IGF-1对HSF细胞分泌近视相关因子的影响及机制探索  

Effects of insulin-like growth factor-1 on the mechanism of myopia-related factors secreted by human scleral fibroblasts

作　　者：晁荣荣 丁芝祥[1] 范晶 郑柳 Chao Rongrong;Ding Zhixiang;Fan Jing;Zheng Liu(Department of Ophthalmology,the Affiliated Hospital of Guilin Medical University,Guilin 541001,Guangxi Zhuang Autonomous Region,China)

机构地区：[1]桂林医学院附属医院眼科,广西壮族自治区桂林市541001

出　　处：《国际眼科杂志》2025年第2期198-205,共8页International Eye Science

基　　金：国家自然科学基金项目(No.82160197)。

摘　　要：目的:探讨胰岛素样生长因子-1(IGF-1)对人巩膜成纤维细胞(HSF)分泌转化生长因子-β2(TGF-β2)、基质金属蛋白酶-2(MMP-2)、缺氧诱导因子-1α(HIF-1α)的影响及其机制。方法:分别使用IGF-1和PI3K/AKT通路抑制剂LY294002培养细胞,采用CCK-8法检测细胞活力,确定药物的最佳作用浓度和时间。采用细胞划痕法观察细胞迁移活性。为明确IGF-1对HSF细胞的影响以及PI3K/AKT通路在其中的调控作用,将细胞分为对照组(不含药物)、IGF-1(80μg/L)组、IGF-1+LY294002(80μg/L+5 mmol/L)组、LY294002(5 mmol/L)组,培养24 h;Western blot检测细胞中TGF-β2、MMP-2、HIF-1α、PI3K、AKT的蛋白表达水平;细胞免疫荧光检测TGF-β2、MMP-2、HIF-1α的荧光表达。结果:CCK-8结果显示不同浓度IGF-1培养的细胞活力以80μg/L IGF-1组最高(均P<0.05),且在不同培养时间下,以24 h时80μg/L IGF-1组细胞活力最高,因此确定后续实验中IGF-1的浓度为80μg/L,作用时间为24 h。不同浓度LY294002培养的细胞活力从6 h起逐渐降低(均P<0.05),根据IC50值确定后续实验中LY294002浓度为5 mmol/L,作用时间为24 h。细胞划痕结果显示,与对照组相比40、80μg/L IGF-1组细胞迁移率均升高(均P<0.05)。与对照组相比2.5、5 mmol/L LY294002组细胞迁移率均降低(均P<0.05)。Western blot结果显示,与对照组相比,IGF-1组细胞TGF-β2、MMP-2、HIF-1α、PI3K、AKT的蛋白表达量均升高,LY294002组的蛋白表达量均下降(均P<0.05);与IGF-1组比较,IGF-1+LY294002组的TGF-β2、MMP-2、HIF-1α、PI3K、AKT蛋白表达量表达均下降(均P<0.05)。细胞免疫荧光结果显示,与对照组相比,IGF-1组细胞TGF-β2、MMP-2、HIF-1α的荧光表达均升高,LY294002组的荧光表达均下降(均P<0.05);与IGF-1组比较,IGF-1+LY294002组的TGF-β2、MMP-2、HIF-1α荧光表达均显著下降(均P<0.05)。结论:IGF-1促进了人HSF细胞增殖与迁移;IGF-1可能通过PI3K/AKT信号通路上调人HSF细胞中TGF-β2、MMP-2、HIF-1α�AIM:To investigate the effects of insulin-like growth factor 1(IGF-1)on the secretion of transforming growth factorβ2(TGF-β2),matrix metalloproteinase 2(MMP-2)and hypoxia-inducible factor 1α(HIF-1α)in human scleral fibroblasts(HSF)and their mechanism.METHODS:The cells were cultured with IGF-1 and PI3K/AKT pathway inhibitor LY294002,respectively.CCK-8 method was used to detect cell viability and determine the optimal concentration and time of drug action.Cell migration activity was observed by cell scratch method.To determine the effects of IGF-1 on HSF cells and the regulatory role of PI3K/AKT pathway,HSF cells were divided into control group(without drugs),IGF-1(80μg/L)group,IGF-1+LY294002(80μg/L+5 mmol/L)group,and LY294002(5 mmol/L)group,and were cultured for 24 h;the protein expression levels of TGF-β2,MMP-2,HIF-1α,PI3K and AKT were detected by Western blot;the fluorescence expression of TGF-β2,MMP-2 and HIF-1αwas detected by cellular immunofluorescence.RESULTS:The results of CCK-8 showed that the cell viability of the 80μg/L IGF-1 group cultured with different concentrations of IGF-1 was the highest(all P<0.05),and the cell viability of the 80μg/L IGF-1 group at 24 h was the highest under different culture times.Therefore,the concentration of IGF-1 was selected as 80μg/L for 24 h.The viability of cells cultured with different concentrations of LY294002 gradually decreased from 6 h(all P<0.05).According to the IC50 value,therefore,the concentration of LY294002 was selected as 5 mmol/L for 24 h.The cell scratch results showed that compared with the control group,the cell mobility of 40μg/L and 80μg/L IGF-1 groups was increased(all P<0.05).Compared with the control group,cell mobility in the 2.5 and 5 mmol/L LY294002 groups was decreased(all P<0.05).Western blot results showed that compared with the control group,the protein expressions of TGF-β2,MMP-2,HIF-1α,PI3K and AKT in the IGF-1 group were increased,while those in the LY294002 group were decreased(all P<0.05).Compared with the IGF-1 group,

关 键 词：近视 人巩膜成纤维细胞(HSF) 胰岛素生长因子-1(IGF-1) 转化生长因子-β2(TGF-β2) 基质金属蛋白酶-2(MMP-2) 缺氧诱导因子-1α(HIF-1α) 

分 类 号：R73[医药卫生—肿瘤]