转基因斑马鱼体内的鼻咽癌移植瘤模型的建立及应用

Nasopharyngeal carcinoma (NPC) exhibits highly invasive and metastatic biological characteristics. However, the mechanism remains unclear. To date, the in vivo models can be used for studying the underlying mechanisms of NPC metastasis are still confined to traditional mammals. Previously, we observed the extravasation of the single micro-injected NPC cell from distal vasculature in transgenic zebrafishes. However, how NPC cells invade into the nearby vessels during the early stage of metastasis is still unknown. The proposed study aims to establish a xenograft model in transgenic zebrafishes by transplanting NPC cells into the peritoneal cavity, investigate the xenograft growth and tumorous angiogenesis, and observe the vasculature-invasion malignant behavior of NPC cells at a single cell level. Store-operated Ca2+ entry (SOCE) has been demonstrated to actively participate in the progression of NPC. Utilizing the newly established zebrafish xenograft model, the in vivo effects of SKF96365, a blocker of SOCE, on the xenograft growth, tumorous angiogenesis and the vasculature-invasion during the early stage of metastasis are examined. The proposed study would offer a powerful means to clarify the mechanisms of NPC metastasis at the early stage through the establishment of a novel in vivo model, and further determine the effects of the candidate drug on the early-stage metastasis in NPC.

鼻咽癌具有高侵袭转移的生物学特性但形成机制尚未清晰。目前可用于鼻咽癌转移机制研究的在体模型只局限于哺乳动物。我们前期在转基因斑马鱼体内观察到显微注射进入循环的单个鼻咽癌细胞在远端血管的翻出行为，但鼻咽癌细胞在转移早期如何侵入周围血管尚未明。本课题拟通过腹腔种植方式在斑马鱼体内建立鼻咽癌移植瘤模型，观察移植瘤的生长及肿瘤相关血管新生，在单细胞水平上记录鼻咽癌细胞血管侵袭的恶性行为。钙池操纵钙内流(Store-operated Ca2+ entry, SOCE) 已被我们证实积极地参与了鼻咽癌的进展，本研究拟进一步在斑马鱼移植瘤模型上检验SOCE阻断剂SKF96365对鼻咽癌移植瘤的生长，血管生成及在转移早期对血管的侵犯等行为的影响。本研究通过建立全新的斑马鱼体内鼻咽癌研究模型，为在细胞水平上阐明鼻咽癌的早期转移机制提供有力的研究手段，并应用该模型验证抗鼻咽癌早期血行转移的候选药物的在体效果。

鼻咽癌是中国南方最常见的头颈部恶性肿瘤，具有显著的地域集中性，在广西普遍高发。鼻咽癌发病机制复杂，与遗传，EB病毒感染及环境因素均密切相关。鼻咽癌具有明显的局部淋巴结侵袭及远处转移倾向。然而，鼻咽癌细胞的生物学行为，特别是高侵袭转移的恶性细胞行为形成机制尚未清晰。在传统的哺乳动物如裸鼠上研究鼻咽癌的恶性行为获得的信息很有限, 往往只能对某一个或几个连续的时间点进行截面评价分析，缺乏真正的动态观察，尤其是在单细胞水平上的动态观察。建立新型的鼻咽癌在体研究模型，不仅可进一步揭示鼻咽癌发生发展的潜在机制，还可利用此模型探索治疗鼻咽癌的新对策新方法。在本研究中，我们通过构建转基因斑马鱼鼻咽癌异体移植瘤模型，在单细胞水平上实现对体内鼻咽癌细胞侵袭的动态观察。通过研究我们发现：（1）钙池操控钙内流(SOCE)的药理阻断剂SKF96365通过抑制EGF激发的钙信号传导，引起鼻咽癌细胞凋亡增加、细胞周期停滞、克隆形成能力降低、及异体瘤增殖的抑制；（2）SOCE的特异性阻断剂2-APB通过拮抗EB病毒感染所重塑的钙信号，抑制EB病毒感染鼻咽癌细胞的促肿瘤血管生成能力；（3）通过利用转基因斑马鱼移植瘤模型，证实了沉默SOCE的关键蛋白STIM1后可明显抑制鼻咽癌移植瘤的局部转移扩散。本项目的实施为研究鼻咽癌的侵袭转移提供了崭新的动物体内模型，同时进一步揭开了EBV如何参与鼻咽癌细胞转移侵袭恶性生物学行为形成的潜在机制。