摘要目的 建立稳定抑制β-eatenin基因表达的入神经母细胞瘤BE2C细胞株,为探讨Wnt/-eatenin信号在神经母细胞瘤发生中的作用提供细胞模型.方法 使用Real-time PCR、Westernblot的方法检测BE2C细胞中β-catenin在基因及蛋白水平的表达.于β-catenin基因编码区选择4个siRNA靶点及1个非编码序列(NC)合成5对shRNA干扰序列,分别与质粒PgLV-H1-GFP+ Puro载体连接,构建重组质粒；将重组质粒与慢病毒包装质粒共转染BE2C细胞,筛选出干扰效果最佳的shRNA序列,经嘌呤霉素筛选并扩大培养后得到稳定克隆株.RT-PCR、Western blot检测干扰组β-catenin抑制效率.结果 RT-PCR、Western blot检测显示BE2C细胞中β-catenin在基因及蛋白水平均有较好的表达；慢病毒的滴度达到1×108 TU/ml时,侵染BE2C细胞的效率可达到90％左右；通过RT-PCR和Western blot检测结果显示G1为最有效的干扰靶点；通过嘌呤霉素筛选得到稳定靶向干扰β-catenin细胞株,抑制效率可达约90％.结论 成功构建了β-catenin shRNA慢病毒表达载体,建立了稳定抑制β-eatenin基因表达的人神经母细胞瘤BE2C细胞株,为进一步研究β-catenin在神经母细胞瘤发生中的作用提供了可靠的细胞模型.

abstractsObjective To establish a neuroblastoma cell line by inhibition of β-catenin with siRNA interference technique.Methods RT-PCR and western blotting were used to test the expression of β-catenin in human neuroblastoma BE2C cells.Four siRNA interference sequences targeting β-catenin gene CTNNB1 and one non-interference sequence were designed and synthesized.Double-strand shRNA hairpins were synthetized and separately cloned into PgLV-H1-GFP + Puro vector to produce five plasmids.The lentiviral packaging plasmids and lentiviral RNAi plasmid were co-transfected into 293T cells.The most effective interference sequences were screened by RT-PCR and western blotting.After puromycin selection and culture expansion,stable cell clones were established.The inhibition efficiency of interference was analyzed by RT-PCR and western blotting.Results RT-PCR and western blotting showed that β-catenin was constantly expressed by human neuroblastoma cell lines BE2C.When the lentiviral titer was 1 × 108TU/ml,infection efficiency can be up to 90％.RT-PCR and western blotting showed that G1 was the most effective target.And efficiency of siRNA interference of β-catenin could be up to about 90％.Conclusions A stable neuroblastoma cell line is established by inhibition of β-catenin with siRNA interference technique.