基于分子影像技术的结外NK/T细胞淋巴瘤肿瘤干细胞侵袭机制的研究

Extranodal natural killer/T-cell lymphoma, nasal type, is most common extranodal non-B-cell lymphoma associated with aggressive characteristics. Recently, the incidence of the disease is increasing, and the invasive mechanism is still unclear. Our previous works demostrated that CD34+CD38+ phenotypic tumor cells have the features of cancer stem cells, and the expression of CXCL9, CXCL11, CXCR3 and MMP-9 in the tumor were observed using microarray analysis and immunohistochemistry methods. Based on our previously works, we suggest that chemotaxis of CXCL9, CXCL11 and their ligand CXCR3, and MMP-9 are the two independent pathways for invasion and metastasis, they may play important roles for invasion and metastasis by acting on cancer stem cells. In present study, we established a natural kill/T-cell lymphoma cell lines stably expressing luciferase, and will develop a nude mouse model using the cell lines, and will establish two nanometer fluorescence probes bonding anti-CXCR3 and anti-CD34, respectively. Furthermore, using bioluminescent living imaging, we try to clarify the mechanism of CXCR3 and MMP-9 for invasion and metastasis in the tumor by invasion inhibition experiments in vitro and in vivo, and reveal the relationship between the two genes and cancer stem cells. The present study maybe provide novel targets for treatment of extranodal natural killer/T-cell lymphoma, nasal type.

结外鼻型NK/T细胞淋巴瘤是我国最常见的淋巴结外非B细胞淋巴瘤，是最具侵袭性的恶性肿瘤之一。然而，目前对其侵袭机制的了解甚少。我们前期的研究证实该肿瘤CD34+CD38+瘤细胞具有肿瘤干细胞的特性，基因芯片检测发现CXCR3/CXCL9/CXCL11趋化轴和MMP-9在该肿瘤的特异表达，并进行验证。基于以上研究，假设CXCR3/CXCL9/CXCL11趋化轴和MMP-9在NK/T细胞淋巴瘤是两条独立的侵袭途径，主要通过作用于肿瘤干细胞而发挥促进侵袭转移的作用。本课题拟构建稳定高表达荧光素酶的NK/T细胞淋巴瘤细胞株，建立裸鼠动物模型，制备抗CXCR3和CD34的荧光纳米探针，借助于分子成像手段，通过CXCR3抑制剂和MMP-9抑制剂的体内和体外侵袭转移实验，阐明CXCR3和MMP-9在该肿瘤侵袭转移过程中的作用，以及与肿瘤干细胞之间的关系，进而为该肿瘤的靶向治疗提供理论依据。

本课题通过将Luciferase基因稳定转染至结外NK/T细胞淋巴瘤细胞株（NK92和SNK6）达到实时观察裸鼠肿瘤模型瘤细胞生长与扩散的目的，再通过纳米探针制作并与特异性抗体偶联（CXCR3和CD34），能够在裸鼠肿瘤模型进行活体成像，实时监测特殊表型的肿瘤细胞群在肿瘤生长过程中的分布与扩散特征。.同时，通过流式细胞术和细胞免疫荧光检测，印证了CXCR3和CD34纳米探针能够成功地标记NK/T细胞淋巴瘤的瘤细胞，表明CXCR3细胞亚群中包含肿瘤干细胞样细胞。进一步的体外趋化实验和侵袭实验证实，趋化因子CXCL9和CXCL11对表达其配体CXCR3的瘤细胞具有诱导作用，该作用可以被CXCR3抑制剂（NBI-74330）所阻断；裸鼠肿瘤模型体内实验表明，CXCR3抑制剂和MMP9抑制剂（SB-3CT）均能够抑制肿瘤细胞的生长和扩散；纳米探针成像表明，在CXCR3抑制剂作用下，CXCR3阳性细胞群分布面积减少，主要聚集于肿瘤周边区；Western blot和real-time PCR实验分别检测CXCR3阳性区组织和其周边区域组织的CXCR3表达水平，证实CXCR3阳性区域高水平表达CXCR3基因和蛋白。.结论：通过本课题的研究工作，成功地将分子影像学观察手段和纳米探针技术应用于肿瘤生物学行为研究，证实趋化因子轴CXCR3/CXCL9/CXCL11在结外NK/T细胞淋巴瘤增殖和扩散过程中的促进作用，并证实MMP9对该肿瘤的侵袭有明显促进作用。