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。结果成功构建了携带OPN-siRNA的重组慢病毒。该重组慢病毒可使SW480细胞内OPN的mRNA和蛋白的表达降低,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LVsiRNA-OPN慢病毒可以有效抑制SW480细胞内OPN的mRNA和蛋白表达,并抑制SW480细胞的增殖。
Objective To construct a 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 recom binant lentivirus vector was constructed and used to challenge SW480 cells. The expression level of osteopontin mR NA and protein was detected by real time PCR and Western blot. The effects on SW480 cells proliferation were an alyzed by MTI＂ assay. Results The restriction enzyme digestion, DNA sequencing and detection of GFP expression demonstrated that recombinant lentivirus vector was successfully constructed. Real time PCR and Western blot anal ysis 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 ）. Conclu sions The recombinant lentivirus vector could effectively inhibit the expression of osteopontin in SW480 cells and depress the proliferation of SW480 cells.
出处
《基础医学与临床》
CSCD
北大核心
2014年第3期350-354,共5页
Basic and Clinical Medicine
基金
宁夏高等学校科学技术研究项目(2011)
