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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 couple the neuraminidase inhibitor oseltamivir on antisense oligonucleotide Flutide (FT) in order to improve the cellular uptake of flutadex by the modification of specific small molecule compounds, Anti-flu effect. Methods Fluorescein-3-fluorophore-labeled glass beads (3’FAMCPG) were used in the synthesis of all-thiol-modified fludetad and their 5 ’end was modified with mercapto to connect with structurally modified oseltamivir molecules to obtain flu-thalidomide (FTO). The antisense drugs before and after modification were directly transfected into human lung cancer A549 cells infected with influenza virus. Fluorescence microscopy and flow cytometry were used to detect the intracellular fluorescence intensity of FTO and FT To compare antisense drug conjugate small molecule compounds before and after intracellular uptake; using CCK8 cell proliferation detection technology FT and FTO in vitro anti-influenza virus activity evaluation. Fluorescence microscopy results showed that the fluorescence intensity of FTO was stronger than that of FT. The results of flow cytometry showed that the fluorescence intensity of modified FTO was significantly higher than that of FTO The average fluorescence value of virus-infected cells + FTO (BFTO) group increased by (102.3 ± 56.0)% and that of normal cells + FTO (ZFTO) The mean fluorescence value of BFTO group was (89.4 ± 44.7)% higher than that of normal cells + FT group (115.2 ± 28.4%). The average fluorescence value of BFTO group was (115.2 ± 28.4)% higher than that of BFT group (102.9 ± 12.1)%, (P <0.05), respectively. The anti-influenza virus activity in vitro showed that the IC50 values of FTO and FT were (1.57 ± 0.08) μmol / L and (4.44 ± 0.11) μmol / L. Conclusion Fluteta modified with oseltamivir helps to increase its intracellular uptake and anti-influenza activity.