Gd-DOTA-hTERT ASON磁共振肿瘤端粒酶靶向显像实验研究

摘要

Background and purpose:Researches had indicated that about over 85%of malignant tumors highly express telomerase activity. So telomerase has become one of the important methods in the research field of tumor diagnosis and treatment. Nowadays, several reports about malignant tumor which over expresses hTERT targeting imaging with radionuclide labeled hTERT ASON had been published. In these reports, high quality of pictures can hardly be acquired because of poor anatomical and spacial resolution in nuclear imaging itself. Accordingly, in this study, we developed a method of detecting human telomerase in vivo with magnetic resonance imaging (MRI) and evaluate its feasibility. Methods:Firstly, Uniformly phosphorothioate-modified human telomerase reverse transcriptase antisense oligonucleotide (hTERT ASON) was labeled with Gd3+ through the bifunctional chelator 1, 4, 7, 10-tetraazacyclododecane-N, N’, N’’, N’’’-tetraacetic acid (DOTA) and iv vitro experiments were performed to characterize the antisense probes (for biodistribution and cellular uptake, 99mTc-DOTA-ASON was used in stead of Gd-DOTA-ASON). Then Gd-DOTA-ASON was injected intraperitoneally in pulmonary adenocarcinoma A375 nude mice tumor-bearing BALB/c for in vivo imaging using 7.0 T Micro MRI periodically, tumors and their surrounding tissues were defined as region of interest (ROI) to calculate the signal to noise ratio (SNR) of tumor to muscle using Gd-DTPA as control. Finally, immunohistochemical analysis of telomerase activity of each xenograft was operated 2 days after imaging. Results:The binding efficiency of Gd-DOTA-ASON reached was as high as 65%(63.2±2.4, n=6). And it can maintain 61%in fresh human serum and normal saline at 37℃over 24 h;A375 cells showed an uptake of 8.5%when incubated with 99mTc-DOTA-ASON;In comparing with DOTA-ASON and Gd-DTPA, cells transected with Gd-DOTA-ASON had higher SI when performed MRI with T1WI. The hTERT-expressing xenografts were obviously enhanced by Gd-DOTA-ASON at 0.5-6 h after injection and the SNR can reach 2.37, whereas obvious enhancement only could be found within 2 h after injection of Gd-DTPA. Both labeled and non-labeled antisense probes can suppress the activity of telomerase of A375 cells either in vitro or in vitro. Conclusion:Our research offers proof that Gd-DOTA-ASON can be used as tumor specific targeting MR probe for diagnosing malignant tumors with high expression of telomerase.%背景与目的：研究表明，约有超过85%的恶性肿瘤高表达端粒酶活性。因此，端粒酶已成为肿瘤诊断和治疗研究领域的热点之一。目前，以放射性核素标记人端粒酶逆转录酶反义寡核苷酸(human telomerase reverse transcriptase antisense oligonucleotide，hTERT ASON)对高表达hTERT的恶性肿瘤进行靶向显像在国内外也有相关报道，但核医学图像在解剖和空间分辨率上存在着不足，难以获得高质量的图像。因此，本研究拟以磁共振成像方法对活体内肿瘤端粒酶的进行检测，并对其进行可行性评价。方法：以DOTA(1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid)为螯合剂对全链硫代修饰的hTERT ASON(3’末端连接一个伯氨)进行Gd3+标记制备反义探针并进行体外实验，以99mTc标记的DOTA-hTERT ASON生物分布实验；建立人黑色素瘤A375裸鼠模型，分别于腹腔注射前及注射后0.5、1、2、4、6、24 h在7.0T Magnetic Resonance Imaging(MRI)下行T1WI显像，以肿瘤及其周围正常组织作为感兴趣区(region of interest，ROI)计算信噪比(signal to noise ratio，SNR)并与Gd-DTPA进行比较；显像后48 h处死小鼠，以免疫组织化学方法检测肿瘤组织端粒酶活性。结果：Gd-DOTA-hTERT ASON的标记率约为65%，在37℃新鲜人血清中温育24 h后，探针的降解率低于3%；A375细胞对探针的摄取约为8.5%，Gd-DOTA-hTERT ASON转染的A375细胞信号强度明显高于Gd-DTPA和DOTA-hTERT ASON组；肿瘤在注射反义探针和Gd-DTPA均表现为明显强化，反义探针组SNR最高可达2.37，最大强化时间为注射后2~6 h，且可被DOTA-hTERT ASON所抑制(P＜0.05)，而Gd-DTPA组强化时间为注射后2 h；Gd-DOTA-hTERT ASON可抑制肿瘤端粒酶活性。结论：Gd-DOTA-hTERT ASON显示了良好的肿瘤靶向性，对端粒酶阳性表达的肿瘤具有潜在的定性诊断价值。