UVA致人皮肤成纤维细胞急性和慢性光损伤模型的建立  

Establishment of acute and chronic photodamge models in human dermal fibroblasts by UVA-irradiation

作　　者：侯巍[1] 郑跃[2] 许庆芳[2] 刘晨[2] 陆春[2] 赖维[2] 

机构地区：[1]新疆医科大学第一附属医院皮肤科,新疆乌鲁木齐830054 [2]中山大学附属第三医院皮肤科,广东广州510630

出　　处：《临床皮肤科杂志》2014年第12期703-706,共4页Journal of Clinical Dermatology

基　　金：国家自然科学基金(81171523);广东省自然科学基金(10151008901000117)资助项目

摘　　要：目的:探讨人皮肤成纤维细胞(human dermal fibroblasts,HDFs)急性和慢性光损伤模型建立的方法。方法:体外培养原代人皮肤成纤维细胞,选取第4~8代的细胞进行实验。用长波紫外线(UVA)单次照射建立HDFs急性光损伤模型,荧光倒置显微镜观察不同剂量UVA照射后第1、2、3天HDFs的形态变化;CCK-8法检测照射后HDFs的增殖活性。慢性光损伤HDFs模型采用8-甲氧沙林(8-MOP)避光孵育细胞24h,随后以含8-MOP的磷酸盐缓冲液(PBS)置换培养基,行9J/cm^2 UVA照射,照射完成后换Dulbecco改良Eagle培养基(DMEM)(含10%胎牛血清)避光传代培养,21d后显微镜下观察HDFs形态;衰老相关-β-半乳糖苷酶(SA-β-Gal)染色法计算衰老细胞率。结果:单次UVA照射导致HDFs增殖率下降,与UVA剂量呈正相关。UVA在剂量为≤10J/cm^2时,细胞存活率保持在>85%;而UVA剂量≥15J/cm^2时细胞存活率明显降低;≥20 J/cm^2时存活率为50%左右,至25 J/cm^2时仅为约25%。慢性光损伤HDFs诱导组(即UVA+MOP组)几乎所有细胞均出现体积变大、细胞颗粒增加等细胞老化的特征性改变;SA-β-Gal染色细胞的阳性率>95%。结论:UVA单次照射可成功建立HDFs急性光损伤模型,UVA联合8-MOP构建HDFs慢性光损伤模型。Objective： To explore the methods of establishing acute and chronic photodamage models in human dermal fibrob- lasts （HDFs） by UVA irradiation. Methods： Primary HDFs were cultured in vitro, 4 to 8 generation cells were chosen for the experiment. Single dose UVA irradiation was used to establish the models of acute photodamage. Cell morphological changes were observed by inverted fluorescence microscope and photographs were taken at days 1, 2, 3 after different doses of UVA irradiation. Cell proliferation was analysed by CCK-8. The HDFs in the chronic photodamage group were treated with 8-methoxypsoralen of 50 mg/L for 24 h. Then the medium were replaced by PBS containing 8-methoxypsoralen, followed by 9 J/cm2 UVA exposure to induce photodamage. After irradiation the cells were cultured under the protection of light in DMEM （containing 10% fetal bovine serum） for 21 days. The mophorlogical changes of HDFS were observed and photographs were taken. Senescence-associated-β- galactosidase （SA-β-gal） activity was measured. Results： Single dose UVA irradiation inhibited HDFs cell proliferation in a dose--dependent manner. At the dose of ≤10J/cm2, cell viability was maintained more than 85%. Viable cells markedly decreased at the dose of 15J/cm2. At the dose of ~≥20J/cm2, cell viability was 50%. Viable cells only were 25% at the dose of 25 J/cm2. Most of HDFs of the chronic photodamage group manifestated the following characteristics： large cytoplasmic volume, vacuolization and increased number of granules. The positive rate of SA-β-gal staining cells was over 95%. Conclusion： Acute photodamage model in HDFs could be established successfully by UVA single irradiation, and UVA combined with 8-methoxypsoralen can construct chronic photodamage model in HDFs.

关 键 词：光老化 急性光损伤 皮肤成纤维细胞 

分 类 号：R751[医药卫生—皮肤病学与性病学]