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1. 军事医学科学院放射医学研究所
2. 重庆医科大学病毒性肝炎研究所,北京,100850
3. ,重庆,400010
4. 北京100850研究员
纸质出版日期:2005,
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[1]郑素军,林汝仙,夏云,黄爱龙,钟森,王升启,任红.KDR为靶的反义寡核苷酸、siRNA活性比较[J].重庆医科大学学报,2005(03):340-344+382.
ZHENG Sujun, et al. The comparison of antisense oligonucleotides、small interferingRNA targeting KDR for gene silencing[J]. Journal of Chongqing Medical University, 2005,(3):340-344.
[1]郑素军,林汝仙,夏云,黄爱龙,钟森,王升启,任红.KDR为靶的反义寡核苷酸、siRNA活性比较[J].重庆医科大学学报,2005(03):340-344+382. DOI: 10.13406/j.cnki.cyxb.2005.03.009.
ZHENG Sujun, et al. The comparison of antisense oligonucleotides、small interferingRNA targeting KDR for gene silencing[J]. Journal of Chongqing Medical University, 2005,(3):340-344. DOI: 10.13406/j.cnki.cyxb.2005.03.009.
目的:观察反义寡核苷酸(antisenseoligonucleotides
asONs)
细胞内源性表达的短发卡状小干扰RNA(shorthairpinsmallinterferingRNA
siRNA)对血管内皮生长因子受体2(VEGFR2/KDR:Vascularendothelialgrowthfactorreceptor2/kinaseinsertdomain-containingreceptor)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KDRmRNA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
Objective:To investigate and compare the inhibitory effects on KDR gene expressinon of antisense oligonucleotides(asONs)
short hairpin small interfering RNA(siRNA) targeting the mRNA of VEGFR2/KDR(vascular endothelial growth factor receptor 2/kinase insert domain-containing receptor)in MCF-7 cells.Methods:3 antisense oligonucleotides were designed.According to the target KDR mRNA accessible sites of antisense oligonucleotides
3 DNA vector-based pPUR/U6/KDR-siRNA for intracellular transcription KDR specific short hairpin small interfering RNA were constructed.The inhibitory effects on KDR protein expression of antisense oligonucleotides
and pPUR/U6/KDR-siRNA for stable transfection were evaluated using Western-blotting assay.The antisense oligonucleotides inhibitory effects on cell proliferation were determined with MTT assay
while the growth curve of MCF-7 cells stably transfected with pPUR/U6/KDR-siRNA was measured using cell counting.Results:3 antisense oligonucleotides significantly reduced the KDR protein expression
with the inhibitory rates being 66.16%
50.21% and 58.35% at 0.8μmol/L
respectively.They also decreased the proliferation of MCF-7 cells in a dose dependent manner.Compared to the cells transfected with vacant pPUR/U6 control vector
two of three pPUR/U6/KDR-siRNA plasmids caused effcient down-regulation of KDR protein expression in stable transfected MCF-7 cells.At the 15th day after stable transfection of pPUR/U6/KDR-siRNA
the inhibitory rates were 75.21% and 80.08%
respectively;and at the 30th day the inhibitory rates were 59.29% and 60.15%
respectively.The stable transfection of pPUR/U6/KDR-siRNA also slowed MCF-7 cells growth.Conclusion:It is an effective approach to design siRNA according to the antisense oligonucleotides target accessible sites.The endogenous short hairpin siRNA produced by DNA plasmid can more significantly inhibit KDR gene expression than the antisense oligonucleotides for a long time.
反义寡核苷酸siRNA血管内皮生长因子受体2
Antisense oligonucleotidesSmall interfering RNAKDR
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