胰岛素样生长因子1对人RPE细胞分泌TGF-β2、MMP-2的影响及机制研究  

Effects and mechanism of insulin-like growth factor 1 on the secretion of transforming growth factor β2 and matrix metalloproteinase 2 in human retinal pigment epithelial cells

作　　者：晁荣荣 郑柳 范晶 丁芝祥[2] CHAO Rongrong;ZHENG Liu;FAN Jing;DING Zhixiang(Guilin Medical University,Guilin 541100,Guangxi Zhuang Autonomous Region,China;Affiliated Hospital of Guilin Medical University,Guilin 541001,Guangxi Zhuang Autonomous Region,China)

机构地区：[1]桂林医学院,广西壮族自治区桂林市541100 [2]桂林医学院附属医院,广西壮族自治区桂林市541001

出　　处：《眼科新进展》2024年第7期512-517,共6页Recent Advances in Ophthalmology

基　　金：国家自然科学基金项目(编号:82160197)。

摘　　要：目的研究胰岛素样生长因子1(IGF-1)对人视网膜色素上皮细胞(ARPE-19)表达转化生长因子β2(TGF-β2)、基质金属蛋白酶2(MMP-2)的影响,并探索其作用机制。方法ARPE-19细胞分别按不同浓度IGF-1和不同浓度LY294002培养6 h、12 h、24 h、48 h,采用CCK-8法检测细胞活力,确定IGF-1、LY294002的最佳作用浓度与时间。细胞划痕法检测细胞迁移活性。ELISA法检测细胞培养上清液中TGF-β2浓度。将ARPE-19细胞分为对照组、IGF-1组(80μg·L^(-1) IGF-1)、IGF-1+LY294002组(80μg·L^(-1) IGF-1+30 mmol·L^(-1) LY294002)、LY294002组(30 mmol·L^(-1) LY294002),使用无血清DMEM/F12培养基培养,对照组不做任何处理,分别采用RT-PCR、Western blot检测细胞中TGF-β2、MMP-2、磷脂酰肌醇-3-激酶(PI3K)、蛋白激酶B(AKT)的mRNA和蛋白表达量。结果与0μg·L^(-1) IGF-1比较,80μg·L^(-1) IGF-1的细胞活力24 h变化显著(P<0.05),故确定其为IGF-1最佳作用浓度和时间。与0 mmol·L^(-1) LY294002比较,24 h的30 mmol·L^(-1) LY294002接近半数抑制浓度,故确定其为LY294002最佳作用时间和浓度。细胞划痕法检测结果显示,0μg·L^(-1) IGF-1组、40μg·L^(-1) IGF-1组、80μg·L^(-1) IGF-1组细胞迁移率整体比较及两两比较差异均有统计学意义(均为P<0.05)。ELISA检测结果显示,0μg·L^(-1) IGF-1组、40μg·L^(-1) IGF-1组、80μg·L^(-1) IGF-1组细胞上清液中TGF-β2浓度整体比较及两两比较差异均有统计学意义(均为P<0.05)。RT-PCR、Western blot检测结果显示,IGF-1、LY294002培养24 h,与对照组比较,IGF-1组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均升高,而LY294002组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均下降(均为P<0.05);与IGF-1组比较,IGF-1+LY294002组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均下降(均为P<0.05)。结论IGF-1能促进ARPE-19细胞增殖、迁移;IGF-1可能通过PI3K/AKT信号通路上调ARPE-19细胞中TGF-β2、MMPObjective To investigate the effects of insulin-like growth factor 1(IGF-1)on the expression of transforming growth factor β2(TGF-β2)and matrix metalloproteinase 2(MMP-2)in human retinal pigment epithelial cells(ARPE-19)and related mechanisms.Methods ARPE-19 cells were cultured for 6 h,12 h,24 h and 48 h,respectively,with different concentrations of IGF-1 and LY294002.The cell viability was detected using the cell counting kit-8 to determine the optimal action concentration and time of IGF-1 and LY294002.The cell migration activity was detected using the cell scratch assay.The concentration of TGF-β2 in cell culture supernatant was detected using the enzyme-linked immunosorbent assay(ELISA).ARPE-19 cells were divided into the control group,IGF-1 group(80μg·L^(-1) IGF-1),IGF-1+LY294002 group(80μg·L^(-1) IGF-1+30 mmol·L^(-1) LY294002),and LY294002 group(30 mmol·L^(-1) LY294002)and cultured with serum-free DMEM/F12 medium,while cells in the control group received no treatment.The mRNA and protein expression levels of TGF-β2,MMP-2,phosphoinositide 3-kinase(PI3K)and protein kinase B(AKT)in the cells were measured using the reverse transcription-polymerase chain reaction(RT-PCR)and Western blot,respectively.Results Compared with the 0μg·L^(-1) IGF-1 group,the cell viability in the 80μg·L^(-1) IGF-1 group changed the most significantly at 24 h(P<0.05);thus,the optimal concentration of IGF-1 was 80μg·L^(-1) and the optimal culture time was 24 h.Compared with the 0 mmol·L^(-1) LY294002 group,the inhibition concentration in the 30 mmol·L^(-1) LY294002 group at 24 h was close to half;thus,the optimal concentration of LY294002 was 30 mmol·L^(-1) and the optimal culture time was 24 h.The cell scratch assay results showed that the cell migration rate was significantly different among the 0μg·L^(-1) IGF-1 group,40μg·L^(-1) IGF-1 group,and 80μg·L^(-1) IGF-1 group(all P<0.05).ELISA results showed that there was a statistically significant difference in the concentration of TGF-β2 in cell supernatant amon

关 键 词：近视 视网膜色素上皮细胞 胰岛素样生长因子1 磷脂酰肌醇-3-激酶/蛋白激酶B通路 转化生长因子Β2 基质金属蛋白酶2 

分 类 号：R778.1[医药卫生—眼科]