miRNA-29c-3p对体外慢性光损伤皮肤成纤维细胞COL1A1和COL3A1基因表达及Ⅰ、Ⅲ型胶原蛋白合成的影响  

Effects of miRNA-29c-3p on the of collagen typeⅠα1 and collagen type Ⅲ α1 genes and the synthesis of collagenⅠand Ⅲ in chronically photodamaged human dermal fibroblasts in vitro

作　　者：宋晓婧[1] 彭亚婷[1] 陈海燕[1] 郑跃[1] 许庆芳[1] 龚子鉴[1] 陆春[1] 赖维[1] 

机构地区：[1]中山大学附属第三医院皮肤科,广州510630

出　　处：《中华皮肤科杂志》2017年第12期869-874,共6页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81673047）

摘　　要：目的 研究miRNA？29（miR？29）家族对人慢性光损伤（光老化）皮肤Ⅰ、Ⅲ型胶原蛋白的影响。方法 将人皮肤成纤维细胞分为未照射组和慢性光损伤组，后者给予长波紫外线（UVA）连续照射以构建慢性光损伤细胞模型，并用流式细胞仪及β半乳糖苷酶染色法验证模型。Western印迹检测两组Ⅰ型和Ⅲ型胶原蛋白的表达，实时荧光定量PCR（qRT？PCR）检测miR？29家族3个成员（miR？29a？3p、miR？29b？3p和miR？29c？3p）在两组细胞中的表达，选择表达具有显著差异的miR？29c？3p进行功能试验。将人皮肤成纤维细胞分为4组，分别采用荧光标记的miRNA？29c？3p模拟物（过表达组）、抑制物（抑制组）及二者各自的对照RNA寡核苷酸（阴性对照组和抑制对照组）转染各组细胞，观察各组荧光细胞比例，qRT？PCR法检测各组miR？29c？3p的相对表达量，判定干扰效率。采用qRT？PCR法检测转染后4组COL1A1和COL3A1 mRNA水平，Western印迹法检测转染后Ⅰ和Ⅲ型胶原蛋白表达水平。结果 与未照射组相比，慢性光损伤组衰老染色细胞阳性率显著升高（36.47% ± 3.20%比12.56% ± 1.46%，P 〈 0.01），G1期细胞比例明显升高，S期细胞比例明显降低（均P 〈 0.01），慢性光损伤模型成功建立。慢性光损伤组Ⅰ和Ⅲ型胶原蛋白表达（0.40 ± 0.19和0.52 ± 0.10）明显低于未照射组（1.00 ± 0.12和1.00 ± 0.10，均P 〈 0.01），而miR？29c？3p（4.42 ± 2.05）明显高于未照射组（0.89 ± 0.10，P 〈 0.05），两组间miR？29a？3p和miR？29b？3p表达水平差异无统计学意义（均P 〉 0.05）。转染后24 h，过表达组和抑制组的转染效率均大于90%，达到干预要求。过表达组miR？29c？3p的表达（224.17 ± 2.00）明显高于阴性对照组（2.45 ± 0.34），而抑制组（0.20 ± 0.08）明显低于抑制对照组（2.24 ± 0.14），干扰模型构建成功。评价转染效率显示Song Xiaojing, Peng Yating, Chen Haiyan, Zheng Yue, Xu Qingfang, Gong Zijian, Lu Chun, Lai Wei Department of Dermatology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China Corresponding author： Lai Wei, Email： [e-mail] 【Abstract】 Objective To evaluate the effect of miRNA-29 （miR-29） family on the synthesis of collagen Ⅰand Ⅲ in chronically photodamaged （photoaged） skin. Methods Some cultured human dermal fibroblasts （HDFs） were divided into 2 groups： non-irradiated group receiving no treatment, and chronic photodamage group treated with repetitive ultraviolet A （UVA） radiation, which served as a chronically photodamaged cell model and was verified by flow cytometry and β-galactosidase staining. Western blot analysis was performed to determine the protein of collagenⅠand Ⅲ, and real-time fluorescence-based quantitative PCR （qRT-PCR） to measure of 3 members of the miR-29 family （miR-29a-3p, miR-29b-3p and miR-29c-3p） in the above 2 groups. The differentially expressed miR-29c-3p between the above 2 groups was chosen for further functional tests. Some HDFs were divided into 4 groups to be transfected with fluorescein-labelled miR-29c-3p mimics （over group）, inhibitors （inhibition group）, and their control RNA oligonucleotides （negative control group and inhibitor control group） respectively. The transfection efficiency was evaluated by the proportion of fluorescent cells, and the relative of miR-29c-3p in the above 4 groups was measured by qRT-PCR for evaluating the RNA interference efficiency. qRT-PCR was conducted to determine the mRNA of collagen typeⅠα1 （COL1A1） and collagen type Ⅲ α1 （COL3A1） genes, and Western blot analysis to measure the protein of collagenⅠand Ⅲ. Results Compared with the non-irradiated group, the chronic photodamage group showed significantly increased proportion of senescent cells （36.47% ± 3.20% vs. 12.56% ± 1.46%, P 〈 0.01） and G1-phase cells （71.70% ± 2.43% vs. 41.89%

关 键 词：长波紫外线 光老化 成纤维细胞 MIR-29 胶原 

分 类 号：R758.1[医药卫生—皮肤病学与性病学]