shRNA-CTGF重组质粒对瘢痕疙瘩Ⅰ型胶原蛋白表达的影响  

Effects of shRNA-CTGF on the expression of collagen-Ⅰ in keloid

作　　者：沈才齐[1] 金培生[1] 李雪阳[1] 张爱君[1] 陶常波[1] 李强[1] 马志兵[1] 

机构地区：[1]徐州医学院附属医院整形外科,266021

出　　处：《中华医学美学美容杂志》2014年第5期376-380,共5页Chinese Journal of Medical Aesthetics and Cosmetology

基　　金：江苏省卫生厅资助课题（Z200705）

摘　　要：目的 构建pGPU6/GFP/Neo-shRNA-CTGF重组质粒（简称shRNA-CTGF）,通过RNA干扰（RNAi）技术对结缔组织生长因子（connective tissue growth factor,CTGF）进行体内、体外干预,分析其对瘢痕疙瘩Ⅰ型胶原蛋白（collagen-Ⅰ,COL-Ⅰ）表达的影响.方法 设计并构建特异性shRNA-CTGF重组质粒,RNAi转染体外培养瘢痕疙瘩成纤维细胞（keloid fibroblast,KFB）,检测CTGF基因沉默情况及KFB分泌的COL Ⅰ量;将shRNA-CTGF重组质粒对构建的裸鼠瘢痕疙瘩动物模型进行体内转染,检测CTGF基因沉默情况及COL-Ⅰ基因、蛋白表达水平.结果 成功构建了CTGF重组质粒,其在体内、体外环境下均可成功沉默CTGF表达,其抑制率为86.8％和54.1％;RNA体外干扰KFB定向沉默CTGF的同时可显著抑制COL-Ⅰ表达,其mRNA、蛋白水平抑制率分别为76.8％和65.6％;RNA体内干预实验也可显著降低COL-Ⅰ表达,其mRNA、蛋白水抑制率分别为52.7％和48.0％.结论 shRNA-CTGF重组质粒在体外及体内环境下均可成功沉默CTGF基因表达,沉默CTGF能显著降低瘢痕疙瘩中COL-Ⅰ蛋白含量,提示CTGF基因是病理性瘢痕治疗的潜在靶点.Objective To explore the effects of recombinant plasmids of pGPU6/GFP/NeoshRNA-CTGF （shRNA-CTGF） on the type Ⅰ collagen （COL-Ⅰ） protein expression in keloid,through RNA interference on connective tissue growth factor （CTGF） in vivo and in vitro.Methods Recombinant plasmids were designed and constructed by specific shRNA-CTGF; After transfeced human keloid fibroblast with shRNA-CTGF in vitro,RT-PCR was used to detect the CTGF mRNA level,and Western blot to detect the secretion of COL-Ⅰ.After transfected the keloid of nude mice with shRNA-CTGF in vivo,RT-PCR was used to detect the CTGF and COL-Ⅰ mRNA level,and Western blot was used to detect the protein expression of COL-Ⅰ.Results Recombinant plasmids of CTGF were successfully constructed,and the CTGF gene expression was significantly decreased in vivo and in vitro by 86.8% and 54.1 %,respectively; Down-regulation of CTGF in vitro significantly inhibited the mRNA and protein level of COL-Ⅰ by 76.8% and 65.6%,respectively; Down-regulation of CTGF in vivo significantly reduced the COL-Ⅰ mRNA and protein level by 52.7% and 48.0%,respectively.Conclusions CTGF gene expression is successfully down-regulated by the recombinant plasmid of shRNA-CTGF in vivo and in vitro.shRNA-CTGF significantly reduces the COL-Ⅰ protein level in keloid.It implies that CTGF gene is a potential target in the therapy of pathological scar.

关 键 词：瘢痕疙瘩 结缔组织生长因子 成纤维细胞 RNA干扰 CONNECTIVE tissue growth factor (CTGF) RNA interference (RNAi) 

分 类 号：R622[医药卫生—整形外科]