy of corneal fibrotic disorders.Methods Bioinformatic methods were used to analyze the GSE287915 dataset in the Gene Expression Omnibus database, screen for differentially expressed genes, and focus on SOX9.Quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR) was performed to verify the expression of SOX9 in the mouse KCs(m-KCs),mouse CFs(m-CFs),and mouse CMFs(m-CMFs).The Gene Transcription Regulation Database(JASPAR) was used to predict the potential binding sites between SOX9 and Smad4.qRT-PCR was applied to detect the expression levels of Smad4 in the said three types of cells, and finally, co-immunoprecipitation(Co-IP) was used to verify the interaction between SOX9 and Smad4 proteins.Results Bioinformatic analysis showed that compared with KCs, SOX9 was significantly highly expressed in CFs and CMFs(both P<0.05).The qRT-PCR detection results revealed that the expression levels of Smad4 and SOX9 followed the trend: m-KCs group < m-CFs group < m-CMFs group, with statistically significant differences among the three groups(all P<0.001).The JASPAR database predicted that there were potential binding sites between SOX9 and the Smad4 promoter region.Co-IP results demonstrated that when SOX9 and Smad4 antibodies were used as the capture antibodies, the binding amount of Smad4 and SOX9 in the m-CMFs group was significantly higher than that in the m-CFs group and the m-KCs group(both P<0.05),while no obvious binding was detected in the m-KCs group.Conclusion The expression levels of SOX9 and Smad4 in in vitro cultured m-KCs, m-CFs, and m-CMFs are elevated with the degree of cellular activation and fibrosis progression.Moreover, a specific interaction between them has been identified in fibrotic phenotype m-CFs and m-CMFs induced in vitro.The upregulation of their expression and their protein-protein interaction may contribute to the fibrosisn-related activation of keratocytes in vitro,providing an experimental basis for further mechanism studies on corneal fibrotic diseases.更多还原