摘要
目的构建人C-erbB2干扰载体pGenesil-erbB2,并稳定转染入CerbB-2高表达的人结肠癌细胞株HT-29细胞,观察其对人结肠癌HT-29细胞从转录后水平对Her-2蛋白表达的影响。方法利用免疫组化方法筛选出CerbB-2高表达细胞株HT-29,以人C-erbB2cDNA基因为靶标设计RNA干扰片段和阴性对照片段,退火形成双链并克隆进入载体pGenesil-erbB,进行鉴定及测序,并稳定转染至HT-29细胞。结果测序证明人C-erbB2siRNA表达载体质粒pGenesil-erbB构建成功,pGenesil-erbB稳定转染入人结肠癌HT-29细胞,采用Westernblot实验证明pGenesil-erbB可显著抑制Her-2蛋白的表达。结论成功构建了人CerbB2干扰载体质粒pGenesil-erbB,稳定转染至结肠癌HT-29细胞株,并能抑制HT-29细胞的Her-2蛋白的表达,为靶向CerbB2的siRNA对结肠癌的放射增敏研究奠定了基础。
Objective To construct a plasmid carrying small interfering RNA （siRNA） targeting human C-erbB2 gene （pGenesil- erbB2） and test its effect on Her-2 expression at the post-transcriptional level in human colon cancer cell lines HT-29 cells that highly express erbB2. Methods A HT-29 cell line that highly expressed CerbB-2 was selected using immunohistochemical method. The double-stranded siRNA targeting human CerbB-2 cDNA and the negative control fragment were cloned into pGenesil-1 vector, and after identification and sequence analysis, the constructed pGenesil-erbB2 plasmid was transfected into the selected HT-29 cell line. Results The pGenesil-erbB2 plasmid was successfully constructed and stably transferred into HT-29 cells. The transfection resulted in significant inhibition of Her-2 protein expression in the HT-29 cells, as shown by Western blotting. Conclusion The pGenesil-erbB plasmid we constructed can be stably transfected into HT-29 cells to inhibit the expression of Her-2 protein, and can be useful in further studies of increasing the radiosensitivity of HT-29 cell lines.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2009年第9期1870-1873,共4页
Journal of Southern Medical University
