【作者】 程飚； 刘宏伟； 唐建兵； 朱江婷； 彭艳； 项晓飞；

【Author】 Cheng Biao1,Liu Hongwei2,Tan Jianbing1,Zhu Jiangting1,Peng Yan1,Xiang Xiaofei1 1,Department of Plastic Surgery,Guangzhou General Hospital of Guangzhou Military Region,GuangDong,The Key Laboratory of Trauma Treatment & Tissue Repair of Tropical Area,PLA,510010,P.R.China; 2,Department of Plastic Surgery,The First Affliated Hospital of Jinan University,Guangzhou 510630, Guangdong,China;

【机构】 暨南大学华侨医院整形外科组织再生教育部重点实验室； 广州军区总医院整形外科·全军激光整形中心全军热区创伤救治与组织修复重点实验室；

【摘要】 目的:构建大鼠mu-、delta-和kappa-型阿片受体(MOR、DOR和KOR)基因干扰RNA的表达,为研究皮肤瘙痒的分子机制奠定基础。方法:以含大鼠全长mu-、delta-和kappa-型阿片受体基因的载体为模板,分别用软件(http:.//www.ambion.com/techlib/misc/siRNA finder.html)在线设计3对针对MOR、DOR和KOR的siRNA引物和各自的阴性对照引物,将各片段插入BglⅡ,HindⅢ之间。载体转染成纤维细胞中,应用荧光显微镜观察GFP表达情况。结果:转染的成纤维细胞都可以观察到绿色荧光。应用免疫荧光法可观察到目的基因M、D和K型阿片受体的表达。结论:成功构建MOR、DOR和KOR基因干扰RNA在成纤维细胞中实现了高效表达。更多还原

【Abstract】 Objective:To construct iRNA expression vector opiate receptor(MOR,DOR,and KOR).Accordingly it can establish foundation for exploring the molecular mechanism of itch.Methods:The sequence of MOR, DOR,and KOR were found in Gene Bank.We designed and synthesized SiRNA using the software(http://www. ambion.com/techlib/misc/siRNA_finder.html),an online tool to design SiR RNA,and the design rules include a proprietary algorithm.Then,pSUPERretro-GFP/Neo positive expression vector were transfected into fibroblasts. The expression of pSUPERretro-GFP/Neo positive in fibroblasts was examined by fluorescence microscopy. Results The high expression of GFP and MOR,DOR,and KOR gene was detected in fibroblasts after transfection with fluorescence microscopy..Conclusions iRNA expression vector of opioid receptor gene(MOR,DOR,and KOR) are constructed successfully and expressed in high level in fibroblasts.更多还原