Quantitation of CXCR4 expression in myocardial infarction using 99mTc-labeled SDF-1α
Quantitation of CXCR4 expression in myocardial infarction using 99mTc-labeled SDF-1α
复制标题
DOI：
10.2967/jnumed.107.050054
复制
发表时间：
2008-06-01
影响因子：
9.3
通讯作者：
Frangioni, John V.
中科院分区：
文献类型：
--
作者：
Misra, Preeti;Lebeche, Djarnel;Frangioni, John V.
趋化因子基质细胞衍生因子 -1α（SDF -1α，CXCL12）及其受体CXCR4被认为是造血干细胞滞留、癌症转移和艾滋病感染的关键介质。它们在心肌梗死（MI）中的作用尚不明确。CXCR4表达的体内无创定量对于理解其在这些不同过程以及心脏对损伤的反应中的重要性至关重要。
方法：在非质子条件下对重组SDF -1α进行放射性标记，并使用通过固相预载制备的高比活度Tc -99m - S -乙酰巯基乙酰三丝氨酸 - N -羟基琥珀酰亚胺（[Tc -99m - MAS(3)] - NHS）通过凝胶过滤色谱（GFC）进行纯化。通过GFC对放射性示踪剂在恶劣条件下的稳定性和金属转移进行定量。使用高通量活细胞结合试验，对在不表达细胞上腺病毒表达的CXCR4以及大鼠新生心肌细胞上的内源性受体，定量其亲和力、特异性和最大结合位点数量（B - max）。在实验性诱导心肌梗死前后，对Sprague - Dawley大鼠静脉注射[Tc -99m - MAS(3)] - SDF -1α后的血液半衰期、生物分布和清除进行定量。
结果：[Tc -99m - MAS(3)] - SDF -1α总共可在2小时内制备完成，比活度为8.0×10⁷ MBq/mmol（2166 Ci/mmol），放射化学纯度大于98%。在有和没有1 mM二硫苏糖醇的情况下煮沸5分钟后放射性示踪剂的降解以及在37℃下于100%血清中放置4小时的金属转移均可忽略不计。[Tc -99m - MAS(3)] - SDF -1α对活的大鼠新生心肌细胞表面的CXCR4表现出高特异性，亲和力为2.7 ± 0.9 nM，每个细胞的B - max为4.8×10⁴个结合位点。静脉注射后，Tc -99m标记的SDF -1α血液半衰期为25.8 ± 4.6分钟，肾脏快速清除，2小时时尸体中仅残留26.2 ± 6.1%的注射剂量，大多数器官的摄取量持续较低（<每克0.1%注射剂量），且没有血脑屏障穿透的证据。在诱导心肌梗死后，使用[Tc -99m - MAS(3)] - SDF -1α定量并通过共聚焦免疫荧光证实，心肌中CXCR4表达水平增加了5倍以上。
结论：我们描述了一种Tc -99m标记的SDF -1α放射性示踪剂，它可作为体内CXCR4表达的灵敏且特异的探针，并证明该放射性示踪剂能够定量不同生理和病理状态下CXCR4表达的变化。综上所述，现在应该能够在多种人类疾病的动物模型系统中对体内CXCR4水平进行定量。
The chemokine stromal-derived factor-1 alpha (SDF-1 alpha, CXCL12) and its receptor CXCR4 are implicated as key mediators of hematopoietic stem cell retention, cancer metastasis, and HIV infection. Their role in myocardial infarction (MI) is not as well defined. The noninvasive in vivo quantitation of CXCR4 expression is central to understanding its importance in these diverse processes as well in the cardiac response to injury. Methods: Recombinant SDF-1 alpha was radiolabeled under aprotic conditions and purified by gel-filtration chromatography (GFC) using high-specific-activity Tc-99m-S-acetylmercaptoacetyltriserine-N-hydroxysuccinimide ([Tc-99m-MAS(3)]-NHS) prepared by solid-phase preloading. Radiotracer stability and transmetallation under harsh conditions were quantified by GFC. Affinity, specificity, and maximum number of binding sites (B-max) were quantified, with adenoviral-expressed CXCR4 on nonexpressing cells and endogenous receptor on rat neonatal cardiomyocytes, using a high-throughput live-cell-binding assay. Blood half-life, biodistribution, and clearance of intravenously injected [Tc-99m-MAS(3)]-SDF-1 alpha were quantified in Sprague-Dawley rats before and after experimentally induced MI. Results: [Tc-99m-MAS(3)]-SDF-1 alpha could be prepared in 2 h total with a specific activity of 8.0 x 10(7) MBq/mmol (2,166 Ci/mmol) and a radiochemical purity greater than 98%. Degradation of the radiotracer after boiling for 5 min, with and without 1 mM dithiothreitol, and transmetallation in 100% serum at 37 degrees C for 4 h were negligible. [Tc-99m-MAS(3)]-SDF-1 alpha exhibits high specificity for CXCR4 on the surface of living rat neonatal cardiomyocytes, with an affinity of 2.7 +/- 0.9 nM and a B-max of 4.8 x 10(4) binding sites per cell. After intravenous injection, Tc-99m-labeled SDF-1 alpha displays a blood half-life of 25.8 +/- 4.6 min, rapid renal clearance with only 26.2 +/- 6.1 percentage injected dose remaining in the carcass at 2 h, consistently low uptake in most organs (< 0.1 percentage injected dose per gram), and no evidence of blood-brain barrier penetration. After MI was induced, CXCR4 expression levels in the myocardium increased more than 5-fold, as quantified using [Tc-99m-MAS(3)]-SDF-1 alpha and confirmed using confocal immunofluorescence. Conclusion: We describe a Tc-99m-labeled SDF-1 alpha a radiotracer that can be used as a sensitive and specific probe for CXCR4 expression in vivo and demonstrate that this radiotracer is able to quantify changes in CXCR4 expression under different physiologic and pathologic states. Taken together, CXCR4 levels should now be quantifiable in vivo in a variety of animal model systems of human diseases.