靶向Wnt10b的小干扰RNA抑制胚胎皮肤毛囊发育的初步研究  

Preliminary study on inhibition of the hair follicle development by siRNA targeting WntlOb in the cultured rat embryonic skin

作　　者：纪影畅[1] 李宇[1] 鲁峰[2] 胡志奇[2] 王森[1] 林常敏[1] 高建华[2] 

机构地区：[1]汕头大学医学院第一附属医院整形外科,汕头515041 [2]南方医科大学南方医院整形外科

出　　处：《中华整形外科杂志》2012年第1期39-43,共5页Chinese Journal of Plastic Surgery

基　　金：基金项目：国家自然科学基金（81071577）;广东省自然科学基金（9151008901000195）;广东省科技计划项目（20098030801320）;广东省医学科研基金（A2009433）

摘　　要：目的 利用小干扰RNA（siRNA）抑制皮肤Wnt10b基因的表达,观察Wnt10b基因沉默能否抑制毛囊的发育并探讨其潜在机制.方法 化学合成siRNA-Wnt10b,将siRNA转染体外培养的胎鼠背部皮肤,荧光定量PCR检测转染后不同时间段皮肤组织Wnt10b和p-连环蛋白（β-catenin） mRNA的表达,Western blot检测转染后72 h皮肤组织的Wnt10b和β-catenin蛋白含量.将转染后72 h的皮肤组织石蜡包埋、切片,HE染色,镜下观察各组毛囊发育情况并做统计学处理.结果 siRNA-Wnt10b转染后的24、48 h,胎鼠背部皮肤Wnt10b mRNA的表达呈不同程度下降；但β-catenin mRNA表达未随Wnt10b mRNA水平的起伏而明显波动；转染后72 h,Wnt10b蛋白和β-catenin的蛋白表达同时减少,新形成毛囊数量也随之减少（t=3.254,P=0.002）.结论 在体外器官培养的环境中,siRNA-Wnt10b能够抑制胎鼠背部皮肤组织Wnt10b蛋白的表达,减少新形成毛囊的数量；Wnt10b可能只在蛋白水平上调控细胞β-catenin的表达.Objective To investigate whether the suppression of Wnt10b by siRNA could prevent the development of hair follicle in the cultured rat embryonic skin. Methods siRNA-Wnt10b was synthesized by chemosynthesis method.The dorsal skin of SD rat at embryos were cultured in DMEM in the presence of different percentage of interfering RNA targeting Wnt10b. Wnt10b/β-catenin expression was analyzed by real-time PCR everyday and by Western blot on the third day. The cultured embryonic skin underwent paraffin embedding,section,HE staining on the third day,in which the number of de novo hair follicle was calculated and statistically analyzed. Results Wnt10b gene in the cultured embryonic skin could be knocked down with the siRNA-based method.β-catenin mRNA was not greatly influenced by the downregulation of Wnt10b mRNA.The number of de novo hair follicle placode in cultured embryonic skin decreased,along with the downregulation of Wnt10b and β-catenin proteins expression.Conclusions The downregulation of Wnt10b mRNA and protein by siRNA reduces the number of de novo hair follicle placode in the cultured rat embryonic skin.Wnt10b may control cytoplasm β-catenin concentration at the protein level.

关 键 词：毛囊 形态发生 器官培养技术 RNA干扰 

分 类 号：R622[医药卫生—整形外科]