中文摘要：

目的：研究人端粒酶催化亚单位（hTERT）反义分子探针（^99mTc-hTERT ASON）在体外靶细胞摄取动力学情况,以及特异识别靶基因的生物学性质。方法：以双功能螯合剂法,制备放射性核素^99mTc标记的针对hTERT mRNA的反义分子探针（^99mTc-hTERTASON）。培养hTERT表达阳性的人肝细胞癌细胞株（HepG2细胞）。观察在有/无脂质体介导下,细胞对^99mTc-hTERT ASON的摄取动力学情况。阳离子脂质体介导^99mTc-hTERTASON转染细胞,逆转录聚合酶链反应（reverse transcriptase polymerase chain reaction,RT-PCR）分析其特异识别靶基因的生物学活性。结果：^99mTc-hTERT ASON的标记率达到（76±5）%（n=5）,放射性化学纯度达到96%,比活度为1.850×10^6Bq/μg。不论脂质体介导与否,^99mTc-hTERT ASON在不同时相的细胞摄取率均高于对照组（P〈0.05）;脂质体明显提高细胞对^99mTc-hTERT ASON的摄取（P〈0.05）;通过脂质体介导,细胞对^99mTc-hTERT ASON的摄取高峰值提前至2h,并保持稳定水平至3h。RT-PCR结果显示^99mTc-hTERT ASON转染细胞后,hTERT mRNA表达量明显降低。结论：脂质体在体外能有效提高^99mTc-hTERT ASON的细胞摄取率。分子探针^99mTc-hTERT ASON能特异识别靶基因。为进一步开展^99mTc-hTERT ASON体内研究奠定了基础。

英文摘要：

Objective：To observe the cell uptake kinetics and specific gene expression effect of hTERT antisense molecular probe in vitro. Methods： Antisense molecular probes targeting hTERT mRNA were radiolabeled with technetium-99m by the method of bifunctional chelator. HepG2 cells expressing hTERT were cultured. The uptake kinetics of molecular probes mediated by liposome or not in cells were examined in vitro. RT-PCR （reverse transcriptase polymerase chain reaction） method was performed to assay the specific gene expression effect of the molecular probes. All data were analyzed by the statistic software of SPSS 12.0. Results： The labeling efficieneies of molecular probe reached （76±5 ） % （ n = 5 ）, the specific activity was up to 1. 850×10^6 Bq/μg, and the radioehemieal purity was above 96% after purification. The absolute accumulation of ^99mTc, whether on antisense or sense molecular probe, was clearly higher with liposome-mediated than without liposome-mediated （ P 〈 0. 05 ）. Furthermore, liposome advanced the peak time of cellular uptake of antisense molecular probe, with the highest accumulation occurring at the end of 2 h, and remaining at the same level till 3 h later. In comparison with sense molecular probe, antisense molecular probe preserved the capacity to bind living hTERT-expressing cells specifically and inhibited the expression of hTERT mRNA significantly as well as ASON. Conclusion： Liposome-mediated method could increase cell uptake of molecular probes in vitro. Antisense molecular probe preserved the capacity to inhibit the expression of targeting mRNA specifically. All results provided the basis for further in vivo study.