CXCR6介导弥漫大B细胞淋巴瘤sCXCL16自/旁分泌双重作用及分子机制研究

CXCL16/CXCR6 may affect the progress of tumor by its interaction with the microenvironment. Our previous study indicated the high expression of soluble CXCL16 (sCXCL16) and the receptor CXCR6 in tissues and cell lines of diffuse large B cell lymphoma(DLBCL) as well as the autocrine and paracrine effects of sCXCL16 possibly inducted by CXCR6. The present study aims to analyze the clinical features on the basis of verifying the expression of CXCL16 and CXCR6 firstly, then positively or negatively regulate the expression of CXCR6 by lentivirus and level of CXCL16 by endogenously using recombinant CXCL16, to observe the biological phenotypes of DLBCL cells and explore the inter-reaction between tumor cells and the macrophages by co-culturing and detecting of cytokines arrays, thus to verify the autocrine or paracrine mode conducteded by CXCR6 in vitro. Second, as the mechanism is concerned, the genome-wide expression profile chips will be used according to tumors under different conditions respectively, the differently expressed genes will be selected, the related signal pathways be analyzed and the key proteins be verified. Finally, CXCR6 knockout transgenic mouse will be applied to confirm the above phenomena and further elucidate the mechanisms in vivo. .The present study reports the autocrine and paracrine effect of sCXCL16 through its receptor CXCR6 for the first time, in which the elucidation of the molecular mechanisms would provide experimental data for the research into the chemokine-receptor inter-reaction during the inflammation, tumor and microenvironment. The related signal pathways and the key proteins noted in the present study would also provide new targets for biological therapy into diffuse large B cell lymphoma.

CXCL16及受体CXCR6可能通过构筑肿瘤微环境影响肿瘤进程，但在淋巴瘤领域研究极为有限。本组预实验发现sCXCL16(可溶性)及CXCR6在弥漫大B细胞淋巴瘤(DLBCL)高表达并可能介导自分泌和旁分泌双重作用。本项目将首先深入分析sCXCL16/CXCR6表达的临床意义；通过分别正/负调控sCXCL16和CXCR6，观察肿瘤细胞恶性表型变化以明确自分泌作用；通过共培养及细胞因子功能检测等，观察肿瘤细胞与巨噬细胞的相互作用以明确旁分泌作用。进而运用全基因组表达谱芯片，筛选自/旁分泌作用所涉及的基因，分析相关信号通路并验证关键靶蛋白。最后利用基因敲除动物模型研究自分泌及旁分泌作用的体内机制。该研究围绕炎症、肿瘤、微环境等热点领域，针对CXCL16/CXCR6功能轴率先在DLBCL展开研究，将为炎症性肿瘤的机制研究提供重要理论依据；亦将为DLBCL生物治疗提供新的分子靶点。

CXCL16及受体CXCR6与多种肿瘤关系密切，可能通过影响肿瘤微环境干预肿瘤进程，但在淋巴瘤领域研究极为有限。本项目1）在表达检测方面：通过病人组织标本和临床资料检测发现sCXCL16/CXCR6在DLBCL组织中共表达且与预后不良呈正相关；2）在自分泌方面：体外生物学实验中分别正/负调控sCXCL16和CXCR6，发现sCXCL16可显著促进DLBCL细胞的迁徙、促进其分泌TNF-α细胞因子，而对DLBCL细胞的增殖、凋亡和免疫表型等影响不显著；3）在旁分泌方面：①在DLBCL病例组织中发现M1型巨噬细胞表达CXCR6；②sCXCL16可促进由DLBCL病人单核细胞诱导的M1型巨噬细胞的迁徙；③与M1型巨噬细胞共培养可抑制DLBCL细胞增殖；④DLBCL肿瘤细胞可促进共培养的单核/巨噬细胞向M2型分化，并提高其sCXCL16和TNF-α的分泌和表达；⑤单独使用sCXCL16也可促进单核/巨噬细胞向M2型分化；4）在自分泌和旁分泌的机制方面：①运用全基因组表达谱芯片，发现外源性施加sCXCL16重组蛋白后，424个基因上调、788个基因下调；②sCXCL16通过CXCR6的自分泌作用涉及了17个通路，其中直接影响NF-κB信号通路并促进TNF-α的表达；③发现并验证了sCXCL16/CXCR6可以促进DLBCL细胞的FN14基因和蛋白的上调；④通过与TNF-α、IFN-γ、IL-3、IL-6等细胞因子的联合实验，发现sCXCL16与TNF-α及IFN-γ对DLBCL肿瘤细胞的增殖存在协同抑制作用；⑤利用基因编辑技术构建了CXCR6-/-基因敲除小鼠及鼠源性弥漫大B细胞淋巴瘤A20细胞株的Balb/c小鼠模型，为深入探索自分泌和旁分泌现象的体内机制奠定基础。.本项目针对CXCL16/CXCR6功能轴系统性的在DLBCL展开研究。产生的成果包括：发表论文6篇、获得省级项目1项、申请专利1项，建设淋巴瘤系统生物学团队1个、指导青年团队成员6名、其中2人获得国家自然科学基金；组织参加国内学术会议21人次、国际会议2人次；与南方医科大学合作培养博士生1名（已毕业）。