基于新型锰卟啉的凋亡成像分子影像探针的构建及其在早期预测移植瘤放疗后疗效中的意义

It is well known that the levels of early apoptosis after tumor radiotherapy is a predictor of long-term efficacy of radiotherapy. How to early and effectively image apoptosis in vivo become a hot focus. Because of its advantages in high spatial resolution, MRI has been given more and more attention in molecular imaging of apoptosis. Recently, a new type of contrast agent, Mn-TCP, has a huge potential for clinical application, because its biodistribution and metabolic characteristics in vivo is greatly similar to Gd-DTPA which has been widely used in clinical application and its T1 relaxation rate was significantly higher than Gd-DTPA .This study will use the linear short-peptide, targetedly binding the phosphatidylserine (PS) in surface of apoptotic cells, combined with Mn-TCP, a novel T1 MRI contrast agent, to synthetize a small imaging agent of apoptosis with good biocompatibility and high thermodynamic stability. And then, the built probe is to be used for verifying its specificity and sensitivity of imaging apoptosis in vivo and in vitro, as well as for detecting apoptosis in transplanted tumor after x-ray irradiation to figure out the relationship between the levels of early apoptosis in tumor after radiotherapy with the long-term efficacy. Our aim in study is to provide a novel method to early monitor and forecast long-term efficacy of radiotherapy for cancer.

目前认为，在肿瘤放疗后早期的凋亡水平是一种放疗远期疗效的预测指标。如何无创性地在体内显像凋亡成为了研究的热点。MRI因其空间分辨率高等优点，在凋亡分子成像中受到了越来越多的关注。最近，一种新型的Mn造影剂Mn-TCP，因其具有体内生物学分布和代谢特点与临床上广泛应用的Gd-DTPA具有完全相似的特点，以及其T1弛豫率较Gd-DTPA的明显提高，使其具备有巨大的临床应用潜力。本研究将采用靶向凋亡细胞表面磷脂酰丝氨酸（PS）的线性短肽与新型的磁共振T1造影剂Mn-TCP共价结合构建出生物相容性好以及热动力学稳定的小分子凋亡显像剂，接着将构建好的探针在体内、外验证其显像凋亡细胞的特异性和灵敏性，以及用于探测移植瘤x射线照射后凋亡的情况，并探究肿瘤放疗后早期凋亡水平与远期疗效的相关性，目的是为临床上提供一种早期监测和预测肿瘤放疗后的远期疗效的新方法。

目前，凋亡是恶性肿瘤治疗后引起细胞死亡的主要方式，治疗后凋亡水平变化可用于监测和预测治疗疗效，利用分子影像学技术无创显像凋亡成为了研究热点。因MRI具有高空间分辨力，其用于检测放疗后肿瘤内凋亡水平具有独特的解剖学优势，故本研究着重探讨MR凋亡分子成像早期预测、评估恶性肿瘤放疗疗效的可行性。本课题研究以靶向凋亡细胞表面磷脂酰丝氨酸（PS）的特异性结合短肽LIKKPF和新型Mn螯合剂为基础，成功构建凋亡小分子MR探针，体外3T 核磁共振仪检测Mn螯合剂的纵向弛豫率约两倍于Gd剂（7.2 vs. 3.4 mM-1S-1）;探针中的Mn螯合剂具有很强的稳定性，体内外毒性实验证实高剂量（200μM、5mM/kg）螯合Mn剂对细胞活性、增殖和体内重要脏器没有损伤；体内实验证实小分子MR探针有效与特异地对宫颈癌、结肠癌、淋巴瘤与肺癌移植瘤治疗后凋亡显像，同时在肺癌移植瘤内显像研究发现小分子螯合剂Mn探针和MnCl2体内具有相似的代谢速率，两者注射体内后均在30min左右达到成像高峰，2h后迅速从肾脏排出体外。然而，靶向探针在高水平凋亡移植瘤内成像高峰可往2h后延迟，因此选择2h左右是一个合适的特异性凋亡成像时间点；体内TUNEL检测发现靶向探针高信号组与肿瘤组织内凋亡水平呈正相关，验证了成像的特异性。本研究结果表明以线性短肽合成的凋亡MR成像小分子探针可以有效地检测放化疗后凋亡水平，为临床上预测治疗疗效提供一种无创的分子成像技术。