RNA干扰沉默Smad3对人肺成纤维细胞Ⅰ型胶原产生的影响  

Effects of RNA Interference Silencing Smad3 on Production of Type Ⅰ Collagen of Human Lung Fibroblast

作　　者：焦娇[1,2] 谢华[2] 陈萍[2] 马壮[2] 孙文武[2] 

机构地区：[1]第四军医大学西京医院,陕西西安710032 [2]沈阳军区总医院,辽宁沈阳110840

出　　处：《现代生物医学进展》2013年第11期2018-2021,2048,共5页Progress in Modern Biomedicine

基　　金：全军"十二五"医学科研基金项目(BWS12J007);辽宁省自然科学基金项目(201202244)

摘　　要：目的:转化生长因子β1(transforming growth factor-β1,TGF-β1)通过Smad3(drosophila mothers against decapentaplegic)依赖的细胞因子信号转导通路,促进Ⅰ型胶原的合成和分泌,在哮喘气道重塑中起重要作用。近年来RAN干扰技术在基因治疗及功能研究取得很大进步,本实验探讨RNA干扰(RNA interference,RNAi)技术沉默Smad基因表达对人肺成纤维细胞(human lungfibroblast-1,HLF-1)合成I型胶原的影响。方法:设计并合成Smad3特异性小发卡干扰RNA(short hairpin RNAs,shRNA),用脂质体转染入培养的人HLF-1细胞,反转录聚合酶链反应(reverse transfection PCR,RT-PCR)法和蛋白质印迹(Western blot)法观察基因沉默效果,酶联免疫吸附试验(enzyme-linked immunoadsordent assay,ELISA)检测RNA干扰对人HLF-1细胞合成I型胶原的影响。结果:shRNA-Smad3成功转染入人HLF-1细胞并有效沉默Smad3的表达,RT-PCR结果显示shRNA-Smad3转染后Smad3mRNA表达与正常对照组和阴性对照组比较,分别降低68.2%和69.5%,差异有统计学意义(P<0.05);Western blot结果显示shRNA-Smad3转染后Smad3蛋白表达与正常对照组正常对照组和阴性对照组比较,分别降低了67.1%和64.7%,差异有统计学意义(P<0.05)。ELISA检测结果显示shRNA-Smad3转染后I型胶原为正常对照组和阴性对照组的59.3%和61.1%,差异有统计学意义(P<0.05)。结论:shRNA-Smad3能有效沉默HLF-1细胞中Smad3 mRNA和Smad3蛋白的表达,并减少HLF-1细胞Ⅰ型胶原的合成。为进一步应用干扰RNA技术治疗哮喘患者气道重塑提供新的思路。Objective： Transforming growth factor β1 plays an important role in the asthmatic airway remodeling, through Smad3 dependent signal transduction pathway to promote the synthesis and secretion of type I collagen. RAN interference technology has made great progress in gene therapy and functional studies in recent years. This study was to investigate the effects of RNA interference （RNAi） mothed silencing the expression of Smad3 and inhibiting the type I collagen production of human lung fibroblast-1 （HLF-1）. Method： Short hairpin RNA（shRNA） was designed and synthesized, shRNA was transferred into HLF-1 using Lipofectamine. Reverse transfection PCR （RT-PCR） and western blot analysis were used to detect the expression of Smad3 inhibited by shRNA. Enzyme-linked immunoadsordent assay （ELISA） was performed to detect the production of type I e.ollagen. Results： RT-PCR results indicted expression of Smad3 mRNA in experimental group significantly decreased 68.2% and 69.5% （P〈0.05） as compared with normal control group and negative group, respectively; Western blot indicated expression of Smad3 protein in experimental group significantly decreased 67.1% and 64.7%（P〈0.05） as compared with normal control group and negative group, respectively; ELISA results showed type ! collagen significantly decreased 59.3% and 61.1% （P〈0.05）, compared with normal control group and negative group, respectively. Conclusion： The expression of Smad3 mRNA and protein were efficiently silenced by shRNA-Smad3, while the production of type I collagen was also inhibited. RNA interference have potential for clinical therapy of asthma in future.

关 键 词：SMAD3 小发卡干扰RNA 人肺成纤维细胞 I型胶原 

分 类 号：R56[医药卫生—呼吸系统] Q78[医药卫生—内科学]