腺病毒-阴离子脂质体复合物的制备和体外表征  

The complexes of adenovirus and anionic liposomes:preparation and in vitro characterization

作　　者：钟志容[1,2] 万瑜[1] 石三军[1] 张志荣[1] 孙逊[1] 

机构地区：[1]四川大学华西药学院,四川成都610041 [2]泸州医学院,四川泸州646000

出　　处：《药学学报》2012年第1期116-123,共8页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81173011)

摘　　要：为了制备腺病毒-阴离子脂质体复合物(AL-Ad5),首先利用HEK 293细胞大量扩增了带有绿色荧光蛋白报告基因的重组腺病毒,并以氯化铯两步密度梯度离心法进行纯化,再通过细胞病变效应(CPE)法、壳蛋白免疫法和定量聚合酶链反应(Q-PCR)法分别测定腺病毒滴度。以中心组合设计法优化处方和制备条件,通过钙离子融合法制备目标复合物AL-Ad5,以透射电镜和动态光散射法分别对其外观形态、粒径和zeta电位进行考察;并制备双色荧光标记的复合物,通过激光共聚焦技术观察该复合物被MDCK细胞摄取后的胞内定位,以考察阴离子脂质体对腺病毒的包合情况。结果表明,目标复合物分布均匀,粒径和zeta电位分别为(211±10)nm和(-41.2±2.2)mV。激光共聚焦实验结果表明,红色荧光标记的腺病毒和绿色荧光标记的脂质体在MDCK细胞内的定位一致。由此说明,阴离子脂质体能够较好地包裹腺病毒以形成腺病毒-阴离子脂质体复合物。This study is to report the preparation of complexes of Ad5 and anionic liposomes （AL-Ad5）, the amplification of adenoviruses with enhanced green fluorescent protein （eGFP） reporter gene performed by HEK 293 cells, the adenoviral vectors purified by cesium chloride gradient centrifugation, and the titer of adenovirus determined by cytopathic effect （CPE） method, hexon capsid immunoassay and quantitative-PCR （Q-PCR）, separately. The prescription and experiment conditions were optimized by central composite design （CCD）. The complexes of Ad5 and AL-Ad5 were formulated by the calcium-induced phase change method. The morpholopy, particle size and zeta potential were detected by dynamic light scattering （DLS） and transmission electron microscopy （TEM）, respectively. Additionally, the bicolourable fluoresce-labeled complexes （Flabeled- AL-Ad5） were prepared and their intracellular location in MDCK cells was detected by confocal laser scanning microscopy （CLSM）. The results indicate that the complexes of AL-Ad5 exhibited a uniform distribution with a particle size of 211 ± 10 nm and a zeta potential of-41.2±2.2 mV. The result of CLSM demonstrates that the intracellular location of red fluoresce-labeled adenovirus was consistent with that of green fluoresce-labeled liposomes suggesting that the naked adenovirus was well encapsulated by the anionic liposomes in complexes of AL-Ad5.

关 键 词：腺病毒 阴离子脂质体 中心组合设计 钙离子融合法 激光共聚焦 壳蛋白免疫染色 定量聚合酶链反应 

分 类 号：R943[医药卫生—药剂学]