CAJ | 학술논문

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。
배경여목적：연구표명，약유초과85%적악성종류고표체단립매활성。인차，단립매이성위종류진단화치료연구영역적열점지일。목전，이방사성핵소표기인단립매역전록매반의과핵감산(human telomerase reverse transcriptase antisense oligonucleotide，hTERT ASON)대고표체hTERT적악성종류진행파향현상재국내외야유상관보도，단핵의학도상재해부화공간분변솔상존재착불족，난이획득고질량적도상。인차，본연구의이자공진성상방법대활체내종류단립매적진행검측，병대기진행가행성평개。방법：이DOTA(1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid)위오합제대전련류대수식적hTERT ASON(3’말단련접일개백안)진행Gd3+표기제비반의탐침병진행체외실험，이99mTc표기적DOTA-hTERT ASON생물분포실험；건립인흑색소류A375라서모형，분별우복강주사전급주사후0.5、1、2、4、6、24 h재7.0T Magnetic Resonance Imaging(MRI)하행T1WI현상，이종류급기주위정상조직작위감흥취구(region of interest，ROI)계산신조비(signal to noise ratio，SNR)병여Gd-DTPA진행비교；현상후48 h처사소서，이면역조직화학방법검측종류조직단립매활성。결과：Gd-DOTA-hTERT ASON적표기솔약위65%，재37℃신선인혈청중온육24 h후，탐침적강해솔저우3%；A375세포대탐침적섭취약위8.5%，Gd-DOTA-hTERT ASON전염적A375세포신호강도명현고우Gd-DTPA화DOTA-hTERT ASON조；종류재주사반의탐침화Gd-DTPA균표현위명현강화，반의탐침조SNR최고가체2.37，최대강화시간위주사후2~6 h，차가피DOTA-hTERT ASON소억제(P＜0.05)，이Gd-DTPA조강화시간위주사후2 h；Gd-DOTA-hTERT ASON가억제종류단립매활성。결론：Gd-DOTA-hTERT ASON현시료량호적종류파향성，대단립매양성표체적종류구유잠재적정성진단개치。
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.