CAJ | 학술논문

目的:观察反义寡核苷酸(antisense oligonucleotides,asONs),细胞内源性表达的短发卡状小干扰RNA(short hairpinsmall interfering RNA,siRNA)对血管内皮生长因子受体2(VEGFR2/KDR:Vascular endothelial growth factor receptor 2/kinaseinsert domain-containing receptor)表达的抑制作用,探讨阻断DKR蛋白表达对MCF-7细胞增殖、生长的影响,并对两者作了比较.方法:设计了3条反义寡核苷酸,并根据其相应靶KDR mRNA杂交位点,构建了3条以质粒pPUR/U6为载体的可内源性表达短发卡状siRNA的pPUR/U6/KDR-siRNA.分别用Western-blotting方法检测了MCF-7细胞经反义寡核苷酸处理,pPUR/U6/KDR-siRNA稳定转染后对KDR蛋白表达的抑制作用;用MTT法观察了反义寡核苷酸对MCF-7细胞增殖的影响,并观察了pPUR/U6/KDR-siRNA稳定转染细胞的生长曲线.结果:3条反义寡核苷酸于0.8μmol/L时,均有效抑制了细胞KDR蛋白表达,抑制率分别为66.16%,50.21%,58.35%;3条反义寡核苷酸对MCF-7细胞增殖也显示了剂量依赖性下调作用.相应靶位点3条pPUR/U6/KDR-siRNA中,有2条在细胞稳定转染时显著下调了KDR蛋白表达,15天抑制率分别为75.21%,80.08%;30天时为59.29%,60.15%;细胞稳定转染表达siRNA后生长变慢.结论:可根据有效反义寡核苷酸靶位点设计siRNA,稳定转染内源性表达的短发卡状siRNA可长期抑制靶基因表达,并起了更为高效的抑制作用.
목적:관찰반의과핵감산(antisense oligonucleotides,asONs),세포내원성표체적단발잡상소간우RNA(short hairpinsmall interfering RNA,siRNA)대혈관내피생장인자수체2(VEGFR2/KDR:Vascular endothelial growth factor receptor 2/kinaseinsert domain-containing receptor)표체적억제작용,탐토조단DKR단백표체대MCF-7세포증식、생장적영향,병대량자작료비교.방법:설계료3조반의과핵감산,병근거기상응파KDR mRNA잡교위점,구건료3조이질립pPUR/U6위재체적가내원성표체단발잡상siRNA적pPUR/U6/KDR-siRNA.분별용Western-blotting방법검측료MCF-7세포경반의과핵감산처리,pPUR/U6/KDR-siRNA은정전염후대KDR단백표체적억제작용;용MTT법관찰료반의과핵감산대MCF-7세포증식적영향,병관찰료pPUR/U6/KDR-siRNA은정전염세포적생장곡선.결과:3조반의과핵감산우0.8μmol/L시,균유효억제료세포KDR단백표체,억제솔분별위66.16%,50.21%,58.35%;3조반의과핵감산대MCF-7세포증식야현시료제량의뢰성하조작용.상응파위점3조pPUR/U6/KDR-siRNA중,유2조재세포은정전염시현저하조료KDR단백표체,15천억제솔분별위75.21%,80.08%;30천시위59.29%,60.15%;세포은정전염표체siRNA후생장변만.결론:가근거유효반의과핵감산파위점설계siRNA,은정전염내원성표체적단발잡상siRNA가장기억제파기인표체,병기료경위고효적억제작용.