MDM2反义寡核苷酸的^(99)Tc^m标记及在乳腺癌细胞中的摄取动力学研究

Labeling of MDM2 antisense oligonucleotide with ～(99)Tc～m and the research of the uptake kinetics in human breast cancer cells

目的:研究放射性核素99Tcm标记MDM2(小鼠双微体扩增基因)反义寡核苷酸(ASON)的方法及在乳腺癌细胞中的摄取动力学,探讨用于乳腺癌反义显像的可能性。方法:人工合成一段针对MDM2 mRNA的ASON及错义寡核苷酸(ASONM),以HYNIC作为双功能螯合剂进行99Tcm标记,检测标记物的标记率,放化纯及反义探针与互补正义寡核苷酸(SON)的结合能力。不同时间检测脂质体2000包裹的探针在乳腺癌细胞中的摄取和清除情况,并与未经脂质体2000包裹的探针在乳腺癌细胞中的摄取及清除情况比较。结果:ASON的标记率为(57.2±2.98)%(n=5),ASONM的标记率为(56.3±3.01)%(n=5),经纯化后放化纯达95%以上,且反义探针仍保持与互补链的结合能力。22℃条件下,MCF-7细胞对脂质体2000包裹的反义探针和错义探针的摄取峰值分别为(34.62±2.91)%和(14.57±1.62)%,反义探针的摄取峰值明显高于错义探针的摄取峰值,但反义探针的清除率低于错义探针的清除率。脂质体2000包裹探针的摄取率高于未经脂质体2000包裹的探针的摄取率。结论:在双功能螯合剂HYNIC的作用下,寡核苷酸能成功地得到标记,脂质体2000包裹的反义探针能被代谢旺盛MCF-7细胞特异性摄取,有望被进一步用于乳腺癌反义显像的研究。

Objective： To investigate the possibility of using mouse double minute 2 （MDM2） mRNA antisense oligonu cleotide （ASON） labeled with ^99Tc^m for the antisense imaging study, the uptake kinetics of the lipesome-coated antisense probe and the mistatch probe were studied in the MCF-7. Methods： An ASON and a mismatch oligonucleotide（ASONM） targeted to mRNA MDM2 were synthesized and radiolabeled by ^99Tc^m with the bifunctional chelator HYNIC, and investigate the labeling efficiency, radiochemical purity and the ability of the labeled ASON to hybridize to the sense oligonucleotide （SON）. The uptake kinetics of the liposome-coated antisense probe and the mismatch probe were studied in the MCF-7 at different times, compared with uptake kinetics of the antisense probe and the mismatch probe in the MCF-7. Results： The labeling efficacy of ASON and ASONM were （57.2±2.98）%（n=5） and （56.3±3.01）%（n=5） with the bifunctional chelator HYNIC. The radiochemical purity was above 95% after purification. The labeled ASON still has the ability to hybridize to the SON. At 22℃, the uptake peak value of the liposome-coated antisense probe and the mismatch probe were （34.62±2.91）% and （14.57±1.62）%, respectively. The antisense probe showed significantly higher accumulation and effluxed much slowly than mismatch probe. The MCF-7 uptake of the lipesome-coated probe was much higher than that of non-coated probe. Conclusion： MDM2 mRNA antisense oligonucleotide can be radiolabeled successfully with the bifunctional chelator HYNIC. The antisense probe can accumulate in the proliferating MCF-7 cells more specifically which provided a basis for it to be used in antisense imaging study.