脂质体包裹^(99)Tc^m标记Survivin反义寡核苷酸的制备和体外研究  

The preparation and in vitro study of liposome-coated Survivin antisense oligonucleotide labeled with ^(99)Tc^m

作　　者：沈艳霞[1] 高再荣[1] 张永学[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院核医学科,武汉430022

出　　处：《中华核医学杂志》2006年第3期177-179,共3页Chinese Journal of Nuclear Medicine

基　　金：湖北省科技厅自然科学基金(2002AB121)

摘　　要：目的研究脂质体包裹(99)~Tc^m 标记 Survivin 反义寡核苷酸(ASON)的制备方法及其在肝癌细胞中的表达。方法合成20碱基单链 Survivin ASON,全程硫代修饰,5’末端经氨基修饰,以双功能联结剂联肼尼克酰胺(HYNIC)为螯合剂,ASON 与 HYNIC 偶联后用(99)~Tc^m 标记,经 P4柱纯化后,用阳离子脂质体(lipofectamine2000)对纯化产物进行包裹,并对其标记率、放化纯、比活度、稳定性、细胞摄取率进行研究。结果 (99)~Tc^m O_4^-比活度为18.5、14.8和7.4 MBq/μg时,标记率分别为(63.10±1.15)%、(62.40±1.05)%和(58.90±3.01)%。脂质体包裹的(99)~Tc^m-HYNIC-ASON 放化纯为(94.47±3.32)%,以生理盐水、新鲜人血清孵育4 h 后放化纯分别为92.51%和92.70%。转染后6 h,经脂质体包裹(99)~Tc^m-HYNIC-ASON 的肝癌细胞摄取率达(66.21±0.46)%,明显高于未经脂质体包裹的 ASON[(25.17±1.08)%,t=23.979～98.858,P<0.01)]。结论以 HYNIC 作为螯合剂标记 ASON 方法可行,脂质体能明显增加细胞摄取 ASON。Objective To study the preparation of liposome-coated ^99Tc^m labeled Survivin antisense oligonucleotide （ASON） and its expression in hepatocellular tumor cells. Methods A 20 mer single-stranded ASON sequence against Survivin mRNA was synthesized and linked with a primary amine on the 5＇- end. The Survivin ASON was labeled with ^99Tc^m by conjugating the bifunctional chelator-HYNIC, and purified through P4 column. The product was then encapsulated with cationic （liposome lipofectamine2000）. The radiolabeling efficiency, radiochemical purity, specific activity, stability and cell uptake of the product were also studied. Resdts When the specific activities of ^99Tc^mO4^- were 18.5, 14.8 and 7.4 MBq/μg, the radiolabeling efficiencies of ^99Tc^m-HYNIC-ASON were （63. 10 ±1. 15）%, （62.40 ± 1.05）% and （ 58.90 ± 3.01 ） % , respectively. The radiochemical purity was （ 94.47 ± 3.32 ） % in average. After incubation with normal saline or fresh human serum for 4 h, the radiochemical purities were 92.51% and 92.70%. Six-hour after transfection, the cell uptake of the liposome-coated ^99Tc^m-HYNIC-ASON reached （66.21 ± 0.46）% in human hepatocellular tumor cell and was much higher than that of non-coated ^99Tc^m- HYNIC-ASON[（25.17±1.08）%, t=23.979-98.858, P〈0.01）]. Conclusions The Survivin ASON conjugated with HYNIC labeled with ^99Tc^m is a successful method. The liposome has highly efficient for increasing the cellular uptake of ASON.

关 键 词：寡核苷酸类 反义 同位素标记 锝 脂质体 Survivin 体外研究 

分 类 号：R817[医药卫生—影像医学与核医学]