VMAT2在肺大细胞神经内分泌癌中的作用机制及作为PET显像靶点的实验研究

Pulmonary large cell neuroendocrine carcinoma (LCNEC) is a rare lung cancer with high malignancy and poor prognosis. The low accuracy of early diagnosis is the main factor affecting the prognosis. Molecular imaging with PET targeting to specific tumor marker could greatly promote the diagnosis and treatment of malignant tumors. Previously, we found that vesicular monoamine transporter 2 (VMAT2) was specifically highly-expressed in pulmonary LCNEC cells among several different lung cancer cells. A high and specific uptake of 18F-FP-(+)-DTBZ (18F-AV133), a reported 18F labeled VMAT2 ligand, was also observed in the lung LCNEC cell line. Furthermore, inhibition of VMAT2 suppressed the proliferation and migration ability of pulmonary LENEC cells. These results suggested that VMAT2 may play a role in the progression of pulmonary LENEC, and VMAT2 is likely a potential target for pulmonary LCNEC molecular imaging. In this study, we will further comprehensively reveal the underlying mechanism and validate the scientific feasibility of VMAT2 as an imaging target for the pulmonary LCNEC in series of lung cancer cell lines and athymic nude mouse tumor model with the techniques of both molecular biology and cell biology. Then we will systematically evaluate the potential of 18F-AV133 for pulmonary LCNEC imaging using autoradiography, microPET imaging and other nuclear medicine technologies. Our objective is aimed at revealing the role of VMAT2 and exploring the application potential of its 18F labeled ligand in pulmonary LCNEC. We hope our research could provide a new perspective for clinical PET imaging and treatment of pulmonary LCNEC.

肺大细胞神经内分泌癌（LCNEC）是恶性程度高、预后差的罕见癌症，早期诊断准确率低是影响其预后的主要因素。靶向特异性分子的PET分子影像可推动恶性肿瘤的诊断和治疗。前期，我们研究发现肺LCNEC细胞系特异性高表达II型囊泡单胺转运体(VMAT2)，且对VMAT2的示踪药物18F-AV133具有特异性高摄取，而抑制VMAT2降低了肺LCNEC细胞的增殖和迁移能力，提示VMAT2可能在肺LCNEC的发生发展中有重要作用，是其PET显像的潜在靶点。在本项目中，我们将进一步在细胞模型和裸鼠肿瘤模型中，运用分子生物学和细胞生物学技术，揭示VMAT2在肺LCNEC中的作用及分子机制，从而验证其作为肺LCNEC显像靶点的可行性；并用放射自显影和microPET显像等技术手段评价18F-AV133 PET显像在肺LCNEC影像学研究中的应用潜力，以期为肺LCNEC的临床PET显像或干预研究提供新的思路。

神经内分泌肿瘤是一种起源于神经内分泌细胞的肿瘤。基于神经内分泌细胞合成生物胺类物质或相关激素的能力，众多的PET/SPECT分子探针被开发并运用于神经内分泌肿瘤的诊断和治疗中。II 型囊泡单胺转运体（VMAT2）是主要存在于中枢神经系统中的跨膜转运蛋白，其作用是将细胞质中的单胺类物质转运到囊泡内储存。我们的研究表明，VMAT2在包括肺大细胞神经内分泌癌、嗜铬细胞瘤和胰岛素瘤等神经内分泌肿瘤中高表达，这提示我们VMAT2是神经内分泌肿瘤显像的潜在靶点。本研究中我们在细胞和裸鼠皮下肿瘤动物模型中考察了[18F]FP-(+)-DTBZ对肺大细胞神经内分泌癌、嗜铬细胞瘤和胰岛素瘤显像的潜在运用价值。我们发现肺大细胞神经内分泌癌H661细胞高表达VMAT2，且对[18F]FP-(+)-DTBZ具有高摄取。用DTBZ抑制VMAT2活性降低了H661细胞的克隆形成能力和迁移能力。由于皮下接种H661细胞在BALB/c Nude和NCG两种免疫缺陷鼠中均不能得到稳定的足够显像的皮下肿瘤。因此，我们探索了[18F]FP-(+)-DTBZ在嗜铬细胞瘤中的运用潜能。VMAT2在嗜铬细胞瘤细胞和皮下瘤中均存在表达，细胞摄取和microPET显像及其阻断实验证实PC12细胞及其皮下肿瘤对[18F]FP-(+)-DTBZ有一定的特异性摄取。此外，microPET和放射自显影的结果一致表明：胰岛素瘤细胞RINm5F的皮下肿瘤对[18F]FP-(+)-DTBZ具有特异性摄取。综上所述，本项目对[18F]FP-(+)-DTBZ在神经内分泌肿瘤显像中的运用潜能进行了积极的探索，为肺大细胞神经内分泌癌、嗜铬细胞瘤和胰岛素瘤提供了一种新的诊断或鉴别诊断思路。不仅如此，由于VMAT2与帕金森病有关，对多巴胺能神经元的存活至关重要。研究表明VMAT2基因水平的降低增加了多巴胺能神经变性的脆弱性。利用分子生物学方法和基于[18F]FP-(+)-DTBZ的microPET显像方法，系统研究了VMAT2抑制剂对1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）诱导的C57BL/6小鼠神经毒性的增强作用。