淀粉样肽前体蛋白17肽对紫外线照射后皮肤成纤维细胞内活性氧簇和基质金属蛋白酶-1 mRNA表达的作用

Effects of APP17-mer peptide on oxidative damage and expression of MMP-1 mRNA in cultured human skin fibroblasts irradiated with ultraviolet light

目的通过建赢体外培养人皮肤成纤维细胞的紫外线损伤模型，探讨淀粉样肽前体蛋白319～335肽段（APP17肽）对紫外线（ultraviolet，UV）照射后人皮肤成纤维细胞的保护作用及其可能的机制。方法从健康青年男性的包皮组织原代培养成纤维细胞。UV照射培养的成纤维细胞，并给予不同浓度的APP17肽保护。MTT法检测细胞活性。激光共聚焦显微镜检测活性氧（ROS）的水平，实时定最RTPCR方法检测基质金属蛋白酶-1（MMP-1）mRNA的表达。结果成功培养原代人皮肤成纤维细胞，并建立体外UV损伤模型。UV照射后细胞活性下降（P〈0．05），细胞内ROS产生增多（P〈0．05），MMP-1 mRNA表达增加1．78倍（P〈0．01）。APP17肽可增加UV照射后细胞活性（P〈0．05），降低细胞内ROS水平（P〈0．05），抑制MMP-1 mRNA表达（P〈0．05）。结论APP17肽对成纤维细胞的UV损伤有保护作用，APP17肽的光保护作用可能与其抑制ROS产生有关。

Objective To establish an ultraviolet irradiation damage model in cultured fibroblasts derived from human skin and to explore the potential protective effects and mechanisms of amyloid precur sor protein 17 mer peptide （APP17 mer peptide） on the oxidative damage and collagen metabolism in cul tured fibroblasts after ultraviolet irradiation. Methods Human dermal fibroblast cultures were estab lished by outgrowth from foreskin biopsies of a healthy donor and were irradiated by a single exposure to ultraviolet rays and cultured in a series of concentrations of APP17-mer peptide （0, 20, 40, 80μmol/L）. The activity of fibroblasts was detected by the assay of MTT. The intracellular ROS level was measured with a confocal microscope. The expression of MMP 1 mRNA was analyzed real-time quantitatively following RT-PCR. Results Primary cultures of human skin fibroblasts were established from human fore skin in DMEM supplemented with 10 % fetal bovine serum. UV irradiation depressed cellular activity and increased intracellular level of ROS （P〈0.05）. 40μmol/L and 80μmol/L APP17 mer peptide increased the cellular activity in both UVirradiated fibroblasts and unirradiated fibroblasts （P〈0.05）, however, 20 μmol/L did not show such protective effects （P〈0.05）. 40μmol/L APP17-mer peptide could depress the level of ROS in irradiated fibroblasts. A single exposure of fibroblasts to UV irradiation resulted in 1.78 foldup regulation of MMP-1 mRNA compared with unirradiated sample, 40gmol/L and 80μmol/L APP17 mer peptide decreased the expression of MMP-1 mRNA （P〈0.05 and P〈0.01, respectively）. Conclusion APP17 mer peptide can enhance cellular activity under UV induced oxidative stress and inhibit collagen degradation in fibroblasts irradiated with ultraviolet rays. Inhibition of ROS production may be involved in the protective mechanism of APP17 peptide.