NFκB核定位基序增强超声微泡介导的外源基因转染效率
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摘要
目的评价NFκB核定位基序增强超声靶向微泡破坏(UTMD)介导的外源基因转染效率的价值。方法构建人基质细胞衍生因子1α(phSDF-1α)质粒(phSDF-1α组),并插入NFκB核定位基序,构建具有核输入功能的目的基因质粒phSDF-1α-NFκB(phSDF-1α-NFκB)。用Cy3核酸染料标记两种质粒后在优化的UTMD条件下分别转染人脐静脉血管内皮细胞。CCK-8检测细胞存活率,流式细胞仪和荧光显微镜分别检测质粒的入胞效率和入核效率,RT-PCR和ELISA检测SDF-1α基因和蛋白表达水平。比较两种质粒的入核效率和目的基因表达率。结果在优化的UTMD条件下phSDF-1α组和phSDF-1α-NFκB组细胞存活率和质粒入胞效率差异均无统计学意义(P均>0.05),两组质粒入核率分别为(12.96±4.46)%和(83.25±12.15)%,SDF-1α基因相对表达量分别为(39.25±10.14)%和(118.25±27.57)%,SDF-1α蛋白表达量分别为(14.11±5.74)ng/mg和(65.35±11.12)ng/mg蛋白,后者均显著高于前者。结论 NFκB核定位基序能显著增强质粒入核,提高UTMD介导的外源基因转染效率。
Objective To explore the value of nuclear factor kappa B(NFκB)nuclear localization motif improving the gene transfection efficiency of ultrasound targeted microbubbles destruction(UTMD).Methods Human stromal cell derived factor-1alpha plasmid(phSDF-1α)was constructed as the target gene plasmid(phSDF-1αgroup).The NFκB nuclear localization motif was inserted into phSDF-1αplasmid to construct the novel target gene plasmid phSDF-1α-NFκB which had the nuclear import function(phSDF-1α-NFκB group).Human umbilical vein endothelial cells were transfected with Cy3 labeled phSDF-1αand phSDF-1α-NFκB plasmid with optimized UTMD parameters.CCK-8was used to detect the cell viability.Flow cytometry and fluorescence microscope were employed to detect the cellular and nuclear import efficiency of gene.RT-PCR and ELISA were used to detect the expression of SDF-1αgene and protein.The nuclear import efficiency and gene expression of phSDF-1α-NFκB was compared with phSDF-1α.Results Under optimized UTMD conditions,there were no significant difference between the two groups in cell viability and cellular intake of pDNA(both P>0.05).The nuclear import efficiency in phSDF-1αgroup and phSDF-1α-NFκB group were(12.96±4.46)%and(83.25±12.15)%.The SDF-1αgene and protein expression in phSDF-1αgroup and phSDF-1α-NFκB group were(39.25±10.14)% and(118.25±27.57)%,(14.11±5.74)ng/mg protein and(65.35±11.12)ng/mg protein,respectively.The latter were significantly higher than those of the former.Conclusion NFκB nuclear localization motif can enhance the nuclear import of target gene and increase the efficiency of UTMD mediated gene transfection.
Objective To explore the value of nuclear factor kappa B(NFκB)nuclear localization motif improving the gene transfection efficiency of ultrasound targeted microbubbles destruction(UTMD).Methods Human stromal cell derived factor-1alpha plasmid(phSDF-1α)was constructed as the target gene plasmid(phSDF-1αgroup).The NFκB nuclear localization motif was inserted into phSDF-1αplasmid to construct the novel target gene plasmid phSDF-1α-NFκB which had the nuclear import function(phSDF-1α-NFκB group).Human umbilical vein endothelial cells were transfected with Cy3 labeled phSDF-1αand phSDF-1α-NFκB plasmid with optimized UTMD parameters.CCK-8was used to detect the cell viability.Flow cytometry and fluorescence microscope were employed to detect the cellular and nuclear import efficiency of gene.RT-PCR and ELISA were used to detect the expression of SDF-1αgene and protein.The nuclear import efficiency and gene expression of phSDF-1α-NFκB was compared with phSDF-1α.Results Under optimized UTMD conditions,there were no significant difference between the two groups in cell viability and cellular intake of pDNA(both P>0.05).The nuclear import efficiency in phSDF-1αgroup and phSDF-1α-NFκB group were(12.96±4.46)%and(83.25±12.15)%.The SDF-1αgene and protein expression in phSDF-1αgroup and phSDF-1α-NFκB group were(39.25±10.14)% and(118.25±27.57)%,(14.11±5.74)ng/mg protein and(65.35±11.12)ng/mg protein,respectively.The latter were significantly higher than those of the former.Conclusion NFκB nuclear localization motif can enhance the nuclear import of target gene and increase the efficiency of UTMD mediated gene transfection.
引文
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[2]Chen ZY,Yang F,Lin Y,et al.New development and application of ultrasound targeted microbubble destruction in gene therapy and drug delivery.Curr Gene Ther,2013,13(4):250-274.
[3]Kang ST,Yeh CK.Ultrasound microbubble contrast agents for diagnostic and therapeutic applications:Current status and future design.Chang Gung Med J,2012,35(2):125-139.
[4]Miller AM,Munkonge FM,Alton EW,et al.Identification of protein cofactors necessary for sequence-specific plasmid DNA nuclear import.Mol Ther,2009,17(11):1897-1903.
[5]陈金玲,邓倾,郭瑞强,等.NF-κB核定位基序促进SDF-1α质粒转染的入核效率.武汉大学学报:医学版,2013,34(5):858-861.
[6]Deng Q,Chen JL,Zhou Q,et al.Ultrasound microbubbles combined with the NFκB binding motif increase transfection efficiency by enhancing the cytoplasmic and nuclear import of plasmid DNA.Mol Med Rep,2013,8(5):1439-1445.
[7]Miller DL,Averkiou MA,Brayman AA,et al.Bioeffects considerations for diagnostic ultrasound contrast agents.J Ultrasound Med,2008,27(4):611-632.
[8]Zhang C,Zhang X,Liu C,et al.Expression of endostatin mediated by a novel non-viral delivery system inhibits human umbilical vein endothelial cells in vitro.Mol Biol Rep,2010,37(4):1755-1762.
[9]Tanimoto,M,Kamiya,H,Minakawa,N,et al.No enhancement of nuclear entry by direct conjugation of a nuclear localization signal peptide to linearized DNA.Bioconjugate Chem,2003,14(6):1197-1202.
[10]Munkonge FM,Amin V,Hyde SC,et al.Identification and functional characterization of cytoplasmic determinants of plasmid DNA nuclear import.J Biol Chem,2009,284(39):26978-26987.