177Lu-CNP-iRGD纳米探针示踪靶向甲状腺乳头状癌淋巴结转移灶的研究

Cervical lymph node metastasis (CLNM) is commonly observed in papillary thyroid carcinoma (PTC), which might be associated with increased risk of regional recurrence and reduced rate of long-term survival. Until now, although the application of carbon nanoparticles (CNP) probes to trace cervical lymph nodes plays a vital role in guiding surgery, it was always accompanied with both false positives and false negative results. Therefore, how to establish an accurate identification and localization CLNM method in PTC is urgently needed. Our pre-study had shown high-binding-ability of 177Lu-CNP in vitro to PTC tumor cells. Therefore, based on the circular peptide iRGD that specifically targets the tumor neovascularization integrin receptor and CNP nanoparticle, and we design a novel CNP-iRGD dual-targeting multimodal nanoprobe. And then label the probe with 177Lu for both of diagnosis and therapy and.perform the in vitro and in vivo experiments. Such as the HPLC analysis, binding and blocking assays, SPECT/CT imaging, PRRT processing techniques to evaluate the specific stability and targeting. Through biodistribution, histomorphology, shrinkage rate, αvβ3 and TG protein expression, we expect to verify that the probe can accurately identify CLNM. In addition, radiation damage can induce apoptosis and necrosis of tumor tissue. Therefor we can provide a new method for the diagnosis and therapy of CLNM.

乳头状甲状腺癌（PTC）颈部淋巴结转移（CLNM）增加肿瘤复发率并降低患者长期生存率。目前纳米炭（CNP）示踪颈部淋巴结在术中起关键作用，但常伴假阳性和假阴性，因此精准识别和定位清扫CLNM仍是根治PTC亟待解决的难题。我们前期研究发现177Lu-CNP能在体外靶向PTC，据此拟用对肿瘤新生血管整合素受体具有特异性靶向的环状多肽iRGD，设计新颖的177Lu-CNP-iRGD双靶向纳米探针，利用iRGD和CNP双重靶向PTC及CLNM，再结合177Lu诊疗一体化特征，进行体内外实验。通过Radio-HPLC、结合及阻断试验、SPECT/CT显像、PRRT处理等技术，评估探针特异稳定性及靶向性；通过生物分布、组织形态、缩瘤率、αvβ3及TG表达，验证探针可精准识别CLNM，并可针对肿瘤组织进行辐射损伤诱发细胞凋亡坏死达到诊断、治疗的目的，为丰富PTC及CLNM诊治策略提供新的科学方法和思路。

甲状腺乳头状癌（PTC）颈部淋巴结转移是 PTC 患者局部复发的一项独立危险因素，可导致较差的预后。第一部分研究通过转录测序PTC细胞系TPC-1、IHH-4和BCPAP之间的差异基因。结果示PTC细胞之间的基因表达差异较大，且BCPAP细胞存在与肿瘤靶向肽相结合配体基因NRP的表达。总体生存率分析见EGF、CTGF基因在甲状腺癌患者总体生存期显著相关（P＜0.05）。结果表明同一类型的肿瘤所分化出的亚型基因差异甚大，具有重要的科学意义。在第二部分研究中，我们根据转录组蛋白组联合分析结果筛选了肿瘤靶向肽iRGD进行研究设计131I-GO-iRGDs纳米探针并观察其对TPC-1、IHH-4和BCPAP的影响。通过扫描电镜、XPS、傅里叶检测、拉曼光谱对合成的GO-iRGDs各成分进行表征。结果示GO表面成功连接iRGD。37MBq Na131I分别标记不同浓度的GO。结果表明25.6mg/ml的GO的131I标记率最高。通过震荡法把111MBq的131I装载在不同比例的GO：iRGD，结果示GO浓度为25.6mg/ml时，131I-GO100-iRGD标记率最高，为（82.00±1.63）%。体外细胞实验中131I-GO100-iRGD、131I-GO50-iRGD、131I-GO10-iRGD、131I-GO、Na131I 分别处理了TPC-1、IHH-4和BCPAP细胞2h、4h、24h、48h、72h、96h、120h后获得细胞的摄取率。综上，本研究成功构建131I-GO-iRGDs探针；GO与iRGD比例影响131I标记，进而影响探针的靶向性；131I-GO-iRGDs对PTC具有靶向性，可能与PTC细胞存在iRGD相对应的配体及GO具有趋向性有关。而测序研究为精准靶向治疗PTC奠定基础，但仍存在不足，检测细胞株获得的基因与临床差异大，本项目在接下来将用患者组织加以验证，继续研究iRGD作为肿瘤治疗靶点的可行性，为实现临床转化提供依据。