晚期糖基化终末产物降低成纤维细胞增殖和迁移能力的机制  

Mechanisms of advanced glycation end products reducing the proliferation and migration of fibroblasts

作　　者：冯自波[1] 王知 李恭驰[2] 邹利军[1] 张静[1] 祝友鹏 杜烨 李炳辉[1] 郑洁[1] Feng Zibo;Wang Zhi;Li Gongchi;Zou Lijun;Zhang Jing;Zou Youpeng;Du Ye;Li Binghui;Zheng Jie(Department of Wound Repair,Wuhan Liyuan Hospital,Tongji Medical College,Huazhong University of Science & Technology,Hubei chronic wound and diabetic foot medical clinical research center,Wuhan,430077,China;Department of Hand Surgery,Union Hospital,Tongji Medical College,Huazhong University of Science & Technology,Wuhan,430022,China)

机构地区：[1]华中科技大学同济医学院附属梨园医院创面修复科湖北省慢性创面及糖尿病足医学临床中心,武汉430077 [2]华中科技大学同济医学院附属协和医院手外科,武汉430022

出　　处：《中华实验外科杂志》2019年第10期1780-1783,共4页Chinese Journal of Experimental Surgery

基　　金：华中科技大学自主创新研究基金(2016YXMS126);湖北省自然科学基金面上项目(2017CKB892);湖北省慢性创面及糖尿病医学临床研究中心资助项目(2018BCC340).

摘　　要：目的探讨晚期糖基化终末产物(AGEs)对成纤维细胞增殖和迁移的影响及其机制.方法采集2018年1月至2018年12月华中科技大学同济医学院附属梨园医院创面修复科收治的糖尿病足患者足部创面组织和非糖尿病足患者足部正常创面组织各20例,免疫印迹法(Westernblot)检测创面组织中AGEs、波形纤维蛋白(Vimentin)和β-连环蛋白(β-catenin)的表达水平.取包皮成纤维细胞进行原代培养并鉴定,采用0、100、300、500、1 000、2 000 μg/L的AGEs处理成纤维细胞,以300 μg/L牛血清白蛋白(BSA)为对照组,作用48 h后进行细胞计数试剂盒-8(CCK-8)、小室迁移(Transwell)实验以及Western blot检测并采用t检验分析结果.结果与正常组织比较,糖尿病足组织中AGEs明显偏高(t=3.564,P< 0.05),而Vimentin的表达水平偏低(t=8.325,P<0.05),但β-catenin的表达高于正常组织(t=5.738,P<0.05).分离纯化培养的细胞形态呈梭形,细胞免疫荧光检测95%以上细胞表达Vimentin,鉴定为成纤维细胞.随着加入的AGEs浓度从100、300 μg/L增加至500、1 000μg/L时,成纤维细胞的增殖(t=4.336,PA500/BSA<0.01;t=19.562,PA1000/BSA<0.01;t=42.373,PA2000/BSA<0.01;t=5.380,PA1000/A500<0.01;t=23.726,PA2000/A1000<0.01)和迁移(t=12.117,PA300/BSA <0.01;t=14.484,PA500/BSA <0.01;t=21.940,PA1000/BSA <0.01)能力逐渐降低,呈浓度依赖性.并且,加入AGEs浓度大于300μg/L时,成纤维细胞中的Wnt3α和Wnt7α的表达明显增加,β-catenin的表达相较于对照组有明显的升高,与BSA组比较无明显升高.结论 AGEs降低成纤维细胞的增殖和迁移能力与Wnt/β-catenin信号通路有关.Objective To investigate the effects of advanced glycation end products (AGEs) on the proliferation and migration of fibroblasts and its mechanism. Methods 20 diabetic foot wounds and 20 non-diabetic foot normal foot wounds were collected from the Department of Wound Repair, Liyuan Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from January 2018 to December 2018. Western blotting was used to detect the expression of AGEs, Vimentin and β-Catenin in non-diabetic and diabetic foot wound tissue. Prepuce fibroblasts were cultured and identified. Fibroblasts were treated for 48 hours with 0, 100, 300, 500, 1 000, 2 000 μg/L AGEs, 300 μg/L BSA as control group. Cell counting kit-8 (CCK-8), Transwell and Western blotting were performed after treatment and T-test analysis results. Results Compared with normal tissues, AGEs in diabetic foot tissues were significantly higher (t=3.564, P<0.05), while Vimentin expression was lower (t=8.325, P<0.05), but the expression of β-Catenin was higher than that in normal tissues (t=5.738, P<0.05). The morphology of the isolated and purified cells was spindle-shaped. Vimentin was expressed in more than 95% of the cells by immunofluorescence assay and identified as fibroblasts. When the concentration of AGEs increased from 100, 300 μg/L to 500, 1 000 μg/L, the proliferation (t=4.336, PA500/BSA<0.01;t=19.562, PA1000/BSA<0.01;t=42.373, PA2000/BSA<0.01;t=5.380, PA1000/A500<0.01;t=23.726, PA2000/A1000<0.01) and migration (t=12.117, PA300/BSA<0.01;t=14.484, PA500/BSA<0.01;t=21.940 PA1000/BSA<0.01) ability of fibroblasts decreased gradually, showing a concentration-dependent. In addition, the expression of Wnt3α and Wnt7α in fibroblasts increased significantly after AGEs concentration was added more than 300 μg/L, and the expression of β-Catenin was significantly higher than that in control group, but not in BSA group. Conclusion AGEs can decrease the proliferation and migration of fibroblasts, which is related to Wnt/β-Catenin signaling

关 键 词：晚期糖基化终末产物 成纤维细胞 增殖和迁移 糖尿病足 

分 类 号：R587.2[医药卫生—内分泌]