二甲双胍对人增生性瘢痕成纤维细胞增殖及纤维化蛋白表达的影响及其机制  

Effects and mechanism of metformin on the proliferation and expression of fibrotic proteins of human hypertrophic scar fibroblasts

作　　者：谢文博 胡晓龙 魏双 石继红 Xie Wenbo;Hu Xiaolong;Wei Shuang;Shi Jihong(Clinical Medicine Department of Jiangxi Medical College,Nanchang University,Nanchang 330006,China;Department of Burns and Cutaneous Surgery,Burn Center of PLA,the First Affiliated Hospital of Air Force Medical University,Xi'an 710032,China)

机构地区：[1]南昌大学江西医学院临床医学系,南昌330006 [2]空军军医大学第一附属医院烧伤与皮肤外科,全军烧伤中心,西安710032

出　　处：《中华烧伤与创面修复杂志》2025年第3期268-276,共9页Chinese Journal of Burns And Wounds

基　　金：国家自然科学基金面上项目(82172209)。

摘　　要：目的探讨二甲双胍对人增生性瘢痕(HS)成纤维细胞(Fb)增殖及纤维化蛋白表达的影响及其机制。方法该研究为实验研究。收集2021年6月—2022年6月于空军军医大学第一附属医院烧伤与皮肤外科行HS切除术的5例HS患者(男3例、女2例,年龄21~36岁)的HS组织,分离、培养Fb并取第5~7代Fb进行实验。取Fb,分别在其培养基中加入磷酸盐缓冲液(PBS)或终物质的量浓度为5、10、20、40 mmol/L的二甲双胍培养,培养48 h,采用细胞计数试剂盒-8(CCK-8)检测细胞增殖活性并计算细胞增殖抑制率,采用羟脯氨酸测定试剂盒检测细胞培养上清液中羟脯氨酸含量,采用蛋白质印迹法检测细胞中蛋白激酶B(Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)的磷酸化水平并计算磷酸化Akt(p-Akt)与Akt比值、磷酸化mTOR(p-mTOR)与mTOR比值;培养24 h,采用实时荧光定量反转录PCR法检测细胞中Ⅰ型胶原、Ⅲ型胶原和α-平滑肌肌动蛋白(α-SMA)的mRNA表达。另取Fb分为对照组(常规培养)和LY294002组、二甲双胍组、LY294002+二甲双胍组,后3组细胞培养基中分别加入LY294002、二甲双胍、LY294002+二甲双胍培养,LY294002与二甲双胍的终物质的量浓度分别为20μmol/L、10 mmol/L,于培养0(即刻)、24、48 h采用CCK-8检测细胞增殖活性,培养48 h采用蛋白质印迹法检测细胞中Akt、mTOR的磷酸化水平并计算p-Akt与Akt比值、p-mTOR与mTOR比值。细胞增殖抑制率实验样本数为4,其余实验样本数为3。结果培养48 h,与经PBS处理比较,经5、10、20、40 mmol/L二甲双胍处理细胞的增殖抑制率均显著升高(t值分别为10.69、14.20、19.73、52.54,P<0.05),经10、20、40 mmol/L二甲双胍处理细胞培养上清液中羟脯氨酸含量均显著降低(t值分别为8.06、7.86、10.25,P<0.05),经10、20、40 mmol/L二甲双胍处理细胞中p-Akt与Akt比值及经20、40 mmol/L二甲双胍处理细胞中p-mTOR与mTOR比值均显著降低(t值分别为2.82、4.28、9.88及5ObjectiveTo investigate the effects and mechanism of metformin on the proliferation and expression of fibrotic proteins of human hypertrophic scar(HS)fibroblasts(Fbs).MethodsThe study was an experimental study.From June 2021 to June 2022,5 patients with HS were admitted to the Department of Burns and Cutaneous Surgery of the First Affiliated Hospital of Air Force Medical University,including 3 males and 2 females,aged from 21 to 36 years.HS tissue was collected,Fbs were isolated and cultured,and Fbs of passage 5 to 7 were used for experiment.Fbs were taken and cultured in their respective media supplemented with phosphate buffered solution(PBS)or metformin at final molarities of 5,10,20,and 40 mmol/L for 48 hours.The cell proliferation activity was detected using the cell counting kit-8(CCK-8),and the proliferation inhibition rate of cells was calculated.The content of hydroxyproline in the cell culture supernatant was measured using a hydroxyproline assay kit.The phosphorylation levels of protein kinase B(Akt)and mammalian target of rapamycin(mTOR)in the cells were detected by Western blotting,and the ratios of phosphorylated Akt(p-Akt)to Akt and phosphorylated mTOR(p-mTOR)to mTOR were calculated.After 24 hours of culture,the mRNA expressions of typeⅠcollagen,typeⅢcollagen,andα-smooth muscle actin(α-SMA)in the cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction.Another batch of Fbs were divided into control group(with conventional culture),LY294002 group,metformin group,and LY294002+metformin group.LY294002,metformin,and LY294002+metformin were added to the culture media of the last three groups,respectively,with the final molarities of LY294002 and metformin being 20μmol/L and 10 mmol/L,respectively.CCK-8 was used to detect the cell proliferation activity at 0(immediately),24,and 48 hours of culture.After 48 hours of culture,Western blotting was used to detect the phosphorylation levels of Akt and mTOR in the cells,and the ratios of p-Akt to Akt and

关 键 词：二甲双胍 瘢痕 成纤维细胞 细胞增殖 胶原Ⅰ型 胶原Ⅲ型 增生性瘢痕 磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白信号通路 

分 类 号：R622[医药卫生—整形外科]