高糖环境下抑瘤素对人皮肤成纤维细胞增殖和细胞外基质分泌的影响  

Effect of oncostatin M on proliferation and secretion of extracellular matrix in human dermal fibroblasts in high glucose condition

作　　者：刘露露 符学铭 薛碧宇 薛斌[1] LIU Lulu;FU Xueming;XUE Biyu;XUE Bin(Department of Bums and Orthopedic Surgery,the First Affiliated Hospital of Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学附属第一医院烧伤整形外科,重庆400016

出　　处：《第三军医大学学报》2021年第10期955-963,共9页Journal of Third Military Medical University

摘　　要：目的探讨高糖环境下抑瘤素(oncostatin M,OSM)对人皮肤成纤维细胞(human dermal fibroblasts,HDFs)增殖和细胞外基质分泌的影响及其可能的作用机制。方法(1)高糖模型建立:人皮肤成纤维细胞分为5组,分别给予5、10、15、20、25 mmol/L葡萄糖处理;采用MTT检测高糖培养24、36、48 h各组细胞的增殖活力,Western blot检测对照组(5 mmol/L葡萄糖)和高糖组(20 mmol/L葡萄糖)细胞培养24、36、48 h后ERK、p-ERK和CyclinD1蛋白表达。(2)探讨OSM对高糖模型作用的最佳浓度:细胞分为5组,对照组给予20 mmol/L葡萄糖处理,其余各组分别给予20 mmol/L葡萄糖+25、50、100、200 ng/mL OSM处理;采用MTT检测各组细胞培养24、36、48 h后的增殖活力;Western blot检测各组细胞CollagenⅠ、CollagenⅢ、Fibronectin蛋白表达。(3)探讨OSM对高糖模型的作用的机制:将细胞分为对照组、高糖组、高糖+OSM组、高糖+OSM+抑制剂PD98059组。流式细胞仪测定各组细胞周期,Western blot检测ERK、p-ERK、CyclinD1、CollagenⅠ、CollagenⅢ、Fibronectin等相关因子的蛋白表达,q-PCR验证各组细胞ERK1/2、CyclinD1、CollagenⅠ、CollagenⅢ、Fibronectin基因相对表达量的差异。结果与对照组比较,经20 mmol/L葡萄糖处理后,HDFs的增殖活力显著降低(P<0.001),G1期细胞增多(P<0.01),S期细胞显著减少(P<0.01);ERK、p-ERK、CyclinD1、CollagenⅠ、CollagenⅢ、Fibronectin蛋白表达显著降低(P<0.01,P<0.05)。与高糖组比较,100 ng/mL OSM处理高糖组48 h后,细胞的增殖活力显著增高(P<0.001);G1期细胞减少(P<0.05),S期细胞显著增多(P<0.05);ERK、p-ERK、CyclinD1、CollagenⅠ、CollagenⅢ、Fibronectin的蛋白表达显著增高(均P<0.01);q-PCR结果与Western blot结果基本一致,差异均有统计学意义(P<0.05)。当ERK通路特异性小分子抑制剂PD98059阻断ERK信号通路后,上述效应显著减弱(P<0.05)。结论OSM可能通过ERK信号通路逆转高糖对HDFs增殖和细胞外基质分泌的抑Objective To investigate the effects of oncostatin M(OSM)on the proliferation and extracellular matrix secretion of human dermal fibroblasts(HDFs)in high glucose environment and its possible mechanism.Methods(1)Establishment of hyperglycemia model:HDFs were treated with 5,10,15,20 and 25 mmol/L glucose,respectively.MTT assay was used to detect the proliferation activity of cells cultured in high glucose for 24,36 or 48 h.Western blotting was used to detect the protein levels of ERK,p-ERK and CyclinD1 in the HDFs treated with 5 or 20 mmol/L glucose for 24,36 and 48 h.(2)Optimal concentration of OSM on hyperglycemia model:the cells were treated with 20 mmol/L glucose(control group),and 20 mmol/L glucose+25,50,100,200 ng/mL OSM,respectively.MTT assay was used to detect the proliferation of above cells cultured for 24,36 and 48 h.The protein expression of CollagenⅠ,CollagenⅢand fibronectin was detected by Western blotting.(3)Mechanism of OSM on hyperglycemia model:the cells were divided into control group,high glucose group,high glucose+OSM group,and high glucose+OSM+inhibitor PD98059 group.Cell cycle was determined by flow cytometry.The protein levels of ERK,p-ERK,CyclinD1,CollagenⅠ,CollagenⅢ,fibronectin and other related factors were detected by Western blotting.q-PCR was used to measure the levels of ERK1/2,CyclinD1,CollagenⅠ,CollagenⅢand fibronectin in each group.Results Treatment of 20 mmol/L glucose resulted in significantly decreased proliferation activity(P<0.001),increased proportion of G1 cells(P<0.01)and decreased proportion of S-phase cells(P<0.01),and reduced expression of ERK,p-ERK,CyclinD1,CollagenⅠ,CollagenⅢand fibronectin(P<0.01,P<0.05)when compared with the control cells.While treatment of 100 ng/mL OSM for 48 h enhanced the proliferation activity(P<0.001),decreased the proportion of G1 cells(P<0.05)but elevated the proportion of S-phase cells(P<0.05),and raised protein levels of ERK,p-ERK,CyclinD1,CollagenⅠ,CollagenⅢand fibronectin cells(all P<0.01).q-PCR indicated similar resul

关 键 词：抑瘤素 人皮肤成纤维细胞 高糖 细胞增殖 ERK 细胞外基质 

分 类 号：R322.991[医药卫生—人体解剖和组织胚胎学] R341[医药卫生—基础医学]