含气微泡介导反义寡核苷酸转染实验的参数优化  

Optimizing the operating variables that affect the transfection experiment of antisense oligodeoxyribonucleotide by gas-filled microbubbles

作　　者：赵应征[1] 罗渝昆[2] 鲁翠涛 许景峰[1] 梅兴国[4] 王虎军[1] 张梅[1] 

机构地区：[1]北京军区总医院药理科,北京100700 [2]解放军总医院超声科,北京100853 [3]北京麦德姆医学管理集团,北京100055 [4]军事医学科学院毒物药物研究所,北京100850

出　　处：《药学学报》2007年第12期1323-1326,共4页Acta Pharmaceutica Sinica

摘　　要：To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide（AS-ODNs） by insonated gas-filled lipid microbubbles,SF6-filled microbubbles were prepared by sonication-lyophilization method.An AS-ODNs sequence and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of SF6-filled microbubbles.Three levels of mixing speed,different durations of mixing and various delay time before ultrasound were examined,separately.Transfection efficiency was detected by fluorescence microscopy.Transfection results with and without incubation of AS-ODNs and microbubbles before mixing cells were compared.From the results,there is no significant difference between the transinfection efficiency with or without incubation of AS-ODNs and microbubbles before mixing cells.AS-ODNs transfection efficiency showed an increasing trend with mixing speed and mixing duration,but there is a negative relationship with delay time before ultrasound.The optimum parameters for AS-ODNs transfection by SF6-filled microbubbles were found at a mixing speed of 40-50 r·min-1 for 30-60 s with less than 60 s delay before ultrasound.For a successful transfection,long time of incubation with gene is essential for normal nonviral vectors such as liposomes or cationic lipid-polymer hybrids,because these vectors depend on endocytosis and membrane fusion to realize transfection.Unlike liposomes and cationic lipid-polymer hybrids,gas-filled lipid microbubbles depend on sonorporation effect to realize transfection.Therefore,the incubation of gene and microbubbles before mixing cells may not be necessary.Ultrasound-mediated AS-ODNs transfection enhanced by gas-filled lipid microbubbles represents an effective avenue for gene transfer.To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide（AS-ODNs） by insonated gas-filled lipid microbubbles,SF6-filled microbubbles were prepared by sonication-lyophilization method.An AS-ODNs sequence and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of SF6-filled microbubbles.Three levels of mixing speed,different durations of mixing and various delay time before ultrasound were examined,separately.Transfection efficiency was detected by fluorescence microscopy.Transfection results with and without incubation of AS-ODNs and microbubbles before mixing cells were compared.From the results,there is no significant difference between the transinfection efficiency with or without incubation of AS-ODNs and microbubbles before mixing cells.AS-ODNs transfection efficiency showed an increasing trend with mixing speed and mixing duration,but there is a negative relationship with delay time before ultrasound.The optimum parameters for AS-ODNs transfection by SF6-filled microbubbles were found at a mixing speed of 40-50 r·min^-1 for 30-60 s with less than 60 s delay before ultrasound.For a successful transfection,long time of incubation with gene is essential for normal nonviral vectors such as liposomes or cationic lipid-polymer hybrids,because these vectors depend on endocytosis and membrane fusion to realize transfection.Unlike liposomes and cationic lipid-polymer hybrids,gas-filled lipid microbubbles depend on sonorporation effect to realize transfection.Therefore,the incubation of gene and microbubbles before mixing cells may not be necessary.Ultrasound-mediated AS-ODNs transfection enhanced by gas-filled lipid microbubbles represents an effective avenue for gene transfer.

关 键 词：含气微泡 超声 反义寡核苷酸 转染 基因递送 

分 类 号：R943[医药卫生—药剂学]