2-甲氧雌二醇对低氧条件下皮肤成纤维细胞自噬水平及纤维化的影响  

The effect of 2-methoxyestradiol on autophagy and fibrosis of dermal fibroblasts cultured under hypoxia

作　　者：周行 刘超凡 鲁静浩 朱鹭冰 李明 ZHOU Xing;LIU Chao-fan;LU Jing-hao;ZHU Lu-bing;LI Ming(Department of Dermatology,Zhongshan Hospital,Fudan University,Shanghai 200032,Chin)

机构地区：[1]复旦大学附属中山医院皮肤科,上海200032

出　　处：《复旦学报（医学版）》2018年第4期462-466,共5页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金(30901291)~~

摘　　要：目的研究2-甲氧雌二醇(2-methoxyestradiol,2-ME)对低氧条件下人皮肤成纤维细胞自噬水平及纤维化的影响。方法原代培养健康人皮肤成纤维细胞并传代,取第3~6代细胞,在低氧(1%O2)条件下培养。使用2-ME(0、5、10、25μmol/L)和自噬抑制剂Bafilomycin A1(0、5、10、50 nmol/L)干预,采用Western blot法检测I型胶原(collagen typeⅠ)、结缔组织生长因子(connective tissue growth factor,CTGF)、低氧诱导因子-1α(hypoxia-inducible factor 1alpha,HIF-1α)、自噬标志物微管相关蛋白1轻链3(LC3)及P62的水平,采用免疫荧光法检测LC3的水平。结果成纤维细胞低氧培养24 h后,HIF-1α、Ⅰ型胶原、CTGF和LC3Ⅱ蛋白质水平显著升高,P62蛋白质水平降低,LC3免疫荧光信号显著增强。Bafilomycin A1干预组成纤维细胞I型胶原和CTGF水平降低,HIF-1α、LC3Ⅱ和P62水平升高,LC3免疫荧光信号增强,该效应呈浓度依赖性。2-ME干预组成纤维细胞HIF-1α、Ⅰ型胶原、CTGF和LC3Ⅱ蛋白质水平降低、P62蛋白质水平升高,LC3免疫荧光信号减弱,该效应呈浓度依赖性。结论低氧条件下2-ME通过下调细胞自噬抑制皮肤成纤维细胞纤维化。Objective To investigate the effect of 2 methoxyestradiol （2 ME ） on autophagy and fibrosis of dermal fibroblasts cultured under hypoxia. Methods Primary cultured human dermal fibroblasts were established, serially passaged and used for analysis at early passage （3rd to 6th generation）. These fibroblasts were treated with different concentrations of 2 ME （0,5,10,25μmol/L） or the inhibitor of autophagy Bafilomycin A1 （0,5,10,50 nmol/L） and cultured under hypoxia （1%O2）. Then Collagen I, connective tissue growth factor （CTGF）, hypoxia inducible factor 1 alpha （HIF 1%） ,P62 and LC3 were detected by Western blot,and LC3 level was detected by immunofluorescence. Results Compared with those cultured in normoxia, fibroblasts cultured in hypoxia showed increased levels of HIF-1α,collagen Ⅰ ,CTGF, LC3 Ⅱ proteins and fluorescence intensity of LC3, and decreased level of P62 protein. Bafilomycin A1 decreased the levels of collagen Ⅰ and CTGF and increased the levies of HIF 1%,LC3 Ⅱ and P62 in fibroblasts cultured in hypoxia for 24 h. Meanwhile,2 ME inhibited the levels of HIF-1α, collagen Ⅰ ,CTGF and LC3 Ⅱ and fluorescence intensity of LC3, but improved the level of P62. These effects were in a dose dependent manner. Conclusions 2 ME decreases fibrosis of skin fibroblasts cultivated under hypoxia probably by inhibiting autophagy.

关 键 词：2-甲氧雌二醇 低氧 成纤维细胞 纤维化 自噬 

分 类 号：R751[医药卫生—皮肤病学与性病学]