基于乳粘素类似物的PS靶向性探针18F- (cLac2-C)2的合成及肿瘤凋亡分子成像研究

Induction of apoptosis is the primary mechnasim through which most anti-tumor therapies ,especially molecular targeting therapy, cause tumor cell death, so assessment of apoptosis in vivo is very important and meaningful. 99mTc-Annexin V is the most intensively studied apoptosis detection probe in clinical trial. It is limited for further clinical use because of its suboptimal pharmacokinetics ,micro molar concentration Ca2+ dependent and enhanced binding to membranes containing phosphatidylethanolamine，which result in false positive detection. However, to date, the goal of imaging the apoptosis process in vivo as part of routine clinical practice has not been achieved. It has been widely proven that Bovine lactadherin holds better phosphatidylserine (PS) binding characterizations than Annexin V. In preliminary study, we synthesized a cyclic peptide cLac2-C according to the key residues of the PS-targeting C2 domain of lactadherin and labeled with Cy5.5 and Cu64. We further showed that Cy5.5 and Cu64 labeled cLac2-C are specific PS targeting, with better selectivity, sensitivity ,accuracy for apoptotic cells binding and reasonable biodistribution characterization.The novel probes can detect early apoptosis e?ectively in vivo. The principal objective of the present study is to further optimize and modify the structure of cLac2-C to multimeric (cLac2-C)2 to synthesize 18F- (cLac2-C)2 and get a novel Ca2+ independent probe for PET apoptosis imaging. We will evaluate its efficacy to monitor tumor chemotherapy response comparison with 18F-Annexin V,too. Ultimately the ability to detect apoptosis noninvasively is essential to better understanding the progress of tumor formation and development and monitoring the early response of tumor therapy. Moreover, such imaging studies would help to facilitate the clinical translation of apoptosis imaging in the near future.

诱导细胞发生凋亡是肿瘤治疗，尤其是分子靶向治疗的重要分子机制，所以活体检测细胞凋亡意义重大。目前临床尝试应用的99mTc-膜联蛋白V检测方法存在诸多缺欠：①钙离子依赖；②生物学分布特性不佳；③可与磷脂酰乙醇胺结合而出现假阳性等等 ，故寻找一种理想的临床精确检测肿瘤细胞凋亡的新方法已成为重大科研命题。前期研究表明：乳粘素与凋亡蛋白磷脂酰丝氨酸（PS）结合的特性明显优于膜联蛋白V。预实验合成了乳粘素类似物cLac2-C，并构建了PS靶向的小分子探针Cy5.5-cLac2-C和64Cu-cLac2-C，结果表明其靶向性和特异性很高，体内显像效果非常好。本项目拟通过"二聚化"技术进一步优化并合成18F-（cLac2-C）2，建立一种非钙离子依赖、生物学分布特性更佳，更准确的PET凋亡成像方法。这将为深入研究肿瘤发生发展机制、早期检测肿瘤治疗疗效提供新的方法，并将在凋亡成像临床转化方面实现重大突破。

诱导细胞发生凋亡是肿瘤治疗，尤其是分子靶向治疗的重要分子机制，所以活体检测细胞凋亡意义重大。目前临床尝试应用的99mTc-膜联蛋白V检测方法存在诸多缺欠：①钙离子依赖；②生物学分布特性不佳；③可与磷脂酰乙醇胺结合而出现假阳性等等 ，故寻找一种理想的临床精确检测细胞凋亡的新方法已成为重大科研命题。前期研究表明：乳粘素与凋亡蛋白磷脂酰丝氨酸（PS）结合的特性明显优于膜联蛋白V。.本项目的目的是基于乳粘素C末端结构域，构建一种PS靶向的正电子发射断层成像（PET）小分子探针，建立一种非钙离子依赖、生物学分布特性更佳，更准确的PET凋亡成像方法。.本项目成功合成了乳粘素羧基末端（C2结构域）类似物二聚体（cLac2-C）2，并应用近红外荧光染料和放射性同位素64铜标记，合成了探针Cy5.5-（cLac2-C）2和64Cu-NOTA-(cLac2-C)2，并在细胞、离体和活体水平检测了其对PS的靶向特异性。结果表明其靶向性和特异性很高。本项目建立了Hela细胞裸鼠皮下移植瘤模型，应用喜树碱对肿瘤进行化学治疗，建立稳定的肿瘤细胞凋亡模型，PBS治疗为对照组。应用近红外荧光分子成像技术，活体检测肿瘤对喜树碱治疗的反应。Cy5.5–(cLac-2-C)2近红外荧光成像结果显示：肿瘤治疗后，探针在喜树碱治疗4周后的肿瘤部位高浓度聚集（探针注射后3小时，3h），而在PBS治疗的对照组则未见浓聚。应用64Cu-NOTA-(cLac-2-C)2 PET/CT成像，结果显示肿瘤治疗后，探针在喜树碱治疗后1h的肿瘤部位高浓度聚集，阻断组（大量未标记的cLac-2-C），肿瘤部位未见探针聚集，而在PBS治疗的对照组也未见探针浓聚。以上结果表明，64Cu-NOTA-(cLac-2-C)2探针对PS 有很高的特异性、亲和力、对于肿瘤治疗疗效的早期检测具有很高的敏感性，探针活体生物学分布特征理想，64Cu-NOTA-(cLac-2-C)2可作为一种新型的凋亡成像方法。这将为深入研究肿瘤发生发展机制、早期检测肿瘤治疗疗效提供新的方法，并将在凋亡成像临床转化方面实现了突破。

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