低氧环境对创面修复主要效应细胞表达MMP-9的作用

Hypoxia up-regulates MMP-9 in epidermal cells during wound repair

目的探讨低氧(2%O2)对创面修复主要效应细胞(表皮细胞及真皮成纤维细胞)表达MMP-9的影响。方法常规培养人表皮细胞及人真皮成纤维细胞,分为常氧组和低氧组(设低氧培养3 h和6 h两个组),采用Western blot、明胶酶谱法和荧光定量PCR法检测人表皮细胞和真皮成纤维细胞MMP-9细胞蛋白表达、MMP-9细胞外分泌和MMP-9转录水平变化。结果常氧培养的表皮细胞中,MMP-9呈一定程度的基础表达和细胞外分泌。与常氧组比较,低氧能明显促进表皮细胞MMP-9的蛋白表达(6 h时增强约20%,P<0.01)和MMP-9的细胞外分泌[(44.03±3.00)vs(60.64±3.77),P<0.01]。常氧与低氧条件下真皮成纤维细胞极少表达和分泌MMP-9。荧光实时定量PCR结果显示,低氧培养条件下表皮细胞MMP-9 mRNA水平显著增高,常氧对照组为(1.67±0.69),低氧6 h组为(5.47±0.51),两者比较差异有统计学意义(P=0.002)。结论低氧刺激能显著诱导表皮细胞,而非真皮成纤维细胞,表达和分泌MMP-9,这一作用可能与低氧刺激促进MMP-9的转录有关。

Objective To determine the effect of hypoxia （2% oxygen） on the expression of MMP-9 in main effector cells and epidermal cells and fibroblasts during wound repair. Methods Human epidermal HaCaT cells and primarily cultured fibroblasts from fresh foreskin tissues by circumcision were divided into 2 groups, normoxic group （synchronous culture, no treatment ）, and hypoxia group （including 2 subgroups with 3 and 6 h under hypoxia）. The expressin of MMP-9 in epidermal cells and fibroblasts, extracellular secretion of MMP-9 precursor expressions and changes of MMP-9 at the transcription level were respectively by Western blotting, gelatin zymography and fluorescence quantitative PCR. Results MMP-9 had a certain degree of basal expression and extracellular secretion in epidermal ceils from normoxic group. Hypoxia significantly promoted the expression levels of MMP-9 protein in the epidermal cells （20% increased in 6 h compared with the normoxic group, P 〈 0. 01 ）, and the extracellular secretions of MMP-9 precursor protein in hypoxia group at 6 h and normoxic control group （60.64 ± 3.77 vs 44.03 ± 3.00, P 〈0. 01 ）. However, fibroblasts almost never expressed or secreted MMP-9 under normoxic and hypoxic conditions. Fluorescence real-time quantitative PCR indicated hypoxia increased MMP-9 mRNA expression in epidermal cells （hypoxia group at 6 h 5.47 ±0.51 vs normoxic control group l. 67 ± 0.69, P = 0. 002）. Conclusion Hypoxic stimulation significantly induces the expression and secretion of MMP-9 in epidermal cells, rather than dermal fibroblast, which may be related to the promotion of MMP-9 at transcription level in the wound under local hypoxic environment.