CAJ | 학술논문

目的应用超声靶向微泡破坏技术(UTMD)和核因子κB(NF-κB)结合基序分别促进人基质细胞衍生因子-1α(SDF-1α)质粒进入细胞质和细胞核,提高对血管内皮细胞的转染效率.方法构建含NF-κB结合基序的人SDF-1α质粒(phSDF-1α-NF-κB)和不含NF-κB结合基序的人SDF-1α质粒(phSDF-1α),用核酸染料Cy3标记后在优化的UTMD条件下分别转染人脐静脉血管内皮细胞.流式细胞仪检测质粒入胞效率；荧光显微镜观察质粒入核情况；RT-PCR、Western印迹和ELISA分别从基因水平和蛋白水平检测SDF-1α质粒的表达.比较两种质粒的入胞率、入核率以及表达率以评价UTMD和NF-κB结合基序对转染的作用.结果 UTMD能显著提高质粒的入胞效率(81％±7％),同时保持较高细胞存活率(86％±6％).含NF-κB结合基序组的质粒入核效率和蛋白表达效率均较不含NF-κB结合基序组显著提高[65％±12％比10％±3％,(63±10)比(15±5)μg/g蛋白,均P＜0.01].结论 UTMD联合NF-1κB结合基序转染系统通过提高质粒的人胞和入核效率能显著提高SDF-1α质粒对血管内皮细胞的转染效率.
목적 응용초성파향미포파배기술(UTMD)화핵인자κB(NF-κB)결합기서분별촉진인기질세포연생인자-1α(SDF-1α)질립진입세포질화세포핵,제고대혈관내피세포적전염효솔.방법 구건함NF-κB결합기서적인SDF-1α질립(phSDF-1α-NF-κB)화불함NF-κB결합기서적인SDF-1α질립(phSDF-1α),용핵산염료Cy3표기후재우화적UTMD조건하분별전염인제정맥혈관내피세포.류식세포의검측질립입포효솔；형광현미경관찰질립입핵정황；RT-PCR、Western인적화ELISA분별종기인수평화단백수평검측SDF-1α질립적표체.비교량충질립적입포솔、입핵솔이급표체솔이평개UTMD화NF-κB결합기서대전염적작용.결과 UTMD능현저제고질립적입포효솔(81％±7％),동시보지교고세포존활솔(86％±6％).함NF-κB결합기서조적질립입핵효솔화단백표체효솔균교불함NF-κB결합기서조현저제고[65％±12％비10％±3％,(63±10)비(15±5)μg/g단백,균P＜0.01].결론 UTMD연합NF-1κB결합기서전염계통통과제고질립적인포화입핵효솔능현저제고SDF-1α질립대혈관내피세포적전염효솔.
Objective To increase the transfection efficiency of SDF-1αt plasmids for entry into the cytoplasm and nuclei of vascular endothelial cells by using the ultrasound-targeted microbubbles destruction (UTMD) technique combined with nuclear factor-κB binding motif.Methods We constructed the SDF-1 α plasmid with or without NF-κB binding motif (phSDF-1α-NF-κB and phSDF-1α),which were labeled with Cy3,the nucleic acid dye,for transfection into the human umbilical vein endothelial cells by optimizing the conditions of UTMD.Flow cytometry and fluorescence microscope were employed to detect the cellular import efficiency of pDNA and the nuclear import efficiency of pDNA,respectively.Reverse transcriptase polymerase chain reaction,Western blotting and enzyme-linked immunosorbent assay were used to detect SDF-1 α gene expression.The nuclear import and plasmid expression efficiency were compared to explore the effect of UTMD and NF-κB binding motif on transfection.Results UTMD significantly increased the cytoplasmic intake of pDNA (81％±7％) and maintained high cell viability (86％±6％).Compared with the NF-κB-free plasmids,the abundance of NF-κB plasmids in the nuclei and SDF-1α expression increased significantly [65％ ± 12％ vs 10％ ± 3％ ; (63 ± 10) μg/g protein vs (15 ± 5) μg/g protein,both P＜0.01].Conclusion UTMD combined with NF-κB augments the SDF-1α gene transfection efficiency by enhancing cytoplasmic and nuclear import of plasmid DNA.