高糖与同型半胱氨酸对大鼠皮肤成纤维细胞MMP-9表达及活性的影响

目的: 观察不同浓度糖及同型半胱氨酸对大鼠皮肤成纤维细胞MMP-9表达量及活性的影响。方法: 取1 d龄正常SD大鼠背部皮肤分离成纤维细胞,于含10%胎牛血清的DMEM培养液中培养。2-4代生长状态良好的细胞进行24 h血清剥夺(0.5%FBS)后,分为正常糖浓度组、高糖组、高糖+高同型半胱氨酸组分别培养6 h,之后用RT-PCR、Western blotting和明胶酶谱方法分别检测MMP-9 mRNA、蛋白表达量以及细胞外MMP-9活性。结果: 高糖(22 mmol/L)组MMP-9 mRNA、蛋白表达量及其活性均增高,分别为正常糖浓度组的1.15倍、1.59倍和1.34倍,P<0.05;高糖+高同型半胱氨酸(100 μmol/L)增高组更加明显,依次为正常糖浓度组的1.25倍、2.63倍和2.52倍,P<0.05。结论: 大鼠皮肤成纤维细胞在体外高糖环境下可高表达MMP-9;高同型半胱氨酸在高糖环境下可使MMP-9 表达及酶活性增高更为明显,表明高同型半胱氨酸血症也参与了对MMP-9的调节,可进一步增加高糖环境下成纤维细胞表达MMP-9。

Effects of high glucose and homocysteine on expression of MMP-9 in rat fibroblasts

AIM: To observe the effect of different concentrations of glucose and homocysteine on the expression of MMP-9 in fibroblasts, and to investigate the relationship between homocysteine and diabetic foot.METHODS: Fibroblasts were obtained from the dermis of 1-day-old normal SD rat and cultured in DMEM containing 10% FBS. After deprivation of serum (using 0.5% FBS) for 24 h, the fibroblasts in 2-4 passages were cultured for 6 h under the conditions of normal glucose, high glucose and high glucose with high concentration of homocysteine, respectively. The expression of MMP-9 at mRNA and protein levels was assessed by RT-PCR and Western blotting, and the activity of MMP-9 was determined by gelatin zymography analysis. RESULTS: The expression of MMP-9 at mRNA and protein levels and activity of MMP-9 in high glucose (22 mmol/L) group were 1.15 folds, 1.59 folds and 1.34 folds of those in normal glucose group (P<0.05), respectively. The effect of high glucose became more pronounced when co-treated with high concentration of homocysteine (100 μmol/L), which were 1.25 folds, 2.63 folds and 2.52 folds of those in normal glucose group (P<0.05), respectively.CONCLUSION: The increase in the expression of MMP-9 is concentration-dependen with glucose content in the culture. High concentration of homocysteine promotes this process, suggesting that hypercysteinemia might play an important role in the expression of MMP-9 in fibroblast.