流感泰得小分子化合物修饰前后细胞摄取的比较  

Comparison of cellular uptake of antisense oligonucleotide Flutide modified and unmodified by small molecular compound

作　　者：杨兰兰[1] 鲁丹丹[2] 张玉林[2] 吴小桃[2] 李树[2] 赵海豹[2] 王升启[2] 

机构地区：[1]河南中医学院,郑州450000 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《军事医学科学院院刊》2010年第2期105-110,共6页Bulletin of the Academy of Military Medical Sciences

基　　金："重大新药创制"科技重大专项(2009ZX09503-021);国家自然科学基金(30901825);国家重点研究发展计划(973项目)(2005CB523006)资助

摘　　要：目的在反义寡核苷酸流感泰得(Flutide,FT)上偶联神经氨酸酶抑制剂奥司他韦,以期通过特定小分子化合物的修饰提高细胞对流感泰得的摄取,进而提高其抗流感的作用。方法采用3′荧光标记控孔玻璃珠(3′FAMCPG)合成全硫代修饰的流感泰得,并对其5′端进行巯基修饰,与结构改造后的奥司他韦分子进行连接得到流感泰得与奥司他韦偶联物(FTO);修饰前后的反义药物直接转染流感病毒感染的人肺癌上皮A549细胞,通过荧光显微镜观察及流式细胞技术检测FTO与FT的胞内荧光强度来进行反义药物偶联小分子化合物前后胞内摄取量的比较;采用CCK8细胞增殖检测技术进行FT与FTO体外抗流感病毒活性评价。结果得到一种新的寡核苷酸偶联物FTO;荧光显微镜观察结果显示,细胞给予FTO后荧光强度强于FT;流式细胞检测结果表明,修饰后的FTO胞内平均荧光值明显高于修饰前的FT,其中给药12h时,病毒感染细胞+FTO(BFTO)组的平均荧光值比病毒感染细胞+FT(BFT)组增加了(102.3±56.0)%,正常细胞+FTO(ZFTO)组的平均荧光值比正常细胞+FT(ZFT)组增加了(89.4±44.7)%;给药24h后BFTO组的平均荧光值比BFT组增加了(115.2±28.4)%,ZFTO组的平均荧光值比ZFT组增加了(102.9±12.1)%,(P<0.05);体外抗流感病毒活性评价显示,FTO与FT的IC50值分别为(1.57±0.08)μmol/L与(4.44±0.11)μmol/L。结论流感泰得采用奥司他韦修饰后有助于提高其在细胞内的摄取量以及抗流感病毒活性。Objective To conjugate phosphorothioate antisense oligonucleotide（PS-ASON）Flutide（FT）with neuraminidase inhibitor Oseltamivir,in order to improve its cellular uptake by conjugating with certain small molecular compounds,and thereby,to enhance Flutide＇s anti-influenza virus activity.Methods Flutide was synthesized using 3＇-fluorescence-labeled controlled pore glass（3＇-FAM CPG）,and the 5＇end of Flutide was modified with a thiol group（3＇-FAM,5＇-SH Flutide）.The structure of Oseltamivir was transformed to link with 3＇-FAM labeled Flutide to obtain a new compound FTO.Modified and unmodified antisense oligonucleotides（FT and FTO）were directly transfected into human lung epithelial A549 cells which had been infected by influenza A/H1N1 virus.The cellular uptake was measured by fluorescence microscopy and flow cytometry detection.CCK8 was used to detect the anti-influenza virus activity of FT and FTO.Results In the experiment,a new compound FTO was obtained.The results of fluorescence microscopy observation showed that the fluorescence intensity in culture cells with treatment by FTO was stronger than that by FT.The flow cytometry determination results indicated that,at 12 h,the average fluorescence value of virus infected cell＋FTO group（BFTO）was raised by（102.3±56.0）% compared with viral infected cell＋FT group（BFT）,and the fluorescence value of normal cell＋FTO group（ZFTO）was raised by（89.4±44.7）% compared with normal cell＋FT group（ZFT）;after 24 h,the average fluorescence value of BFTO group was raised by（115.2±28.4）% compared with BFT group,ZFTO group was raised by（102.9±12.1）% compared with ZFT group（P0.05）.In evaluation of in vitro anti-influenza virus activity of FTO and FT,the IC50 were（1.57±0.08）μmol/L and（4.44±0.11）μmol/L respectively.Conclusion Flutide-Oseltamivir conjugates can significantly improve the cellular uptake of FT in the target cells and the anti-influenza virus activity.

关 键 词：反义寡核苷酸 流感泰得 奥司他韦 偶联 细胞摄取 转染 流式细胞术 正黏病毒科 抗流感病毒活性 

分 类 号：R511.7[医药卫生—内科学]