177Lu-DOTA-anti-CITED1-PNA的制备、鉴定及其对甲状腺乳头状癌K1细胞增殖能力的影响

Preparation and identification of 177Lu-labeled DOTA-anti-CITED1-PNA and its effect on proliferation of human papillary thyroid cancer K1 cells

目的研究放射性镥核素(^177Lu)标记的结合转化激活因子(Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 1,CITED1)mRNA反义肽核酸(antisense peptide nucleic acid,asPNA)即^177Lu-DOTA-anti-CITED1-PNA的制备,鉴定标记物的理化性质并探讨其在人甲状腺乳头状癌K1细胞系中的摄取能力及细胞毒性作用。方法实时荧光定量PCR(RT-PCR)鉴定肽核酸(peptide nucleic acid,PNA)的靶向特异性。通过间接标记法得到反义探针^177Lu-DOTA-anti-CITED1-PNA并测定其标记率,检测其体内外的稳定性。体外细胞实验测定反义探针的摄取能力及对K1细胞增殖能力影响。结果 RT-PCR显示反义肽核酸作用于K1细胞后能明显降低CITED1的表达;反义探针的放射化学纯度为(94.5±0.12)%,比活度为(8.7±0.53)MBq/μg,48 h的放射化学纯度仍大于90%;体外细胞摄取、滞留以及细胞增殖实验显示反义探针能被人K1细胞特异性摄取,且相较于未偶联的^177Lu和asPNA,^177Lu和asPNA偶联后联用具有显著的抗肿瘤细胞作用。结论成功制备了^177Lu标记的CITED1mRNA反义肽核酸探针,证实反义探针对K1细胞具有特异性、靶向性且反义探针的联合作用能有效降低细胞活力及增殖能力。

Objective To prepare and characterize ^177Lu-DOTA-anti-CIITED1-PNA, a ^177Lu-labled antisense probe for the antisense peptide nucleic acid(asPNA) of Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 1(CITED1) mRNA and assess its uptake and cytotoxicity in human papillary thyroid cancer K1 cells. Methods The targeting specificity of the peptide nucleic acid was identified by RT-PCR. The antisense probe ^177Lu-DOTA-anti-CITED1-PNA was obtained using an indirect labeling method, and the labeling rate and in vitro and in vivo stability of the probe were assessed. The uptake of the antisense probe and its effect on the proliferation of K1 cells in vitro were evaluated. Results RT-PCR showed that the asPNA could significantly reduce the expression of CITED1 in K1 cells. The prepared antisense probe had a radiochemical purity of(94.5±0.12)% with a specific activity of 3.7±0.53 MBq/μg;its radiochemical purity was still above 90% after 48 h. This antisense probe could be specifically taken up by human K1 cells, and compared with ^177Lu-labeled DOTA and a nonsense-PNA, the antisense probe ^177Lu-DOTA-anti-CITED1-PNA showed a much stronger anti-tumor effect in K1 cells. Conclusion We successfully obtained ^177Lu-labeled CITED1 asPNA probe, which can specifically target K1 cells and effectively reduce the cell viability and proliferation in vitro.