视蛋白3调控人真皮成纤维细胞Ⅰ型胶原蛋白生成  

Opsin 3 Regulating TypeⅠCollagen Synthesis in Human Dermal Fibroblasts

作　　者：牟慧玲 曾雯 董仙 汪宇 张伟 冯江龙 兰应华 罗欢欢 叶藤 陆洪光 MOU Huiling;ZENG Wen;DONG Xian;WANG Yu;ZHANG Wei;FENG Jianglong;LANYinghua;LUO Huanhuan;YE Teng;LU Hongguang(Guizhou Medical University,Guiyang 550025,China;Department of Dermatology,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China)

机构地区：[1]贵州医科大学,贵州贵阳550025 [2]贵州医科大学附属医院皮肤科,贵州贵阳550004

出　　处：《中国皮肤性病学杂志》2023年第3期265-272,共8页The Chinese Journal of Dermatovenereology

基　　金：国家自然科学基金面上项目(81972920);贵州省自然科学基金(黔科合基础-ZK[2022]一般449,黔科合基础-ZK[2022]一般413)。

摘　　要：目的研究视蛋白3在正常人真皮成纤维细胞中对Ⅰ型胶原蛋白生成的影响及其可能的机制。方法取小儿包皮真皮成纤维细胞分离、培养、传代,至3~5代,用荧光定量PCR、蛋白免疫印迹分别检测视蛋白1~5的表达,细胞免疫荧光检测视蛋白3及Ⅰ型胶原蛋白表达及定位。设计针对人视蛋白3的小干扰RNA,利用脂质体转染入正常人真皮成纤维细胞,分别用荧光定量PCR、蛋白免疫印迹检验视蛋白3抑制效率。成功下调视蛋白3后,用荧光定量PCR、免疫印迹法、酶联免疫吸附测定及细胞免疫荧光检测Ⅰ型胶原蛋白表达变化;通过CCK-8及EDU检测细胞增殖活力,流式细胞仪检测细胞周期,Transwell检测细胞迁移能力;通过蛋白免疫印迹法检测PI3K、AKT及磷酸化蛋白p-PI3K、p-AKT的表达情况。最后抑制通道蛋白AKT的磷酸化,通过蛋白免疫印迹法检测Ⅰ型胶原蛋白表达变化。结果视蛋白1~5均在正常人真皮成纤维细胞中表达,其中视蛋白3基因及蛋白表达水平较其他视蛋白表达高,差异有统计学意义(P<0.05)。下调正常人真皮成纤维细胞中视蛋白3后,细胞合成及分泌Ⅰ型胶原蛋白水平降低,差异有统计学意义(P<0.05);细胞增殖、迁移能力减弱,差异有统计学意义(P<0.05);细胞周期中S期比例明显降低,差异有统计学意义(P<0.05);磷酸化蛋白p-PI3K、p-AKT的蛋白表达水平被明显抑制;抑制通路蛋白AKT的活化后,细胞合成Ⅰ型胶原蛋白水平降低。结论视蛋白3参与调控正常人真皮成纤维细胞中Ⅰ型胶原蛋白的合成,可能是创伤愈合、纤维化相关疾病及医疗美容潜在的治疗靶点。Objective To explore the effect of opsin 3 on typeⅠcollagen synthesis in normal human dermal fibroblasts and its possible mechanism.Methods Normal human dermal fibroblasts(NHDFs)in children were isolated,cultured and passaged to 3-5 passages.The expression levels of opsin 1-5 were detected by RT-qPCR and Western blot,and the expression and localization of opsin 3 and typeⅠcollagen were detected by cell immunofluorescence.A small interfering RNA targeting human opsin 3 was designed and transfected into NHDFs by liposomes.The inhibition efficiency of opsin 3 was detected by RT-qPCR and Western blot.After opsin 3 was silenced successfully,the expression level of typeⅠcollagen was detected by RT-qPCR,Western blot,enzyme-linked immunosorbent assay(ELISA)and cellular immunofluorescence,respectively.Cell proliferation activity was detected by CCK-8 and EDU,cell cycle was detected by flow cytometry,and cell migration ability was detected by Transwell.The expression levels of PI3K,AKT and phosphorylated proteins were detected by Western blot.After the phosphorylation of channel protein AKT was inhibited,the protein level of typeⅠcollagen was detected by Western blot.Results All opsins(1-5)were all expressed in NHDFs,and the gene and protein expression level of opsin 3 was higher than that of other opsins(P<0.05).After down-regulation of opsin 3 in NHDFs,the levels of cell synthesis and secretion of typeⅠcollagen were decreased(P<0.05);the ability of cell proliferation and migration was weakened(P<0.05);the proportion of S phase in the cell cycle was significantly decreased(P<0.05);the expression levels of phosphorylated proteins p-PI3K and p-AKT were significantly inhibited.After inhibiting the activation of AKT,the protein level of collagenⅠwas decreased.Conclusion Opsin 3 is involved in the regulation of typeⅠcollagen synthesis in NHDFs,and may be a potential therapeutic target for wound healing,fibrosis-related diseases and medical cosmetology.

关 键 词：视蛋白3 成纤维细胞 Ⅰ型胶原蛋白 

分 类 号：R751[医药卫生—皮肤病学与性病学] R622[医药卫生—临床医学]