中波紫外线诱导人皮肤成纤维细胞衰老及机制研究

目的 探讨中波紫外线（UVB）对人皮肤成纤维细胞的诱导衰老作用及可能机制.方法 使用组织块法分离培养原代人皮肤成纤维细胞,通过观察不同剂量UVB照射后的细胞形态学改变以及β-半乳糖苷酶（p-gal）细胞衰老染色情况,确定后续实验诱导皮肤成纤维细胞衰老的UVB单次照射剂量.于经确定剂量UVB照射后的不同时点（12、24、48、72 h）收集细胞及其细胞培养上清液,Western blotting检测诱导衰老的皮肤成纤维细胞衰老相关蛋白P16表达;ELISA法检测诱导衰老细胞Ⅰ、Ⅲ型胶原及基质金属蛋白酶1、3（MMP1、MMP3）表达.结果 经40 mJ/cm2 UVB单次照射的人皮肤成纤维细胞呈现典型的衰老细胞形态学改变,β-gal细胞衰老染色阳性细胞百分比为（88.75±5.32）%,确定诱导衰老UVB单次照射剂量为40 mJ/cm2.Western blotting显示,诱导衰老皮肤成纤维细胞P16蛋白表达随UVB照射时间的延长而明显升高;ELISA法检测发现诱导衰老细胞Ⅰ、Ⅲ型胶原表达随UVB照射时间的延长而明显减少,MMP1和MMP3表达则随UVB照射时间的延长而显著增加.结论 UVB照射能诱导人皮肤成纤维细胞发生衰老,其机制可能与细胞胶原合成减少而降解增加有关.

Senescence of human skin fibroblasts induced by ultraviolet B and its mechanism

Objective To investigate the role of ultraviolet B (UVB) in induction of senescence of human skin fibroblasts and explore its mechanism. Methods Primary human skin fibroblasts were isolated and cultured by tissue explants adherent method, and UVB irradiation dose for induction of senescence of skin fibroblasts for subsequent experiment was determined through observation of cell morphology and β-galactosidase (β-gal) cell senescence staining after irradiation with different doses of UVB. Cells and supernatant of cell culture were collected at different time points (12, 24, 48 and 72 h) after irradiation with established doses of UVB. The expression of marker P16 protein in induced senescent cells was detected by Western blotting, and expression of typeⅠcollagen, type III collagen, matrix metalloproteinase (MMP)-1 and MMP-3 of senescent cells was detected by ELISA. Results Human skin fibroblasts exhibited typical morphology of cell senescence after irradiation with 40 mJ/cm2 UVB, the percent of cells with positive staining with β-gal for cell senescence was (88.75±5.32)%, and the established irradiation dose for induction of cell senescence was 40 mJ/cm2. Western blotting analysis revealed that the expression of P16 in induced  senescent skin fibroblasts increased with time of UVB irradiation. It was indicated by ELISA that the expression of typeⅠcollagen and type Ⅲ collagen significantly decreased with time of UVB irradiation in induced senescent cells, while the expression of MMP1 and MMP3 significantly increased with time of UVB irradiation. Conclusion Senescence of human skin fibroblasts can be induced by UVB irradiation, and the mechanism may relate to the decrease of collagen synthesis and increase of collagen degradation.