中文摘要：

目的建立β—catenin稳定干扰的人胚胎干细胞系。方法利用β-catenin慢病毒干扰质粒PLKO．1-puro—β—catenin转染包装细胞293FT制备重组慢病毒，并感染人胚胎干细胞，采用嘌呤霉素筛选稳定表达shβ-catenin）,．胚胎干细胞克隆；试验分为稳定转染13-catenin干扰质粒组（Shβ-catenin）、稳定转染空白载体组（VECTOR）和对照未感染组（WT）三组，RT-PCR检测干扰后β-cateninmRNA表达；进一步免疫荧光检测干扰后细胞β-cateninOCT4的蛋白表达。结果β-catenin特异性shRNA的慢病毒感染人胚胎干细胞后，获得稳定表达shβ-catenin的人胚眙干细胞系，Shβ-catenin组β—cateninmRNA表达明显降低，只有WT组mRNA表达的1％，而VECTOR组和WT组之间无明显差异；免疫荧光染色进一步验证shβ—catenin组中基本无βcatenin蛋白的表达，但表达多能性标记OCT4。结论通过β—catenin特异性shRNA慢病毒载体构建了稳定干扰8。catenin的人胚胎干细胞系。

英文摘要：

Objective To establish a human embryonic stem cell line with stably β-catenin gene silencing by lentivirus-mediated shRNA interference. Methods PLKO,1-puro-β-catenin vector, a lentivirus plasmid expressing β-catenin shRNA, was packaged into 293FT cells. Human embryonic stem cells were infected with the lentivirus and the cell clones stably expressing β-catenin shRNA were selected by puromycin, with the uninfected cells and cells infected with the empty vector as the control. Real-time RT-PCR was used to evaluate the efficiency of β-catenin knockeddown; β-catenin and OCT4 protein expression in the infected cells was examined using immunofluorescence assay. Results Infection with ~-catenin-specific shRNA lentivirus resulted in stable interference of β-catenin expression in human embryonic stem ceils, which showed a β-catenin mRNA expression of only 1% of that in the uninfected ceils. Infection with the empty vector produced no obvious effect on β-catenin expression compared to the uninfected cells. In the cells infected with β-catenin shRNA lentivirus, β-catenin protein expression was almost undetectable in immunofluorescence assay, while OCT4 was still expressed after the interference. Conclusion Lentiviral vector-delivered shRNA results in effective and stable β-catenin gene silencing in human embryonic stem cells.