LRG1对人翼状胬肉成纤维细胞纤维化的促进作用及其机制  

Promoting effect of LRG1 on fibrosis of human pterygium fibroblasts and its mechanism

作　　者：文艳杰 魏超群 陈杨[1] 孙莉尧 高轶 贺洁 罗佳林 白宇婧 葛红岩[1] Wen Yanjie;Wei Chaoqun;Chen Yang;Sun Liyao;Gao Yi;He Jie;Luo Jialin;Bai Yujing;Ge Hongyan(Eye Hospital,the First Affiliated Hospital of Harbin Medical University,Harbin 150001,China)

机构地区：[1]哈尔滨医科大学附属第一医院眼科医院,哈尔滨150001

出　　处：《中华实验眼科杂志》2025年第4期315-322,共8页Chinese Journal Of Experimental Ophthalmology

摘　　要：目的探讨亮氨酸α-2糖蛋白1(LRG1)在人翼状胬肉成纤维细胞(HPFs)纤维化中的作用及机制。方法收集2022年1月至2023年3月于哈尔滨医科大学附属第一医院行鼻侧原发性翼状胬肉切除手术的翼状胬肉组织和行斜视矫正手术切除的鼻侧正常结膜组织各30例,分别作为翼状胬肉组和正常对照组。采用免疫荧光染色法检测2个组LRG1蛋白表达,采用实时荧光定量PCR、Western blot法检测2个组LRG1和转化生长因子β1(TGF-β1)mRNA和蛋白表达水平。采用组织块贴壁法从翼状胬肉组织中培养原代HPFs,观察其形态,并进行波形蛋白和角蛋白免疫荧光法鉴定。将HPFs分为rhLRG1组和空白对照组,分别用含或不含10μg/ml的rhLRG1培养24 h,采用细胞划痕实验测定细胞的迁移能力。另将HPFs分为空白对照组、LRG1过表达组和LRG1敲减组,LRG1过表达组和LRG1敲减组分别使用LRG1过表达质粒和小干扰RNA转染HPFs 24 h,采用实时荧光定量PCR检测TGF-β1 mRNA表达水平,采用Western blot检测TGF-β1和纤维化标志物纤维连接蛋白(FN)、Ⅲ型胶原(COL3)和α-平滑肌肌动蛋白(α-SMA)表达水平。结果提取细胞呈梭形、旋涡状排列,波形蛋白鉴定呈阳性,角蛋白免疫荧光染色呈阴性,证实为HPFs。rhLRG1组HPFs细胞迁移率为(83.01±2.56)%,明显高于空白对照组的(50.32±4.97)%,差异有统计学意义(t=9.59,P<0.001)。免疫荧光染色结果显示,相比于正常结膜组织,翼状胬肉组织中LRG1蛋白显著高表达,广泛分布于纤维结缔组织和上皮层中。翼状胬肉组LRG1和TGF-β1 mRNA和蛋白相对表达量明显高于正常对照组,差异均有统计学意义(mRNA:t=10.18、6.15,均P<0.05;蛋白:t=6.83、8.79,均P<0.05)。LRG1过表达组HPFs细胞中TGF-β1 mRNA相对表达量和FN、COL3、α-SMA蛋白相对表达量明显高于空白对照组和LRG1敲减组,差异均有统计学意义(均P<0.05)。结论LRG1可促进HPFs的纤维化并增强其迁移能力,其机制可�Objective To investigate the role and mechanism of leucine-richα-2-glycoprotein 1(LRG1)in the fibrosis of human pterygium fibroblasts(HPFs).Methods A total of 30 nasal primary pterygium tissues from patients who underwent pterygium excision surgery and 30 nasal normal conjunctival tissues from patients who underwent strabismus correction surgery were collected from the First Affiliated Hospital of Harbin Medical University between January 2022 and March 2023,serving as the pterygium group and normal control group,respectively.LRG1 protein expression in both groups was detected by immunofluorescence staining.The mRNA and protein levels of LRG1 and transforming growth factor-β1(TGF-β1)were evaluated by quantitative real-time PCR(qRT-PCR)and Western blot.Primary HPFs were cultured from excised pterygium tissues using tissue block adhesion method,and cell morphology was observed.Vmentin and cytokeratin were identified by immunofluorescence staining.HPFs were divided into recombinant human LRG1(rhLRG1)group and blank control group treated with or without 10μg/ml rhLRG1 for 24 hours,respectively,and cell migration was evaluated via scratch assay.Additionally,HPFs were divided into blank control group,LRG1 overexpression group and LRG1 knockdown group.HPFs in LRG1 overexpression group and LRG1 knockdown group were transfected with LRG1 overexpression plasmids and small interfering RNA for 24 hours,respectively.TGF-β1 mRNA level was evaluated by qRT-PCR and expression of TGF-β1,fibronectin(FN),typeⅢcollagen(COL3),andα-smooth muscle actin(α-SMA)proteins were evaluated by Western blot.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University(No.2022IIT026).Written informed consent was obtained from each subject.Results HPFs were successfully isolated,exhibiting spindle-shaped morphology with whorled arrangement,positive identification for vimentin,and negative immunofluorescence staining for cytokeratin.T

关 键 词：翼状胬肉 纤维化 富亮氨酸α-2糖蛋白1 转化生长因子Β1 迁移 

分 类 号：R777.33[医药卫生—眼科]