摘要
目的建立稳定抑制β-catenin基因表达的人神经母细胞瘤BE2C细胞株，为探讨Wnt／β-catenin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-timePCR、Westernblot的方法检测BE2C细胞中β-eatenin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、Westernblot检测干扰组peatenin^0制效率。结果RT-PCR、Westernblot检测显示BE2C细胞中pcatenin在基因及蛋白水平均有较好的表达；慢病毒的滴度达到1×108TU／m1时，侵染BE2C细胞的效率可达到90％左右；通过RT-PCR和Westernblot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％。结论成功构建了伊cateninshRNA慢病毒表达载体，建立了稳定抑制pcatenin基因表达的人神经母细胞瘤BE2C细胞株，为进一步研究β-eatenin在神经母细胞瘤发生中的作用提供了可靠的细胞模型。
Objective To establish a neuroblastoma cell line by inhibition of [3-catenin with siRNA interference technique. Methods RT-PCR and western blotting were used to test the expres- sion of [3-catenin in human neuroblastoma BE2C cells. Four siRNA interference sequences targeting 13- 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 ef- ficiency of interference was analyzed by RT-PCR and western blotting. Results RT-PCR and western blotting showed that ~]-catenin was constantly expressed by human neuroblastoma cell lines BE2C. When the lentiviral titer was 1 ~ 108 TU/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.
出处
《中华小儿外科杂志》
CSCD
北大核心
2013年第4期290-294,共5页
Chinese Journal of Pediatric Surgery
