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论文摘要

大鼠β-catenin miRNA表达载体的构建及在脂肪干细胞中沉默效果的检测

Construction of β-catenin miRNA-expressing Vectors and Test of Silencing Effect in Adipose-derived Stem from Sprague Dawley Rats

作者:黄海森, 刘爽, 于湄等

Author:HUANG Hai-sen, LIU Shuang, YU Mei.et al

收稿日期:          年卷(期)页码:2014,45(5):749-753

期刊名称:四川大学学报(医学版)

Journal Name:JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION)

关键字:脂肪干细胞 β-catenin miRNA 表达载体

Key words:ADSCs β-catenin miRNA Expression vector

基金项目:

中文摘要

目的 构建具有沉默大鼠脂肪干细胞β-catenin表达的miRNA(miR)表达载体。方法 通过酶切法将合成的miR表达载体寡聚核苷酸引物与质粒pSWH相连,形成pSWH-miR;PCR扩增出增强型绿色荧光蛋白(EGFP)开放阅读框,插入经双酶切处理的pSWH-miR形成pSWH-EGFP-miR。β-catenin miR表达质粒瞬时转染SD大鼠脂肪干细胞 (adipose-derived stem cells derived from rats,rADSCs),72 h后提取细胞全蛋白,Western blot检测β-catenin的表达情况。结果 针对大鼠β-catenin的不同位点,一共构建了5个miRNA表达载体(miR-780、miR-796、miR-1467、miR-1948、miR-1960),其中miR-780、miR-796对β-catenin沉默效果最好(PPP

英文摘要

Obejective To construct miRNA-expressing plasmid vector for interfering the expression of β-catenin in adipose-derived stem cells from Sprague-Dawley rats. Methods The double strands oligonucleotides of miR-expressing cassette were ligated into plasmid pSWH to generate pSWH-miR. Then the PCR product of enhanced green fluorescence protein (EGFP) open reading frame (ORF) fragment was inserted into BamHⅠand XboⅠdigested pSWH-miR to generate pSWH-EGFP-miR. The adipose-derived stemcells from rats (rADSCs) were transfected with pSWH-EGFP-miR respectively. The expression of β-catenin was determined by Western blot at 72 h post-transfection. Results Five miRNA-expressing plasmid vectors were constructed (miR-780, miR-796, miR-1467, miR-1948, miR-1960). miR-780 and miR-796 had the best silencing effect on the expression of β-catenin (PPP

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