中波紫外线诱导的提前衰老成纤维细胞上清液对人真皮成纤维细胞增殖、老化及自噬的影响  

Effects of conditioned medium of prematurely senescent fibroblasts induced by ultraviolet B on cellular proliferation, aging and autophagy of human dermal fibroblasts

作　　者：王申[1] 周炳荣[1] 骆丹[1] 张家安[1] 刘娟[1] 张丽超[1] 易飞[1] 吴红巾 栗丹[1] 胡燕燕[1] 

机构地区：[1]南京医科大学第一附属医院皮肤科,210029

出　　处：《中华皮肤科杂志》2015年第7期485-489,共5页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81371757、81301384、81000700）

摘　　要：目的观察中波紫外线诱导的提前衰老的成纤维细胞条件培养液对人真皮成纤维细胞增殖、老化及自噬的影响。方法取健康青少年男子环切术后包皮进行人真皮成纤维细胞的分离培养。将中波紫外线诱导的提前衰老的成纤维细胞条件培养液培养成纤维细胞设为实验组，并设立对照组即正常成纤维细胞条件培养液培养成纤维细胞。处理20d后，用CCK8法检测细胞增殖活性，EDU（5-乙炔基-2，脱氧尿嘧啶核苷）法检测细胞增殖，流式细胞仪检测细胞周期，β半乳糖苷酶染色计算衰老细胞百分比，吖啶橙染色检测细胞自噬，Western印迹法及间接免疫荧光检测自噬相关蛋白LC3-B的表达水平。数据采用GraphpadPrism5软件分析，两组间比较采用成组t检验。结果实验组成纤维细胞增殖活力（0．831±0．017）明显低于对照组（0．973±0．017），但EDU染色及流式细胞仪检测结果均显示，实验组s期细胞百分比明显高于对照组，差异均有统计学意义（P〈0．05）。β半乳糖苷酶染色显示，实验组成纤维细胞阳性率（25．710％±0．304％）高于对照组（5．257％±1.023％），差异有统计学意义（t=19．170，P〈0．05）。吖啶橙染色结果显示，实验组成纤维细胞红色荧光量（14．287±2．269）低于对照组（29．614±2．650）。Western印迹及间接免疫荧光结果均显示，实验组成纤维细胞LC3-B表达量明显高于对照组，差异均有统计学意义（P〈0．05）。结论中波紫外线诱导的提前衰老成纤维细胞的条件培养液可降低成纤维细胞的自噬及增殖，并加速其老化。Objective To investigate the effects of conditioned medium of prematurely senescent fibroblasts induced by ultraviolet B （UVB） on the proliferation, aging and autophagy of human dermal fibroblasts. Methods Human dermal fibreblasts were isolated from the circumcised foreskin of healthy adolescent males, and subjected to primary culture. Premature senescence was induced in some fibroblasts by UVB radiation at 10 mj/cm： once daily for 5 consecutive days. Some fibroblasts were classified into two groups： an experimental group cultured in conditioned medium of UVB-induced prematurely senescent fibroblasts, and a control group cultured in conditioned medium of normal fibroblasts. After treatment for 20 consecutive days, cell counting kit-8 （CCK8） assay and 5-ethynyl-2＇ .-deoxyuridine （EDU） staining were performed to evaluate cellular proliferation, flow cytometry was conducted to estimate cell cycle, β- galactosidase staining to determine the percentage of senescent ceils, accridine orange staining to detect the autophagy level, and Western blot and indirect immunofluorescence assay were carried out to determine the expression level of the autophagy-related protein LC3-B. Statistical analysis was done by using a two-sample t test with the Graphpad Prism 5 software. Results Compared with the control group, the proliferative activity of fibroblasts was significantly decreased （0.831 ± 0.017 vs. 0.973 ± 0.017, t = 5.850, P 〈 0.05）, while E DU staining and flow cytometry both showed a significant increase in the percentage of S-phase cells （both P 〈 0.05 ）, in the experimental group. The percentage of [3-galactosidase- positive fibroblasts was significantly higher in the experimental group than in the control group （25.710% ± 0.304% vs. 5.257% ± 1.023%, t = 19.170, P 〈 0.05）. Accridine orange staining revealed that the red fluorescence intensity of fibroblasts was significantly lower （ 14.287 ± 2.269 vs. 29.614 ± 2.650, t = 4.390, P 〈 0.05）, while Western blot and indirect i

关 键 词：紫外线 成纤维细胞 细胞衰老 自噬 细胞增殖 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]