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位于线粒体外膜的相对分子质量为18 000的转运蛋白(translocator protein, TSPO),曾被称为外周的苯二氮卓类受体(peripheral benzodiazepine receptor, PBR),是在进化上具有高度保守性的转运蛋白家族成员之一,主要位于肾上腺、睾丸、卵巢等细胞的线粒体外膜上,介导类固醇的合成[1]。在中枢神经系统炎症中,TSPO高表达于激活的小胶质细胞和星形胶质细胞[2]。近年的研究结果显示,在外周组织炎症中,TSPO也高表达于巨噬细胞和单核细胞[3-4],这表明TSPO可作为参与炎症反应的免疫细胞显像的靶点。目前,在TSPO显像中主要是用11C或18F标记TSPO配体行PET显像,如11C-(R)-PK11195、11C-PBR28、11C-DAA1106、18F-FEPPA、18F-PBR06和18F-DPA-714等[5],且有些已进入临床应用。与发射正电子的放射性核素11C和18F相比,发射单光子的放射性核素99Tcm具有价格较低廉,来源、运输方便,半衰期长,操作方便,使用范围较广等优点,且SPECT较PET便宜、普及,因此,研发新型99Tcm标记的TSPO靶向的分子探针具有较重要的临床价值。本研究以具有与TSPO较高亲合力的新型配体CB86[6]为母体,借助其表面的氨基偶联双功能螯合剂DTPA获得DTPA-CB86,进行99Tcm标记后获得新型探针99Tcm-DTPA-CB86,行左踝关节炎症小鼠Micro SPECT/CT显像,并研究其在体内的分布情况,现报道如下。
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经HPLC分析99Tcm-DTPA-CB86具有较高的标记率[(95.86 ±2.45)%]和放化纯度[(97.45 ± 0.69)%]。稳定性实验结果显示,99Tcm-DTPA-CB86在室温下的PBS溶液中稳定性良好,放置4 h后其标记率仍>90%。
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细胞摄取实验结果如图 1所示,小鼠RAW264.7巨噬细胞对99Tcm-DTPA-CB86的摄取率在0.5 h为(28.45 ± 1.56)%,3 h时达到最高峰[(36.45 ± 2.18)%],4 h时摄取率虽然有所下降,仍保持在较高水平[(35.63 ± 2.21)%]。在加入过量未标记的DTPA-CB86时,RAW264.7细胞对99Tcm-DTPA-CB86的摄取明显下降,且随时间的延长而逐渐下降,与未阻断组相比,细胞摄取下降差异具有统计学意义(t=6.217,P < 0.05)。细胞释放实验结果如图 2所示,RAW264.7细胞对99Tcm-DTPA-CB86的摄取随着时间的延长而减少,4.5 h时的摄取率为(33.31±2.34)%,8 h时为(19.32±2.01)%,减少了13.99%。
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尾静脉注射示踪剂后,除了左踝关节炎症组织外,99Tcm-DTPA-CB86在各组织器官中的放射性分布随着时间的延长逐渐下降(表 1)。左踝关节炎症组织的放射性分布0.5 h时为(1.33±0.16)%ID/g,3 h时为最高值(2.35 ± 0.10)%ID/g;肝脏0.5 h时为最高值(5.56 ± 0.76)%ID/g,3 h时仍有较高的摄取,为(2.14 ± 0.23)%ID/g;血液中0.5 h时为(0.78 ± 0.07)%ID/g,3 h时为(0.56 ± 0.09)%ID/g。A/B和A/M的放射性比值随时间的延长逐渐增高,0.5 h时分别为(1.71 ± 0.05)和(1.32 ± 0.43),3 h时为(4.19 ± 0.21)和(3.01 ± 0.16)。
组织或器官 注射后时间/h 0.5 1.5 3 心 1.49±0.17 0.46±0.06 0.33±0.05 肝 5.56±0.76 3.82±0.97 2.14±0.23 肺 1.51±0.14 1.80±0.33 0.68±0.16 肾 2.03±0.25 1.86±0.38 1.71±0.28 脾 1.24±0.43 0.91±0.08 0.58±0.03 胃 2.76±0.36 1.77±0.31 1.34±0.21 骨 0.51±0.16 0.49±0.09 0.41±0.07 肌肉 1.01±0.12 1.47±0.12 0.78±0.02 小肠 3.48±0.59 2.83±0.63 1.97±0.28 血液 0.78±0.07 0.68±0.08 0.56±0.09 左踝关节炎症部位 1.33±0.16 2.01±0.18 2.35±0.10 表 1 99Tcm-DTPA-CB86在左踝关节炎症小鼠体内的生物学分布[x± s(%ID/g)](n= 4)
Table 1. Biodistribution for 99Tcm-DTPA-CB86 in mice models of arthritis[x± s %ID/g)] (n = 4 for each group)
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左踝关节炎症小鼠注射99Tcm-DTPA-CB86后Micro SPECT/CT显像结果如图 3、图 4所示。未阻断组注射99Tcm-DTPA-CB86后0.5 h时左踝关节炎症部位可见放射性分布,且随时间的延长逐渐增浓,3 h时炎症部位显像最清晰(图 3),而正常组织的放射性分布随时间的延长逐渐减淡;竞争性抑制组始终未见明显的炎症部位显影(图 4)。
靶向TSPO显像剂99Tcm-DTPA-CB86的制备及其对关节炎症的SPECT/CT显像研究
Preparation and imaging of arthritis of 99Tcm-DTPA-CB86 for TSPO targeted imaging
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摘要:
目的制备99Tcm标记的转运蛋白(TSPO)配体CB86[99Tcm-DTPA(二亚乙基三胺五乙酸)-CB86],探讨其作为TSPO靶向关节炎症显像新型分子探针的可行性。 方法通过偶联双功能螯合剂制备DTPA-CB86,进行99Tcm标记,经高效液相色谱纯化,测定其放化纯度和体外稳定性。选用巨噬细胞RAW264.7进行体外细胞结合实验,测定99Tcm-DTPA-CB86的结合率和外排率。采用弗氏佐剂建立左踝关节炎症小鼠模型,对其行99Tcm-DTPA-CB86 Micro SPECT/CT显像,并观察探针的体内分布情况。采用SPSS 18.0统计软件对符合正态分布及方差齐性的数据进行t检验。 结果99Tcm-DTPA-CB86的标记率为(95.86 ±2.45)%,放化纯度为(97.45 ±0.69)%,其在室温下的磷酸盐缓冲液中的稳定性良好,放置4 h后,其标记率仍>90%。99Tcm-DTPA-CB86能与巨噬细胞RAW264.7特异性结合,3 h的摄取率达到最高峰[(36.45 ±2.18)%],在加入过量未标记的DTPA-CB86后,RAW264.7细胞对99Tcm-DTPA-CB86的摄取明显受到抑制[(10.43 ±2.01)%],与未阻断时相比差异具有统计学意义(t=6.217,P < 0.05);RAW264.7细胞对99Tcm-DTPA-CB86外排较少,摄取率从4.5 h时的(33.31 ±2.34)%到8 h时的(19.32 ±2.01)%,减少了13.99%。Micro SPECT/CT显像结果显示,99Tcm-DTPA-CB86在小鼠左踝关节炎症部位清晰可见,且能被过量的DTPA-CB86明显抑制;体内生物学分布结果表明,其具有较好的炎症靶向性;注射后3 h踝关节炎症部位的摄取仍可达(2.35 ±0.10)% ID/g。 结论99Tcm标记CB86易于制备,具有较好的理化性质及体内代谢学性质,同时具有较好的关节炎症摄取,有望发展成新的TSPO靶向SPECT关节炎症显像分子探针。 -
关键词:
- 转运蛋白 /
- 关节炎 /
- 同位素标记 /
- 锝放射性同位素 /
- 体层摄影术, 发射型计算机, 单光子 /
- 体层摄影术, X线计算机
Abstract:ObjectiveTo develope a novel radiolabeled translocator protein(TSPO) ligand CB86 targeting agent 99Tcm-diethylene-triaminepentaacetic acid (DTPA)-CB86 and evaluate its biological properties. MethodsDPTA-CB86 was prepared by coupling with a bifunctional chelating agent, and then labeled with 99Tcm to obtain 99Tcm-DTPA-CB86. The labeling efficiency, radiochemical purity, and stability were determined in vitro. In vitro cellular uptake and efflux were performed using RAW264.7 macrophage cells. Biodistribution and micro-SPECT/CT images were investigated on Freund's adjuvant-induced left arthritis in mice. SPSS 18.0 analysis software(t-test) was used to fit the normal distribution and homogeneity of variance. ResultsThe labeling yields and radiochemical purity of 99Tcm-DTPA-CB86 were (95.86±2.45)% and (97.45±0.69)%, respectively. 99Tcm-DTPA-CB86 displayed good stability, with a radiochemical purity of more than 90%, in phosphate-buffered solution(PBS) at 4 h. It also exhibited high specific TSPO binging in RAW264.7 macrophage cells in vitro. The highest uptake ratio was (36.45±2.18)% at 3 h after incubation, which then decreased significantly[(10.43 ±2.01)%; t=6.217, P < 0.05)] after adding excessive unlabeled DTPA-CB86. The difference was significant. Cell efflux analysis showed that 99Tcm-DTPA-CB86 had good cell retention by RAW264.7 cells, with only about 13.99%[decreased from (33.31±2.34)% to (19.32±2.01)% of total input radioactivity] of 99Tcm-DTPA-CB86 efflux observed during 4.5 h to 8 h of incubation. Biodistribution and SPECT/CT imaging demonstrated that the uptake of 99Tcm-DTPA-CB86 in the left arthritic ankles was significantly increased compared with that in contralateral normal ankles. Uptake in the arthritic ankles could be largely blocked by an excess of DTPA-CB86. Conclusion99Tcm-DTPA-CB86 can be readily synthesized and clearly visualize arthritis with low background, thus demonstrating its potential as a promising molecular probe targeting TSPO for arthritic SPECT imaging. -
表 1 99Tcm-DTPA-CB86在左踝关节炎症小鼠体内的生物学分布[x± s(%ID/g)](n= 4)
Table 1. Biodistribution for 99Tcm-DTPA-CB86 in mice models of arthritis[x± s %ID/g)] (n = 4 for each group)
组织或器官 注射后时间/h 0.5 1.5 3 心 1.49±0.17 0.46±0.06 0.33±0.05 肝 5.56±0.76 3.82±0.97 2.14±0.23 肺 1.51±0.14 1.80±0.33 0.68±0.16 肾 2.03±0.25 1.86±0.38 1.71±0.28 脾 1.24±0.43 0.91±0.08 0.58±0.03 胃 2.76±0.36 1.77±0.31 1.34±0.21 骨 0.51±0.16 0.49±0.09 0.41±0.07 肌肉 1.01±0.12 1.47±0.12 0.78±0.02 小肠 3.48±0.59 2.83±0.63 1.97±0.28 血液 0.78±0.07 0.68±0.08 0.56±0.09 左踝关节炎症部位 1.33±0.16 2.01±0.18 2.35±0.10 -
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