| 陈金玲,邓倾,周青,曹省,胡波,宋宏宁,郭瑞强.NFκB核定位基序增强超声微泡介导的外源基因转染效率[J].中国医学影像技术,2014,30(8):1141~1144 |
| NFκB核定位基序增强超声微泡介导的外源基因转染效率 |
| NFκB nuclear localization motif improving the gene transfection efficiency of ultrasound targeted microbubbles destruction |
| 投稿时间：2014-06-10 修订日期：2014-07-21 |
| DOI： |
| 中文关键词: 微泡 核定位信号 NFκB 基因转染 |
| 英文关键词:Microbubbles Nuclear localization signal NFκB Gene transfection |
| 基金项目:国家自然科学基金（81101058）、武汉市科技攻关项目（2013060602010268）。 |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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-1 alpha plasmid (phSDF-1α) was constructed as the target gene plasmid (phSDF-1α group). The NFκB nuclear localization motif was inserted into phSDF-1α plasmid 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-1α and phSDF-1α-NFκB plasmid with optimized UTMD parameters. CCK-8 was 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-1α gene 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-1α group and phSDF-1α-NFκB group were (12.96±4.46)% and (83.25±12.15)%. The SDF-1α gene and protein expression in phSDF-1α group 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. |
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