初步研究（99）Tc（m）-His10- Annexin V 成像紫杉醇诱导的肿瘤细胞凋亡。
A preliminary study of imaging paclitaxel-induced tumor apoptosis with (99)Tc(m)-His10-Annexin V.
机构信息
Department of Nuclear Medicine, China-Japan Friendship Hospital, Beijing 100029, China.
出版信息
Chin Med J (Engl). 2013;126(15):2928-33.
BACKGROUND
In tumors the process of apoptosis occurs over an interval of time after chemotherapy. It is important to determine the best time for detecting apoptosis by in vivo imaging. In this study, we evaluated the dynamics and feasibility of imaging non-small cell lung cancer (NSCLC) apoptosis induced by paclitaxel treatment using a (99)Tc(m)-labeled Annexin V recombinant with ten consecutive histidines (His10-Annexin V) in a mouse model.
METHODS
(99)Tc(m)-His10-Annexin V was prepared by one step direct labeling; radio-chemical purity (RCP) and radio-stability was tested. The binding of (99)Tc(m)-His10-Annexin V to apoptotic cells was validated in vitro using camptothecin-induced Jurkat cells. In vivo bio-distribution was determined in mice by dissection. The human H460 NSCLC tumor cell line (H460) tumor-bearing mice were treated with intravenous paclitaxel 24, 48 and 72 hours later. (99)Tc(m)-His10-Annexin V was injected intravenously, and planar images were acquired at 2, 4 and 6 hours post-injection on a dual-head gamma camera fitted with a pinhole collimator. Tumor-to-normal tissue ratios (T/NT) were calculated by ROI analysis and they reflected specific binding of (99)Tc(m)-His10-Annexin V. Mice were sacrificed after imaging. Caspase-3, as the apoptosis detector, was determined by flow cytometry, and DNA fragmentation was analyzed by the terminal deoxynucleotidytransferase mediated dUTP nick-end labeling (TUNEL) assay. Nonspecific accumulation of protein was estimated using bovine serum albumin (BSA). The imaging data were correlated with TUNEL-positive nuclei and caspase-3 activity.
RESULTS
(99)Tc(m)-His10-Annexin V had a RCP > 98% and high stability 2 hours after radio-labeling, and it could bind to apoptotic cells with high affinity. Bio-distribution of (99)Tc(m)-His10-Annexin V showed predominant uptake in kidney, relatively low uptake in myocardium, liver and gastrointestinal tract, and rapid clearance from blood and kidney was observed. The T/NT was significantly increased after paclitaxel treatment, whereas it was low in untreated tumors (T/NT = 1.43 ± 0.18). The %ID/g activity in Group 2 (24 hours), Group 3 (48 hours) and Group 4 (72 hours) after treatment was 2.55 ± 0.73, 3.35 ± 1.10, and 3.4 ± 0.96, respectively. Whereas in the non-treated group, Group 1, %ID/g was 1.10 ± 0.18. The radiotracer uptake was positively correlated to the apoptotic index (r = 0.852, P < 0.01), as well as caspase-3 activity (r = 0.816, P < 0.01).
CONCLUSION
This study addresses the dynamics and feasibility of imaging non-small cell lung tumor apoptosis using (99)Tc(m)- His10-Annexin V.
背景
在肿瘤中，细胞凋亡是在化疗后一段时间内发生的。因此，通过体内成像确定检测细胞凋亡的最佳时间非常重要。在这项研究中，我们使用一种（99）Tc（m）标记的带有连续十个组氨酸（His10- Annexin V）的重组 Annexin V 对紫杉醇诱导的非小细胞肺癌（NSCLC）凋亡进行了动态评估，并在小鼠模型中进行了可行性研究。
方法
（99）Tc（m）-His10-Annexin V 通过一步直接标记法制备；放射性化学纯度（RCP）和放射性稳定性通过测试得到验证。在体外使用喜树碱诱导的 Jurkat 细胞验证了（99）Tc（m）-His10-Annexin V 与凋亡细胞的结合。通过解剖确定小鼠体内的生物分布。荷人 H460 NSCLC 肿瘤细胞系（H460）的小鼠在紫杉醇静脉注射后 24、48 和 72 小时进行治疗。将（99）Tc（m）-His10-Annexin V 静脉注射，并用配备针孔准直器的双探头伽马相机在注射后 2、4 和 6 小时采集平面图像。通过 ROI 分析计算肿瘤与正常组织的比值（T/NT），反映（99）Tc（m）-His10-Annexin V 的特异性结合。成像后处死小鼠。通过流式细胞术测定凋亡探测器 caspase-3，通过末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）测定分析 DNA 片段化。使用牛血清白蛋白（BSA）估计蛋白质的非特异性积累。将成像数据与 TUNEL 阳性核和 caspase-3 活性相关联。
结果
（99）Tc（m）-His10-Annexin V 的 RCP > 98%，放射性标记后 2 小时稳定性高，与凋亡细胞具有高亲和力。（99）Tc（m）-His10-Annexin V 的生物分布显示，在肾脏中的摄取占主导地位，在心肌、肝脏和胃肠道中的摄取相对较低，在血液和肾脏中的清除速度很快。紫杉醇治疗后 T/NT 显著增加，而未经治疗的肿瘤中 T/NT 较低（T/NT = 1.43 ± 0.18）。治疗后 24 小时（Group 2）、48 小时（Group 3）和 72 小时（Group 4）的 %ID/g 活性分别为 2.55 ± 0.73、3.35 ± 1.10 和 3.4 ± 0.96，而未治疗组（Group 1）的 %ID/g 为 1.10 ± 0.18。放射性示踪剂摄取与凋亡指数呈正相关（r = 0.852，P < 0.01），与 caspase-3 活性呈正相关（r = 0.816，P < 0.01）。
结论
本研究探讨了使用（99）Tc（m）-His10-Annexin V 对非小细胞肺癌肿瘤凋亡进行体内成像的动态和可行性。
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