端粒酶靶向的RNA标记探针在肿瘤活体显像的研究

In-vivo tumor imaging by radiolabeled RNA probe targeting telomerase

目的:探讨端粒酶靶向的小干扰RNA(small interference RNA,siRNA)标记探针在肿瘤靶向活体显像中的应用价值。方法:化学合成端粒酶反转录酶(human telomerase reverse transcriptase,hTERT)靶向和人类基因无关靶向的siRNA双链。通过螯合剂NHS-MAG3的偶联作用对siRNA进行放射性核素锝[99mTc]标记。构建HepG2肝癌荷瘤裸鼠模型,以HE染色和免疫组织化学方法鉴定肿瘤组织的病理情况和hTERT的表达水平。经裸鼠尾静脉注射7.4 MBq放射性标记的siRNA,分别于注射后0.5、1、3、6 h进行显像。通过在肿瘤和对侧正常组织勾画感兴趣区(region of interest,ROI),比较肿瘤与非肿瘤(T/NT)比值。经裸鼠尾静脉注射1.85 MBq探针,分别测定并计算2、4、6 h每克肿瘤及血液的组织放射性分布(%ID/g)。使用t检验方法进行统计学评价。结果:标记率为(73.4±3.0)%,放射化学纯度大于92%,比活度达25.9 GBq/μmol。HE染色中肿瘤组织可见病理性核分裂相。hTERT抗体免疫组织化学显示褐色浓染区域主要集中于细胞核。注射hTERT靶向探针的荷瘤裸鼠显示出清晰的肿瘤影像,而对照组则无法显示肿瘤。注射后6 h,实验组探针的肿瘤影像明显强于对照组。注射后0.5、1、3、6 h后,实验组的T/NT比值由2.68±0.21升至5.86±0.30,而对照组由1.55±0.16降至1.28±0.12。注射后2、4、6h后,实验组的肿瘤放射性分布由(0.71±0.14)%ID/g增加到(0.97±0.15)%ID/g,而对照组的肿瘤分布则不断降低,提示实验组探针的靶向性。结论:端粒酶靶向的小干扰RNA标记探针具备肿瘤体内靶向显像的潜在应用价值。

Objective：To explore the value of telomerase targeted radiolabeled small interference RNA （siRNA） in tumor imaging in vivo. Methods： Human telomerase reverse transcriptase （hTERT） -targe-ted and human gene non-targeted siRNAs were chemically synthesized. Through the conjugation with the chelator N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyhriglyeine （NHS-MAG3 ）, the siRNAs were radiolabeled with technetium-99m （99m^Tc）. HE staining and immunohistochemical staining were used to identify their pathological characteristics. 7.4 MBq of 99m^Tc-siRNAs were injected via the tail vein of hepatocarcinoma bearing mice. At 0.5, 1, 3, and 6 h post injection, the mice were laid on a face-up detector and imaged by single-photon emission computed tomography （SPECT）, respectively. The ratio of radioactive counts in tumor to that in the contralateral equivalent region was calculated by drawing re-gions of interest （ROI） at each time point. After the administration of 7.4 MBq of 99m^Tc-siRNAs, the biodistrlbution （ % ID/g） of tumors and blood was measured at the end of 2, 4 and 6 h. Statistical com-parisons of the variables were performed by t-test. Results： The labeling efficiency reached 73.4% ± 3.0%. After purification, the radiochemical purity was no less than 92% and the specific ac-tivity was up to 25.9 GBq/μmol. HE staining showed pathological mitotic figure in the nucleus of the tumor ceils, hTERT immunohistochemical staining showed deep brown dyed spots in the cell nucleus.hTERT-targeted 99m^Tc-siRNA administrated xenografts showed tumor images clearly after the administra-tion, especially at 6 h. In contrast, 99m^Tc-control-siRNA showed no tumor image. The ratios of uptake in tumor to that in contralateral region of hTERT-targeted siRNA increased from 2.68 ±0.21 to 5.86 ±0.30 at 6 h, whereas those of control siRNA decreased from 1.55 ± 0.16 to 1.28 ± 0.12 （ P 〈 0.01 ）. The biodistribution of tumors in the hTERT-targeted mice increased from 0.71 ±0.14 to 0.97 ±0.15 at 6 h, whereas that in the control mice decreased. Conclusion： 99m^Tc radiolabeled telomerase-targeted siRNA probe allows for noninvasive visualization of tumor telomerase in vivo.