TGFβ1 shRNA对293细胞合成TGFβ1的干扰作用  

The interference effect of TGFβ1 short hairpin RNA on the expression of TGFβ1 in 293 cells

作　　者：柳国胜[1] 王志军[1] 文妍[1] 罗瑶[1] 刘海英[1] 

机构地区：[1]暨南大学附属第一医院儿科,广东广州510630

出　　处：《暨南大学学报（自然科学与医学版）》2009年第4期420-424,共5页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：广东省自然科学基金资助项目(07005966)

摘　　要：目的:探讨自行设计的TGFβ1 shRNA对293细胞TGFβ1基因表达的干扰作用,为研究纤维化病变的基因治疗方法提供技术基础和依据。方法:针对大鼠TGFβ1基因mRNA序列,设计、合成携带3条TGFβ1 shRNA和TGFβ1基因的绿色荧光蛋白融合表达质粒载体,并设阴性质粒组和空质粒组为对照,通过脂质体包裹分别转染293细胞。转染后24、48和72 h收集细胞,在荧光显微镜下观察干扰效果,采用荧光定量PCR检测TGFβ1基因表达情况,并计算干扰效率。结果:荧光显微镜下观察,可见转染后24、48和72 h TGFβ1shRNA质粒组细胞绿色荧光强度均明显弱于阴性质粒组细胞,空质粒载体组未产生绿色荧光;荧光定量PCR检测转染后293细胞TGFβ1mRNA表达量,转染后24、48和72 h TGFβ1shRNA质粒组TGFβ1 mRNA表达量均显著低于阴性质粒组(P<0.01),其基因干扰效率则依次递减,分别为97.2%、97.1%和67.7%。结论:本研究证明自行设计的TGFβ1shRNA转染293细胞后24、48和72 h TGFβ1shRNA均能够高效干扰TGFβ1基因的表达,其基因干扰效率呈现一定的时间依赖性。Aim:The effects of RNA interference on TGFβ1 gene in 293 cells through TGFβ1shRNAs designed by my own were investigated in order to provide the foundation and evidence of a potential siRNA therapy of fibrotic diseases. Methods: Three 21-nucleotides shRNA targeting the TGFβ1 mRNA sequence were designed by myself.Then to construct the green fluorescence plasmid DNA of expressing TGFβ1 shRNA and TGFβ1 fusion genes and identified it.To determine the effect of these shRNAs,the positive plasmid vectors of TGFβ1 were transfected into 293 cells with the aid of lipofectamin,besides using negative and empty vectors were treated as above as controls.After 24,48 and 72 hours,the preliminary effect was evaluated with green fluorescence microscope,and then fluorescent quantitation PCR was used to detect the expression of TGFβ1 gene at mRNA level.Moreover,calculating the efficiency of RNA interference.Results: Observing 293 cells by the green fluorescence microscope.Fluorescence intensity decreased in the TGFβ1 shRNA plasmid groups compared with the negative plasmid groups at 24,48 and 72 hours after transfection.Fluorescence was not observed in the null plasmid groups.Detecting the expression of TGFβ1 gene at mRNA level with fluorescent quantitation PCR.the TGFβ1 gene expression was highly knocking down in the groups of TGFβ1 shRNA plasmid compared with groups of negative plasmid at 24,48 and 72 hours after transfection(P<0.01),and their efficiency of RNA interference reduced by degree following times,were 97.2%,97.1% and 67.7% respectively.Conclusion:the results suggested that the TGFβ1 shRNAs designed by my own can interfere the expreesion of TGFβ1 gene efficiently at 24,48 and 72 hours after transfection and the efficiency of RNA interference reduced by degree following times.

关 键 词：转化生长因子Β1 RNA干扰 293细胞 

分 类 号：R587.1[医药卫生—内分泌] R322.1[医药卫生—内科学]