中波紫外线诱导人皮肤成纤维细胞自噬对凋亡影响的初步研究  

Effects of ultraviolet B-induced autophagy on apoptosis in human skin fibroblasts： a preliminary study

作　　者：陈旭[1] 张青松[2] 鞠梅[1] 任发亮[1] 黄丹[1] 齐蓓[1] 陈崑[1] 常宝珠[1] 李新宇[1] 顾恒[1] 

机构地区：[1]中国医学科学院北京协和医学院皮肤病研究所,南京210042 [2]江苏省常熟市新区医院

出　　处：《中华皮肤科杂志》2012年第7期496-500,共5页Chinese Journal of Dermatology

基　　金：国家自然科学基金面上项目（30872268）;江苏省自然科学基金（BK2010135,BK2011129）;天津市卫生局科技基金（09KZ52）

摘　　要：目的探讨不同剂量中波紫外线（UVB）照射人皮肤成纤维细胞诱导的自噬对细胞凋亡活性的影响。方法人皮肤成纤维细胞来自于23岁健康男性环切术后包皮的原代培养，连续传代培养后取第3～10代的细胞进行实验。通过单丹酰戊二胺（MDC）染色和免疫荧光标记微管相关蛋白1轻链3（LC3）的方法，确定不同剂量3-甲基腺嘌呤（3-MA）对自噬的抑制作用。UVB照射后立即加入0．5mmol／L3-MA孵育细胞4h作为自噬的抑制方法。Hoechst和碘化丙啶（PI）染色结合AnnexinV-异硫氰酸荧光素（FITC）和PI标记细胞后流式细胞仪检测作为细胞凋亡的定性和定量方法。结果0．5mmol／L3-MA能明显抑制人皮肤成纤维细胞自噬活性（饥饿诱导组自噬细胞阳性百分数为63．037％±5．876％，3-MA孵育后下降至34．425％±5．183％）。空白对照组、30、50、100mJ／cm。UVB照射组标记LC3绿色荧光信号逐渐增强，照射后立即对上述4组加入3-MA孵育4h则LC3蛋白表达差异不明显。0．5mmol／L3-MA对细胞活性影响最小。50mJ／cmzUVB照射下加入3-MA孵育较未加3-MA孵育细胞强Hoechst和强PI双染细胞增多；100mJ／cm2UVB照射后加3-MA孵育较未加3-MA孵育细胞强Hoechst和强PI双染细胞减少。AnnexinV—FITC和PI标记细胞后流式细胞仪分析显示，在50mJ／cmzUVB照射下，抑制自噬的细胞中晚期凋亡水平[（10．933±0．839）％]较未抑制细胞[（7．267±0．473）％]上升，两者之间差异具有统计学意义（t=5．20，P〈0．05）；而在100mJ／cmzUVB照射下，抑制自噬的细胞中晚期凋亡水平[（7．100±0．781）％]较未抑制细胞[（10．133±0．681）％]下降，两者之间差异具有统计学意义（t=6．29，P〈0．05）。结论50mJ／cm。UVB照射诱导人皮肤成纤维细胞自噬通过抑制凋亡对细胞起到保护作用，而在100mJ／cm。UVB照射下发生的较高水平自噬可能诱导了自噬．I生细�Objective To observe the effects of autophagy induced by different doses of ultraviolet B （UVB） irradiation on the apoptosis in human skin fibroblasts. Methods Skin fibroblasts were isolated from the circumcision specimen of a 23-year-old healthy male, and subjected to a primary culture. After 3 to 10 passages, the cells were collected and applied in the following experiment. Methyl thiazolyl tetrazolium （MTT） assay was performed to evaluate the proliferation of some fibroblasts treated with different concentrations （0, 0.5, 2.0, 5.0 and 10.0 mmol/L） of 3-methyladenine （3-MA）. To qualitatively and quantitatively detect the autophagy in fibroblasts treated with different concentrations of 3-MA and in fibroblasts treated with 3-MA of 0.5 mmol/L following UVB irradiation, monodansylcadaverine （MDC） staining was carried out, and immunofluorescencc was used to detect the expression of microtubule-associated protein 1 light chain （LC3）. Some fibroblasts were classified into 8 groups to remain untreated, be irradiated with UVB of 30, 50 and 100 mJ/cm2 alone, treated with 3-MA of 0.5 mmol/L alone, or treated with 0.5 mmol/L 3-MA following irradiation with UVB of 30, 50 and 100 mJ/cm2, respectively, then, cell apoptosis was qualitatively detected by Hoechst and propidium iodide （PI） staining, and quantitatively detected by flow eytometry with annexin V and PI. Results The percentage of autophagic cells was （63.037±5.876） % in fibroblasts treated with starvation condition, significantly decreased to （34.425±5.183 ） % in fibroblasts treated with 3-MA of 0.5 mmol/L. The expression of LC3 showed a gradually increasing trend from untreated fibroblasts, to fibroblasts irradiated with UVB of 30, 50 and 100 mJ/cm2, while the increase was attenuated by the 4-hour treatment with 3-MA immediately after the irradiation. Compared with the other concentrations, the 3-MA of 0.5 mmollL showed the least influence on the viability of fibroblasts. The addition of 3-MA of 0.5 mmol/L increased the percent

关 键 词：紫外线 成纤维细胞 自噬 细胞凋亡 3-甲基腺嘌呤 

分 类 号：R751[医药卫生—皮肤病学与性病学]