SOX9在角膜内皮损伤过程中的表达及功能  

Expression and Function of SOX9in Corneal Endothelial Injury

作　　者：孙图南 李晓琦 李宗源 王丽强[1] 黄一飞[1] SUN Tunan;LI Xiaoqi;LI Zongyuan;WANG Liqiang;HUANG Yifei(Department of Ophthalmic Medicine,the Third Medical Center of Chinese People′s Liberation Army General Hospital,Beijing100039,China)

机构地区：[1]解放军总医院第三医学中心眼科医学部,北京100039

出　　处：《联勤军事医学》2023年第8期637-642,690,共7页Military Medicine of Joint Logistics

基　　金：国家自然科学基金面上项目(82070921);北京市自然科学基金面上项目(7212098)。

摘　　要：目的探究性别决定区Y框蛋白9(sex determining region Y box protein 9,SOX9)在角膜内皮损伤过程中的表达及功能。方法通过转录组数据库分析人角膜内皮损伤与SOX9表达的关联。冷冻法构建小鼠角膜内皮损伤模型,每只小鼠左眼造模、右眼空白对照,采用裂隙灯照相、眼前节光学相干断层扫描技术(optical coherence tomography,OCT)、免疫荧光技术评估动物模型质量,角膜铺片免疫荧光染色观察损伤后内皮细胞中SOX9的表达变化。分别用低浓度(30μmol/L)和高浓度(50μmol/L)的甲萘醌对人角膜内皮细胞(B4G12)处理3 h(短时间组)、6 h(中时间组)、12 h(长时间组)、24 h(超长时间组),构建细胞模型。采用显微镜照相、细胞计数试剂盒(cell counting kit-8,CCK-8)实验评估细胞模型质量,实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,qPCR)、Western blot检测SOX9和介导角膜内皮-间质转化(endothelium-mesenchymal transition,EndMT)关键转录因子Snail家族转录抑制因子2(snail family transcriptional repressor 2,SNAIL2)的表达变化。结果数据库资料显示在角膜内皮损伤代表性疾病Fuchs角膜内皮营养不良(Fuchs′endothelial corneal dystrophy,FECD)患者中,FECD3期以上患者角膜内皮SOX9表达量比2期患者明显升高(P<0.05),且SOX9的下游基因富含半胱氨酸的酸性分泌蛋白(secreted protein acidic and rich in cysteine,SPARC)表达量比正常人明显升高(P<0.05)。在动物模型中,对照组角膜内皮铺片免疫荧光染色显示无SOX9荧光,损伤后短期出现大量SOX9荧光,损伤后中期、长期SOX9荧光回归极低水平。在甲萘醌处理的细胞模型中,SOX9表达随处理时间的延长呈现先升高后降低的趋势;同时发现SNAIL2的表达变化也会随甲萘醌处理时间的延长呈现先升高后降低的趋势。结论SOX9的表达随角膜内皮损伤进程产生变化,并与损伤修复中的EndMT紧密相关。Objective To investigate the expression and function of sex determining region Y box protein9(SOX9)in corneal endothelial injury.Methods The association between SOX9 expression and human corneal endothelial injury was analyzed by transcriptome database.Mouse corneal endothelial injury model was constructed by freezing method,the left eyes of each mouse were used to establish the model,and the right eyes were used as blank control,slit lamp photography,anterior chamber optical coherence tomography(OCT)and immunofluorescence were used to evaluate the quality of the animal model,the expression of SOX9 in endothelial cells after injury was observed by immunofluorescence staining.Human corneal endothelial cells(B4G12)were treated with low concentration(30μmol/L)and high concentration(50μmol/L)of menadione for 3 hours(short time group),6 hours(medium time group),12 hours(long time group)and 24 hours(extended long time group)to construct cell models.The quality of cell model was evaluated by microscope photography and cell counting kit-8(CCK-8)experiment,the expression change of SOX9 and cornea endothelium-mesenchymal transition(EndMT)key transcription factor snail family transcriptional repressor 2(SNAIL2)were detected by real-time quantitative polymerase chain reaction(qPCR)and Western blot.Results The database data showed that in patients with Fuchs′endothelial corneal dystrophy(FECD),a representative disease of corneal endothelial injury,cornea endothelium SOX9 expression in patients with FECD stage 3 and above was significantly higher than that in patients with FECD stage 2(P<0.05),the expression level of secreted protein acidic and rich in cysteine(SPARC)in the downstream gene of SOX9 was significantly higher than that of normal controls(P<0.05).In animal models,immunofluorescence staining of corneal endothelium in control group showed no SOX9fluorescence,a large amount of SOX9fluorescence appeared in the short term after injury,and a very low level of SOX9 fluorescence regression in the middle and long term a

关 键 词：角膜内皮细胞 性别决定区Y框蛋白9 Snail家族转录抑制因子2 损伤修复 内皮-间质转化 

分 类 号：R772.2[医药卫生—眼科]