纳米靶向光声层析成像特异性检测早期骨肉瘤机制研究

Osteosarcoma is a malignant tumor with high mortality. Early diagnosis is the prerequisite to improve the prognosis of patients. However, the current commonly used X-ray, CT, MRI and bone scan et al imaging methods can't reach the goal of early diagnosis. Photoacoustic tomography (PAI) is a novel technology developed in recent years which holds the advantages of simple operation, noninvasive, no radiation, easy to determine early lesion characteristics. It was the first time to be introduced into the early osteosarcoma imaging studies by our research team. We have obtained a novel imaging method for higher resolution images, suggesting that the technology is expected to become significant in the early detection of osteosarcoma. Nanoparticles as a new biomedical carrier, has been used as a photoacoustic imaging contrast enhancement agent. It can be connected to relative antibody molecules after chemically modified, thus early osteosarcoma targeting photoacoustic imaging has become possible. This study will be the first to perform UMR106 osteosarcoma cell targeting peptide screening through phage display technology. Then specific photoacoustic molecular probes will be made by connecting single wall carbon nanotubes to UMR106 targeting peptide. The targeting nanoparticles will be applied in detection of early rat osteosarcoma model through PAI. Meanwhile, the artificial synthesis of RGD will be used as a control. Finally, we will analysis the mechanisms on the enhancement of the specific contrast for UMR106 animal model in vivo trials. This research will provide a theoretical basis for the early diagnosis of osteosarcoma through PAI in clinic.

骨肉瘤是一种病死率极高的恶性肿瘤，早期诊断是改善患者预后的前提条件。目前常用的X线、CT、MRI及骨扫描等影像方法均存在不能早期明确诊断的缺陷。光声层析成像是近年来发展起来的新技术，具有操作简便、无创、无辐射、易发现早期病灶等特点，本课题组曾首次将其引入早期骨肉瘤成像研究并获取了较常规影像方法更高分辨率的图像，提示该技术有望成为早期检测骨肉瘤的新方法。纳米微粒作为新型的生物医学载体，已被用作光声成像对比增强剂，可经化学修饰连接相关抗体分子赋予靶向性，因此早期骨肉瘤靶向光声成像已成为可能。本研究将通过噬菌体展示技术进行UMR106骨肉瘤细胞的靶向肽筛查，与修饰的单壁纳米碳管结合，制成特异性光声分子探针，实现对早期大鼠骨肉瘤模型的靶向光声层析成像，同时将人工合成的RGD作为对照，通过动物体内试验阐明靶向探针的成像效应增强机制及代谢规律，为光声层析成像应用于临床早期诊断骨肉瘤提供理论基础。

骨肉瘤是最常见的恶性骨肿瘤，通常累及四肢的骨骼，并与局部复发和远处转移有着密切关系。其局限时患者5年生存率约为65%，转移的患者尽管在手术切除和积极的化疗下，生存率仍下降到约20%。尽管外科技术和化疗的不断发展，转移的骨肉瘤患者在过去的三十年间，临床预后一直未得以改善。预后和治疗方案的选择与诊断时的分期和分级是密切相关的。特异、可靠识别早期阶段的骨肉瘤组织，有望实施个性化的治疗，将有助于改善患者的长期预后。因此，临床迫切需要发展敏感的诊断技术方法，用于特定的识别早期骨肉瘤。无损光声成像技术结合了纯光学成像高选择性和纯超声成像中深穿透性的优点，克服了光散射限制，实现了对活体深层组织的高分辨、高对比度成像。基于功能纳米探针的光声分子成像极大扩宽光声成像的应用范围，可以在活体层面对病理过程进行分子水平的定性和定量研究，将为实现骨肉瘤的早期诊断提供强大的技术支持。本论文主要开展了以下工作：1. 噬菌体七肽库中以靶细胞大鼠骨肉瘤UMR-106做3轮筛查富集得到候选多肽PT6和PT7；鉴定特异性多肽对UMR-106细胞的亲和力；临床骨肉瘤微阵列标本亲和力检测特异性多肽的临床应用潜力；2. 多肽连接纳米金棒，完成特异性探针的构建并表征；3. 分析探针安全无生物毒性；4. 定性定量分析探针能成功特异性靶向结合UMR-106细胞；5. 构建裸鼠皮下UMR-106骨肉瘤早期模型同时搭建光声成像系统；6. 裸鼠静脉注射纳米靶向探针，研究特异性探针高亲和定位骨肉瘤组织。在本研究中，我们用噬菌体展示技术识别两组寡肽（PT6和PT7）并成功验证其能够特异性结合骨肉瘤细胞株 UMR-106，从而构建了特异性寡肽纳米探针用于靶向光声分子成像。相对于随机寡肽的纳米探针，PT6和PT7纳米探针拥有更优异的特异性靶向能力，并通过光声分子成像在早期骨肉瘤动物模型上实现肿瘤增强可视化。据此，高效特异性纳米探针结合光声成像，在早期准确检测骨肉瘤组织应用中蕴含巨大潜力。