以缺氧诱导因子为靶点研究脂氧素“再教育”肿瘤相关巨噬细胞的机制

Tumor associated macrophages (TAMs) are important component of solid tumor and also the connection between inflammation and cancer. Previously, we have confirmed that Lipoxin A4 (LXA4), a crucial endogenous pro-resolution lipid mediator in inflammation, obviously inhibited hepatocellular carcinoma growth and metastasis in H22 transplanted mice. In vitro, it was also found that LXA4 converted the polarization of macrophages to an anti-tumor type. Combining with the theory proposed recently that hypoxia and HIF activation in solid tumor could regulate the polarization of TAM, and the studies we have performed on the effect of LXA4 on tumor angiogenesis, oxidant, anti-oxidant and HIF level,we assume that "LXA4 could re-educate TAM through interfering HIF pathway and then inhibit the progress of tumor". In this study, we will apply tumor tissue from patients with hepatocellular carcinoma, H22 transplanted mice model, TAM/tumor cell co-culture model, hypoxic model, chimeras for HIF-1α or HIF-2α conditional knockout mice, and use advanced techniques like FRET and TPLMS, to systemically explore the key-point between regulation of LXA4 on HIF pathway and its re-education on TAM. The study will help us to make clear how LXA4 exert its inhibitory effect on the progression of tumor. Our research will also provide both theoretical and experimental information for clinical TAM targeting therapy on patients with tumor.

肿瘤相关巨噬细胞(TAM)是联系炎症与肿瘤的关键。我们前期已证实促炎症消退介质脂氧素(LXs)能抑制小鼠移植肝癌的生长转移；在细胞水平能促进TAM表型改变，恢复其免疫监视的功能。结合新近提出的"肿瘤内部氧浓度降低和缺氧诱导因子(HIF)激活能诱导TAM表型变化"的学说，以及申请人所在课题组对LXs与肿瘤血管新生、氧化及抗氧化状态和HIF水平的初步研究结果，我们提出"LXs能通过干预HIF途径进而再教育TAMs，发挥其抗肿瘤作用"的假设。本课题拟采用肝癌患者癌组织，小鼠肝癌细胞移植瘤模型、TAM/肝癌细胞共培养、细胞缺氧模型和HIF-1α或HIF-2α条件性基因敲除嵌合体小鼠，应用荧光共振能量转移、双光子激光扫描显微技术等先进手段，系统研究LXs对TAM内HIF通路的调控与其再教育TAM的关联点，并明确此作用与其抑制肝细胞肝癌的关系，以期为TAM为靶点的肿瘤治疗提供理论和实验依据。

在本课题资助下，我们从肝细胞肝癌患者、小鼠H22皮下移植瘤模型和多种体外培养的正常肝脏和肝癌细胞等几个水平，系统研究了促炎症缓解介质脂氧素(Lipoxin A4, LXA4)对肝癌发展的影响，尤其是缺氧诱导因子-1 α (Hypoxia-inducible factor-1 α )在此过程中的作用。.我们的结果主要发现：1)Lipoxin A4 能通过调节肿瘤相关巨噬细胞对肝癌microRNA的表达谱发生影响, 经过LPS活化巨噬细胞上清液作用24 h的HepG2细胞有35个miRNAs上调、130个miRNAs下调；加入LXA4以后的活化巨噬细胞上清液与之相比， HepG2细胞有185个miRNAs上调、71个miRNAs下调。2) LXA4抑制缺氧状态下肝癌的增殖，在接种H22细胞的7到15天内，小鼠皮下可见清晰肿瘤包块生长；在各相应时间点，LXA4治疗组的肿瘤体积均明显小于模型组，同时LXA4明显降低了肿瘤组织内部Ki-67 和HIF-1a 表达，提示肿瘤组织的缺氧和增殖同时下降。体外培养亦证实200nM LXA4明显抑制了缺氧条件下HepG2细胞的增殖。3) 缺氧和LXA4对肝癌代谢组学的影响。针对常氧、缺氧和缺氧+脂氧素等状态下培养的肝癌细胞，进行全细胞代谢组学检测，找到几种与肿瘤生长和能量代谢相关的因子的变化，正在研究其中几个具体因子的变化机制及意义。4) LXA4对肝癌糖代谢的影响：发现LXA4能抑制H22皮下移植瘤小鼠的肝癌生长，并减少其葡萄糖摄取；LXA4能抑制肝癌细胞内上调的 HIF-1α和葡萄糖转运体表达；LXA4 能抑制转染高表达HIF-1α的腺病毒的肝癌细胞的HIF-1α高表达同时抑制其肿瘤生长。4) 肝癌患者几种HIF-1 羟化酶的变化：组织芯片和RT-PCR显示肝癌组织中PHD3和FIH的水平明显低于正常肝组织，统计分析显示，低PHD3或者FIH的表达同时与较大的肿瘤、不完整的肿瘤包裹、血管侵犯、更高的TNM分期、BCLC分期、血管密度或者更高的增殖相关；同时与患者更短的无病生存时间、更短的总体生存时间、更高的复发率相关。肿瘤内PHD3和FIH同时低表达的患者，其肿瘤包裹形成几率更低，TNM分期更高，总体生存率更低，复发率更高。多因素方差分析显示FIH的低表达是肝癌的独立预后指标。结论：LXA4能抑制肝癌的发展。