UVB照射诱导皮肤成纤维细胞早期衰老的研究

Ultroviolet B exposure triggers premature senescence in hmnan skin fibroblasts

目的探讨UVB诱导的细胞衰老与肿瘤发生的关系。方法噻唑蓝（MTT）法检测UVB辐射后的细胞增殖情况，筛选诱导衰老适宜的亚毒性剂量和照射次数。染色法检测衰老相关的β-半乳糖苷酶（SA β-gal）活性。RT—PCR检测衰老相关基因纤维结合素（FN）、骨结合素（ON）和平滑肌22（SM22）的表达。结果以10mJ／cm^2的亚毒性剂量连续5次照射人成纤维细胞后，衰老的生物学特征得以明显表现：①MTT法检测显示细胞增生能力的减弱。②照射组具有SAB—gal活性的阳性细胞明显增加，照射组和对照组的阳性率分别为82．0％和33．7％（P〈0．01）。③3种衰老相关基因FN、ON和SM22的表达亦明显增强，分别约为对照组细胞的2．7、2．0、2．3倍（P〈0．05）。结论反复亚毒性剂量UVB照射人成纤维细胞，初步建立一种UVB诱导的应激诱导的早期衰老（SIPS）模型。

Objective To develop a model of ultraviolet B （UVB）-induced premature senescence in human skin fibroblasts （HSF） so as to assess the relationship between stress-induced premature senescence and tumorigenesis. Methods The irradiation dose and frequency were optimized for the induction of premature senescence. HSF were irradiated with UVB of 10 mJ/cm^2 once daily for 5 days, and unirradiated HSFs served as the control. After the last irradiation, cell proliferation was determined by MTT assay on day 3, 4, 5, 6 and 7, SA 13-Gal staining was performed to evaluate the senescence state of ceils on day 3, and RT-PCR to detect the expressions of three senescene-associated genes, including fibronectin （FN）, osteonectin （ON） and smooth muscle 22 （SM22） on day 3. Results After five exposures to UVB, HSF showed biological characteristics of senescence. As assessed by MTr assay, there was a loss of replicative potential in irradiated cells. The proportion of SA-β-gal-positive ceils was 82.0% in UVB-stressed HSFs and 33.7% in the control cells （P 〈 0.01 ）. The mRNA levels of FN, ON and SM22 were upregulated by 2.7, 2.0 and 2.3 folds respectively in irradiated HSF compared with the control ceils （all P 〈 0.05）. Conclusion A stress-induced premature senescence model is established using HSF by repeated exposure to subcytotoxic UVB.