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

作　　者：黄海森[1,2] 刘爽[1,3] 于湄[1,2] 罗小昕 郭维华[1,2,5] 田卫东[1,2,6] 

机构地区：[1]四川大学口腔再生医学国家地方联合工程实验室,成都610041 [2]四川大学口腔疾病研究国家重点实验室,成都610041 [3]四川大学生命科学学院,成都610064 [4]四川大学材料科学与工程学院,成都610064 [5]四川大学华西口腔医院儿童口腔科,成都610041 [6]四川大学华西口腔医院口腔颌面外科,成都610041

出　　处：《四川大学学报（医学版）》2014年第5期749-753,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家高技术研究发展计划(No.2011AA030107);国家自然科学基金(No.30973348);四川省科技支撑计划(No.2012SZ0013;No.2012SZ0035;No.2013GZX0158及No.2013SZ0015)资助

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),72h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(P<0.05),miR-1960的沉默效果次之(P<0.05),miR-1467、miR-1948不具有沉默效果(P<0.05)。结论 miR-780、miR-796能够有效沉默SD大鼠脂肪干细胞中β-catenin的表达。Obejective To construct miRNA-expressing plasmid vector for interfering the expression ofβ-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 BamHⅠ and XboⅠdigested pSWH-miR to generate pSWH-EGFP-miR.The adipose-derived stemcells from rats（rADSCs）were transfected with pSWH-EGFP-miR respectively.The expression ofβ-catenin was determined by Western blot at72hpost-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 ofβ-catenin（P〈0.05）,less did the miR-1960（P〈0.05）,but not the miR-1467 and the miR-1948（P〈0.05）.ConclusionmiR-780 and miR-796 could interference theβ-catenin in ADSCs with high efficiency.

关 键 词：脂肪干细胞 Β-CATENIN MIRNA 表达载体 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]