高葡萄糖高游离脂肪酸对皮肤成纤维细胞β-连环蛋白表达的影响

Effects of high glucose level and free fatty acids on expression of β-catenin in skin fibroblasts

目的 观察高浓度葡萄糖、高浓度游离脂肪酸对人皮肤成纤维细胞β-连环蛋白（β-catenin）表达的影响,探讨糖尿病皮肤溃疡难愈合的机制.方法 复苏正常成人皮肤成纤维细胞株,高糖改良伊格尔培养基（DMEM,25 mmol/L）培养基进行培养.首先,噻唑蓝（MTT）法筛选葡萄糖和游离脂肪酸对成纤维细胞最佳作用浓度：将细胞分为葡萄糖处理组和游离脂肪酸处理组,葡萄糖处理组含不同浓度葡萄糖（30、35、40、50、60、70 mmol/L）,对照组不加葡萄糖；游离脂肪酸处理组含不同浓度软脂酸（100、200、400、600、800、1000 μmol/L）,对照组仅为高糖DMEM培养基.以MTT法检测不同培养条件下成纤维细胞吸光度（A）值,结合细胞形态学观察,选择一个对细胞会造成一定损伤,但又不会使细胞大量死亡的浓度作为处理成纤维细胞的最佳浓度.其次,以最佳浓度培养成纤维细胞不同时间,Western blot法检测胞质内β-catenin蛋白的表达.取对数生长期的细胞分为3组：高浓度葡萄糖组（35 mmol/L）、高浓度游离脂肪酸组（200 μmol/L）、高浓度葡萄糖±高浓度游离脂肪酸（35 mmol/L ±200 μmol/L）联合组.于加入葡萄糖和软脂酸培养前（0 h）以及培养后4、8、12、16、20、24、48 h,收集各组细胞,采用Western blot法检测各组细胞3-catenin蛋白的表达.结果 （1）培养24 h后,成纤维细胞A值于葡萄糖浓度为35 mmol/L和游离脂肪酸为200 μmol/L时分别为（0.741 ±0.012、0.676±0.043）,与对照组（0.934 ±0.007）比较开始下降（t值分别为1.70和2.48,P＜0.05）,后随着葡萄糖和软脂酸浓度的增加,各组A值均减小（P＜0.05）.（2）35 mmol/L葡萄糖组、200 μmol/L游离脂肪酸组及两者联合培养组细胞内3-catenin蛋白的表达分别于12、4、8h开始降低（t值分别为7.45、5.12、3.33,P＜0.05）,48 h降至最低（P＜0.05）.结论 高葡萄糖短期内能延缓高游离脂肪酸诱导的细胞β-catenin低表达,但在持续高糖存在的情况下,成纤维细胞β-catenin低表达程度比两者单独作用时更显著,高葡萄糖对高游离脂肪酸诱导的细胞β-catenin低表达呈现出先抑制后促进的作用.

Objective To study the effects of high glucose,high free fatty acids （FFA） and combination on the expression of β-catenin in skin fibroblasts in vivo.Methods Normal human skin firbroblasts （HSFs） were pelleted and grown in high-glucose Dulbecco＆#39; s modified Eagle＆#39; s medium （DMEM,glucose concentration：25 mmol/L） supplemented with 10% fetal bovine serum （FBS）.Cells in passage three were used for the following experiments.They were seeded in 96-well plates and cultured in nutrient solution with different concentrations of glucose and FFA respectively.First,the optimized concentration was selected by methyl thiazol tetrazolium （MTT） assay.Following two groups were set up：high-glucose medium groups （with different glucose concentrations of 30,35,40,50,60 and 70 mmol/L） and highFFA groups （with different concentrations of 100,200,400,600,800 amd 1000 μmol/L）.Neither glucose nor FFA was given in control group.80% confluent fibroblasts were incubated for 24 h and then proliferation was measured by using MTT assay.Second,in another experiment,the expression of β-catenin was detected by using Western blotting.HSFs were harvested before （0 h） or 4,8,12,16,20,24,and 48 h after stimulation with glucose （35 mmol/L）,FFA （200 μmol/L） and both （35 mmol/L ± 200 μmol/L）.Total protein was extracted from those cells.Protein levels of β-catenin were determined using Western blotting.Results （1） As compared with control group,the absorbance values of HSFs in glucose 35 mmol/L group and FFA 200 μmol/L group after culture for 24 h began to reduce （t =1.70 and 2.48respectively,P 〈 0.05）.With the increases of glucose and FFA,the absorbance values were significantly decreased （P 〈 0.05）.（2） HSFs exposed to high-FFA showed a low expression of β-catenin protein at 4.h （P 〈0.05）,and the level of β-catenin protein began to reduce at 12 h after treatment with high-glucose medium （P〈0.05）.However,the β-catenin protein expression in HSFs stimulated with glucose combined with FFA was decreased at 8 h,reaching the minimum at 48 h （P 〈 0.05）.Conclusion Glucose and FFA can both inhibit the expression of β-catenin.In a short time,high FFA exhibit inhibition of the expression on β-catenin,but this suppressive effect of high glucose was not obvious.When combined intervention,high glucose could prevent the low expression of β-catenin induced by FFA.In the case of the presence of sustained high glucose level,the inhibition of β-catenin was more significant than their respective role.