血管生成素抑制雄激素性秃发区毛乳头细胞中Ⅰ型胶原和纤维连接蛋白表达的研究

doi:10.3760/cma.j.issn.0412-4030.2018.09.002

摘要/Abstract

摘要： 目的探讨血管生成素抑制雄激素性秃发区毛乳头细胞中Ⅰ型胶原和纤维连接蛋白表达的作用及可能机制。方法分离培养雄激素性秃发区毛乳头细胞，实时定量荧光PCR检测雄激素受体mRNA在不同传代次数毛乳头细胞中的相对表达量，CCK8法检测0、10、20、40、80、160 μg/L血管生成素对含或不含0.1 nmol/L二氢睾酮培养基培养的毛乳头细胞增殖的影响。取生长融合的第1代毛乳头细胞传代后分为3组：对照组（不用二氢睾酮或血管生成素处理）、0.1 nmol/L二氢睾酮组、0.1 nmol/L二氢睾酮 + 80 μg/L血管生成素组。作用48 h后，用实时荧光定量PCR检测Ⅰ型胶原基因、纤维连接蛋白和转化生长因子β1（TGF?β1）mRNA的表达，Western印迹检测Ⅰ型胶原、纤维连接蛋白、TGF?β1、p?Smad2和p?Smad3蛋白的表达。实验数据采用单因素方差分析、LSD检验和独立样本t检验进行分析。结果体外培养的雄激素性秃发区毛乳头细胞随着传代次数增加，雄激素受体mRNA相对表达量明显降低（P < 0.05）。细胞增殖实验发现，20 ~ 160 μg/L血管生成素明显拮抗0.1 nmol/L二氢睾酮对毛乳头细胞增殖的抑制作用（均P < 0.05）。与对照组相比，二氢睾酮组Ⅰ型胶原基因、纤维连接蛋白和TGF?β1 mRNA显著升高，但二氢睾酮 + 血管生成素组较二氢睾酮组Ⅰ型胶原基因mRNA（1.563 ± 0.143比4.329 ± 0.165）、纤维连接蛋白mRNA（1.290 ± 0.063比2.156 ± 0.115）和TGF?β1 mRNA（1.136 ± 0.098比1.707 ± 0.100）显著降低（均P < 0.05）。而且，血管生成素还能明显抑制由二氢睾酮诱导的毛乳头细胞中Ⅰ型胶原、纤维连接蛋白、TGF?β1、p?Smad2和p?Smad3蛋白的表达（均P < 0.05）。结论血管生成素在体外能够抑制雄激素性秃发区毛乳头细胞中细胞外基质成分Ⅰ型胶原和纤维连接蛋白的表达，其机制可能与其下调TGF?β1表达及抑制TGF?β1/Smad信号通路的活化有关。

关键词: 血管生成素类, 秃发, 细胞外基质, 转化生长因子β1, 毛乳头细胞

Abstract: Zhou Naihui, Li Yan, Zhu Yueqian, Ren Sun, Wang Miaomiao, Liu Ming Department of Dermatology, the First Affiliated Hospital of Soochow University, Suzhou 215006, China Corresponding author: Zhou Naihui, Email: zhounaihui@163.com 【Abstract】 Objective To evaluate effects of angiogenin on the of typeⅠcollagen and fibronectin in dermal papilla cells from androgenetic alopecia areas, and to explore its possible mechanisms. Methods Dermal papilla cells were isolated from androgenetic alopecia areas and cultured. Real-time fluorescence-based quantitative PCR was performed to determine the mRNA of androgen receptor in dermal papilla cells of different passages, and cell counting kit-8 （CCK-8） assay to evaluate the effect of angiogenin at different concentrations of 0, 10, 20, 40, 80, 160 μg/L on the proliferative activity of the dermal papilla cells cultured in a medium with or without 0.1 nmol/L dihydrotestosterone. The confluent first-passage dermal papilla cells were divided into 3 groups: control group receiving no treatment, dihydrotestosterone group treated with 0.1 nmol/L dihydrotestosterone, and dihydrotestosterone + angiogenin group treated with 0.1 nmol/L dihydrotestosterone and 80 μg/L angiogenin. After 48-hour treatment, real-time fluorescence-based quantitative PCR was conducted to measure the mRNA of typeⅠcollagen gene, fibronectin and transforming growth factor-β1 （TGF-β1）, and Western blot analysis to determine the protein of type I collagen, fibronectin, TGF-β1, phosphorylated Smad2 （p-Smad2） and p-Smad3. Statistical analysis was done by one-way analysis of variance （ANOVA）, least significant difference （LSD）-t test and t test for two independent samples. Results The mRNA of androgen receptor significantly decreased during the subcultivation of in vitro cultured dermal papilla cells from androgenetic alopecia areas （P < 0.05）. Cell proliferation assay showed that 20 - 160 μg/L angiogenin could evidently antagonize the inhibitory effect of 0.1 nmol/L dihydrotestosterone on the proliferation of dermal papilla cells （all P < 0.05）. Compared with the control group, the dihydrotestosterone group showed significantly higher mRNA of typeⅠcollagen gene, fibronectin and TGF-β1. However, the mRNA of type Ⅰcollagen gene, fibronectin and TGF-β1 was significantly lower in the dihydrotestosterone + angiogenin group than in the dihydrotestosterone group （type Ⅰcollagen gene: 1.563 ± 0.143 vs. 4.329 ± 0.165; fibronectin: 1.290 ± 0.063 vs. 2.156 ± 0.115; TGF-β1: 1.136 ± 0.098 vs. 1.707 ± 0.100; all P < 0.05）. Moreover, angiogenin could obviously suppress the of typeⅠcollagen, fibronectin, TGF-β1, p-Smad2 and p-Smad3 protein by dihydrotestosterone-induced dermal papilla cells（all P < 0.05）. Conclusion Angiogenin can inhibit the of typeⅠcollagen and fibronectin in dermal papilla cells from androgenetic alopecia areas in vitro, which may be associated with the down-regulated of TGF-β1 and inhibition of TGF-β1/Smad signaling pathway.

Key words: Angiopoietins, Alopecia, Extracellular matrix, Transforming growth factor beta1, Dermal papilla cell

周乃慧李艳朱月倩任孙王淼淼刘铭. 血管生成素抑制雄激素性秃发区毛乳头细胞中Ⅰ型胶原和纤维连接蛋白表达的研究[J]. 中华皮肤科杂志, 2018, 51(9): 642-646.

图/表 1

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