自噬对晚期糖基化终末产物致成纤维细胞损伤的早期保护  

Autophagy has an early protective effect against fibroblast injury induced by advanced glycation end products

作　　者：韩焱福 陶然[2] 孙天骏[3] Han Yanfu;Tao Ran;Sun Tianjun(Department of Plastic Surgery,Beijing Shijitan Hospital,Capital Medical University,Beijing 100038,China;Department of Plastic Surgery,First Medical Center,General Hospital of Chinese PLA,Beijing 100853,China;Department of Burn and Plastic Surgery,Fourth Medical Center,General Hospital of Chinese PLA,Beijing 100048,China)

机构地区：[1]首都医科大学附属北京世纪坛医院整形美容外科,北京市100038 [2]解放军总医院第一医学中心整形修复科,北京市100853 [3]解放军总医院第四医学中心烧伤整形科,北京市100048

出　　处：《中国组织工程研究》2020年第35期5619-5624,共6页Chinese Journal of Tissue Engineering Research

基　　金：2020年度军队医学科技青年培养计划孵化项目(20QNPY097),项目负责人:陶然。

摘　　要：背景:晚期糖基化终末产物与糖尿病创面难愈合密不可分,晚期糖基化终末产物可影响成纤维细胞增殖及迁移,但自噬在晚期糖基化终末产物致成纤维细胞损伤中的早期保护作用尚未见相关研究。目的:研究晚期糖基化终末产物对成纤维细胞活力及自噬的影响,探讨自噬在成纤维细胞损伤中的早期保护作用。方法:以体外传代培养的人成纤维细胞为研究对象,设立正常对照组(DMEM培养液)、晚期糖基化终末产物组(DMEM培养液+100 mg/L晚期糖基化终末产物)、晚期糖基化终末产物抑制剂N-苯乙酰噻唑组(DMEM培养液+100 mg/L晚期糖基化终末产物+100 mg/L N-苯乙酰噻唑)、自噬抑制剂氯喹组(DMEM培养液+100 mg/L晚期糖基化终末产物+10μmol/L氯喹)。培养48 h后,锥虫蓝染色检测各组细胞活力,Western blot法检测LC3及P62蛋白的表达,细胞免疫荧光实验检测自噬体形成,透射电镜观察自噬泡超微结构。结果与结论:①细胞死亡率:与正常对照组比较,晚期糖基化终末产物组细胞死亡率显著增加(P<0.01);与晚期糖基化终末产物组比较,N-苯乙酰噻唑组细胞死亡率显著降低(P<0.05),而氯喹组细胞死亡率显著增加(P<0.05);②LC3及P62蛋白的表达:与正常对照组相比,晚期糖基化终末产物组LC3Ⅱ/LC3Ⅰ比值显著增加,P62表达显著降低(P<0.01);与晚期糖基化终末产物组相比,N-苯乙酰噻唑组LC3Ⅱ/LC3Ⅰ比值明显下降,P62表达明显上升(P<0.05),而氯喹组LC3Ⅱ/LC3Ⅰ蛋白含量变化不明显(P>0.05),P62表达明显上升(P<0.05);③自噬体形成:晚期糖基化终末产物组和氯喹组均可见细胞中LC3绿色荧光点状聚集物增加;晚期糖基化终末产物组可见数目较多的自噬小体和自噬泡结构,而氯喹组细胞中自噬体结构数量和空泡状结构少于晚期糖基化终末产物组;④结果证实,晚期糖基化终末产物可损伤成纤维细胞,降低细胞生存率,早期可激活细胞自噬;�BACKGROUND:Advanced glycation end products(AGEs)are closely related to diabetic wound healing.AGEs can affect the proliferation and migration of fibroblasts,but the early protective effect of autophagy in AGE-induced fibroblast injury has not been explored.OBJECTIVE:To confirm the effect of AGEs on fibroblast cell viability and autophagy,and to explore the early protective effect of autophagy on cell injury.METHODS:Human fibroblasts were cultured in vitro.Normal control group(DMEM medium),AGE group(DMEM medium+100 mg/L AGEs),AGE+PTB(AGE inhibitor)group(DMEM medium+100 mg/L AGEs+100 mg/L PTB),and AGE+chloroquine(autophagy inhibitor)group(DMEM medium+100 mg/L AGEs+10μmol/L chloroquine)were established.After 48 hours of culture,trypan blue staining was used to detect the cell viability of each group,western blot was used to detect the expression of LC3 and P62 protein,cell immunofluorescence assay was used to detect the formation of autophagy using,and transmission electron microscopy was used to observe the ultrastructure of autophagy vesicles.RESULTS AND CONCLUSION:Compared with the control group,cell death rate in the AGE group increased significantly(P<0.01).Compared with the AGE group,the cell death rate in the AGE+PTB group decreased significantly(P<0.05),while that in the AGE+chloroquine group increased significantly(P<0.05).Compared with the control group,the ratio of LC3 II/LC3 I in the AGE group increased significantly,and the expression of P 62 decreased significantly(P<0.01);compared with the AGE group,the ratio of LC3 II/LC3 I in the AGE+PTB group decreased significantly,the expression of P62 increased significantly(P<0.05),whereas the content of LC3 II/LC3 I in the AGE+chloroquine group did not change significantly(P>0.05)and the expression of P62 increased significantly(P<0.05).Both in the AGE group and AGE+chloroquine group,the green fluorescent dot-like aggregation of LC3 was increased.By comparing these two groups,there were more autophagy bodies and autophagy vesicles in the AGE group,whereas the

关 键 词：细胞 成纤维细胞 晚期糖基化终末产物 自噬 N-苯乙酰噻唑 氯喹 细胞活力 免疫 创面 实验 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]