CAJ | 학술논문

目的研究一种质粒DNA荧光标记的简便方法,以便进行DNA递送的示踪.方法质粒经溴激活后,于不同温度下存放不同时间,再与1,10-二氨基葵烷形成氨基衍生物.氨基修饰质粒与荧光素异硫氰酸酯( FITC)反应,制备出荧光标记的质粒,纯化并收集产物,计算其标记效率,评估溴激活质粒在不同温度下存放不同时长对标记反应的影响；观察荧光标记对质粒转染效率的影响,并用激光共聚焦显微镜观察和流式细胞仪检测示踪效果.结果实验数据显示溴活化的质粒随存放时间的延长标记效率反而下降,存放4℃较室温(25℃)的标记效率更高,提示溴活化质粒不适宜存放；而与没有标记的质粒相比,荧光标记的质粒对转染效率几乎没有影响.荧光标记的质粒应用于流式细胞仪检测和激光共聚焦显微观察都取得了较好的示踪效果.结论本研究建立了一种荧光标记质粒DNA的简便方法,并且在基因递送的示踪实验中获得了较好的效果.
목적 연구일충질립DNA형광표기적간편방법,이편진행DNA체송적시종.방법 질립경 추격활후,우불동온도하존방불동시간,재여1,10-이안기규완형성안기연생물.안기수식질립여형광소이류청산지( FITC)반응,제비출형광표기적질립,순화병수집산물,계산기표기효솔,평고추격활질립재불동온도하존방불동시장대표기반응적영향；관찰형광표기대질립전염효솔적영향,병용격광공취초현미경관찰화류식세포의검측시종효과.결과 실험수거현시추활화적질립수존방시간적연장표기효솔반이하강,존방4℃교실온(25℃)적표기효솔경고,제시추활화질립불괄의존방；이여몰유표기적질립상비,형광표기적질립대전염효솔궤호몰유영향.형광표기적질립응용우류식세포의검측화격광공취초현미관찰도취득료교호적시종효과.결론 본연구건립료일충형광표기질립DNA적간편방법,병차재기인체송적시종실험중획득료교호적효과.
Objective Conjugation of fluorescent dye onto plasmid DNA was investigated in order to monitor delivery process of plasmid DNA.Methods Plasmid was activated with bromine,stored for different time intervals at 4 ℃ or room temperature,and subsequently coupled with 1,10-diaminodecane to prepare aminemodified plasmid DNA.Amine-modified plasmid was then reacted with isothiocyanate (FITC) for fluorescent labeling,and the labeling ratio was calculated after purification.The effect of storage conditions (time/temperature) of bromine-actived plasmid (BP) on fluorescent labeling efficacy was estimated,and the cell transfection efficiency of fluorescent plasmid-lipofectamine complex was observed.The fluorescent plasmid delivered by lipofectamine 2000 in A10 cells was observed by laser scanning confocal microscope (LSCM) and flow cytometry.Results The experimental data showed that prolonged storage time of bromine-activated DNA had a negative effect on the labeling ratio,and lower storage temperature had a positive effect on the labeling ratio.It also demonstrated that FITC modification had no effect on the transfection efficiency of plasmid-lipofectamine complex as compared with that of unlabeled plasmid-lipofectamine complex,and FITC modified plasmid had enough fluorescent intensity to monitor cell uptake with flow cytometer and sub-cellular distribution with LSCM.Conclusion A facile method for conjugating fluorescent dye onto plasmid was established in the study,and could be utilized to trace the plasmid delivery for investigating the transfection mechanism.