CAJ | 학술논문

目的探讨超声破坏载重组腺病毒Ad-EGFP/HIF-1α的微泡对Ad-EGFP/HIF-1α生物学活性的影响.方法采用机械振荡法及分层吸附法制备携带重组腺病毒的脂质超声微泡造影剂,用DFY-Ⅱ型超声图像定量分析诊断仪检测微泡粒径.计算其浓度.将脐静脉内皮细胞(HUVEC)接种在24孔板中,随机分为以下5组:①空白对照组;②重组腺病毒Ad-EGFP/HIF-1α组;③载霞组腺病毒Ad-EGFP/HIF-1α微泡组;④超声+重组腺病毒Ad-EGFP/HIF-1α组;⑤超声+载重组腺病毒Ad-EGFP/HIF-1α微泡组.荧光显微镜及流式细胞仪检测各组在细胞内的表达及转染效率,R-PCR法检测HIF-1α的mRNA的表达.结果载重组腺病毒的超声微泡的大小、粒径及分布与普通超声微泡相似.与对照组相比,各组间绿色荧光强度及转染率无明显差别;各组间HIF-1α mRNA的表达也无明显差别.结论利用超声破坏载重组腺病毒Ad-EGFP/HIF-1α的微泡造影剂对腺病毒活性无明显影响,为拓展超声微泡介导的基因治疗领域提供了新的思路和方法.
목적 탐토초성파배재중조선병독Ad-EGFP/HIF-1α적미포대Ad-EGFP/HIF-1α생물학활성적영향.방법 채용궤계진탕법급분층흡부법제비휴대중조선병독적지질초성미포조영제,용DFY-Ⅱ형초성도상정량분석진단의검측미포립경.계산기농도.장제정맥내피세포(HUVEC)접충재24공판중,수궤분위이하5조:①공백대조조;②중조선병독Ad-EGFP/HIF-1α조;③재하조선병독Ad-EGFP/HIF-1α미포조;④초성+중조선병독Ad-EGFP/HIF-1α조;⑤초성+재중조선병독Ad-EGFP/HIF-1α미포조.형광현미경급류식세포의검측각조재세포내적표체급전염효솔,R-PCR법검측HIF-1α적mRNA적표체.결과 재중조선병독적초성미포적대소、립경급분포여보통초성미포상사.여대조조상비,각조간록색형광강도급전염솔무명현차별;각조간HIF-1α mRNA적표체야무명현차별.결론 이용초성파배재중조선병독Ad-EGFP/HIF-1α적미포조영제대선병독활성무명현영향,위탁전초성미포개도적기인치료영역제공료신적사로화방법.
Objective To investigate the biological activity of recombinant adenovirus Ad-EGFP/HIF-1α by microbubble destruction in vitro.Methods The lipid ultrasound microbubbles carried with recombinant adenovirus were prepared using mechanical vibration method and stratified absorption method.The diameter and concentration of microbubbles were determined by DFY-Ⅱ software.Human umbilical vein endothelial cell(HUVEC)was seeded in the 24-well plates,recombinant adenovirus or microbubbles carried with recombinant adenovirus was added in every well,with or without ultrasound exposure.Expression of the report gene EGFP was observed with fluorescent microscope and quantified by flow cytometry analysis.The expression of HIF-1α mRNA was detected by RT-PCR.Results The average diameter and distribution of microbubbles carried with recombinant adenovirus were similar to that of common microbubbles.There was no significant difference of green fluorescence intensity,transfection efficiency and expression of HIF-1α mRNA in each group compared with the control group.Conclusions It has no significant effective on the biological activity of recombinant adenovirus after microbubble destruction,which may be taken as a potenttial way of gene therapy in future.