基础医学与临床 ›› 2014, Vol. 34 ›› Issue (3): 350-354.

• 研究论文 • 上一篇    下一篇

OPN siRNA慢病毒载体的构建及其对大肠癌细胞增殖抑制作用

邢译文,白静,范维宁,刘鑫,周林林,徐广贤   

  1. 宁夏医科大学
  • 收稿日期:2013-06-26 修回日期:2013-10-25 出版日期:2014-03-05 发布日期:2014-02-27
  • 通讯作者: 徐广贤 E-mail:guangxianxu2004@yahoo.com.cn

Construction of osteopontin siRNA expression lentivirus vector and itsinhibitory effect on the proliferation of SW480 cells

  • Received:2013-06-26 Revised:2013-10-25 Online:2014-03-05 Published:2014-02-27

摘要: 目的 构建OPN-siRNA的慢病毒载体,探讨其对结肠癌SW480细胞增殖的调节作用。 方法 设计合成针对OPN的siRNA序列,退火形成双链DNA并克隆到pSicoR质粒后,包装重组慢病毒。应用Real Time PCR及Western blot检测病毒刺激SW480细胞后OPN的mRNA和蛋白的表达水平,应用MTT法检测慢病毒对SW480细胞增殖的调节作用。结果 经酶切、测序以及GFP表达证实,成功构建了携带OPN-siRNA的重组慢病毒。Real Time PCR及Western blot结果显示该重组慢病毒可使SW480细胞内OPN的mRNA和蛋白的表达降低,MTT法结果显示LV-siRNA-OPN慢病毒对SW480细胞增殖有明显的抑制作用(P<0.05)。 结论 成功构建的LV-siRNA -OPN慢病毒可以有效的抑制SW480细胞内OPN的mRNA和蛋白表达, 并抑制SW480细胞的增殖。

关键词: [关键词] OPN, siRNA, HEK-297T, 慢病毒载体

Abstract: Objective To construct the recombinant lentivirus vector and explore its effects on colon cancer cell line SW480. Methods The siRNA interfering sequence targeting to osteopontin gene was designed and synthesized,then DNA segment was gained through annealing after chemosynthesis,and then was cloned to pSicoR vector, the recombinant lentivirus vector was constructed and used to challenge SW480 cells.The expression level of osteopontin mRNA and protein was detected by Real Time PCR and Western blotting. The effects on SW480 cells proliferation were analyzed by MTT assay. Results The restriction enzyme digestion,DNA sequencing and detection of GFP expression demonstrated that recombinant lentivirus vector was constructed successfully. Real Time PCR and Western blot analysis confirmed that the expression of OPN mRNA and protein were inhibited.The MTT assay showed that the anti-proliferation effect of lentivirus vector of osteopontin-siRNA on SW480 cells were significant(P<0.05). Conclusions The recombinant lentivirus vector could effectively inhibit the expression of osteopontin in SW480 cells and depress the proliferation of SW480 cells.

Key words: [Key words] osteopontin, siRNA, HEK-293T, lentivirus vector