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

中文关键词:  脂肪干细胞 β-catenin miRNA 表达载体

英文关键词:ADSCs β-catenin miRNA Expression vector

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（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。

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 (P<0.05), less did the miR-1960 (P<0.05), but not the miR-1467 and the miR-1948 (P<0.05). Conclusion miR-780 and miR-796 could interference the β-catenin in ADSCs with high efficiency.