EphA1/EphrinA1通路调控EPCs归巢肝癌新生血管的分子机制研究

Endothelial progenitor cells(EPCs) can specific target tumor's neovascularization and participate in it's angiogenesis. And as a carrier and target of tumor anti-angiogenic therapy which has also made successful progress. However, the regulation mechanism of their mobilization and homing to tumor neovascularization is not yet clear. Our preliminary experiments have confirmed EphA1/EphrinA1 signal axis has participate in the process of liver cancer's angiogenesis, however, whether its involved in the regulation of EPCs homing to liver cancer's neovascularization remains to be further confirmed. The subject combined with our previous build plasmid of EphA1SiRNA, First, blocking EphA1/EphrinA1 signal axis in EPCs use EphA1SiRNA, then test the migration, adhesion, chemotaxis, and in vitro vasculogenesis capacity, followed used of cDNA microarray analysis the downstream signaling pathway protein of EphA1/EphrinA1 signal axis, and verificated by signaling pathway inhibitors, In vivo, established xenografts in nude mice via the tail vein transfusion of green fluorescent labeled EPCs to explore the impact of regulation EphA1/EphrinA1 signaling axis on EPCs homing to liver cancer's neovascularization and analysis its molecular mechanism. This subject aimed to clarify the molecular mechanism of EPCs homing to liver cancer's neovascularization and analysis its signal pathway, at the same time search the key regulatory sites of EPCs homing to liver cancer's neovascularization, Our ultimate purpose is expected to yield a new target for anti-angiogenic therapy of liver cancer.

内皮祖细胞(EPCs)可特异性的靶向肿瘤新生血管，以EPCs为载体和靶点的肿瘤抗血管生成治疗也取得了卓有成效的进展，然而调控其归巢肿瘤新生血管的机制尚未明确。我们的前期实验已证实EphA1/EphrinA1通路参于肝癌的血管生成，然而其是否参与调控EPCs归巢肝癌新生血管尚有待进一步证实。本课题结合我们前期构建的EphA1SiRNA质粒，在体外通过阻断EphA1/EphrinA1通路检测对EPCs迁移、粘附、趋化及体外血管生成能力的影响，利用基因芯片技术筛选其下游通路蛋白，并利用信号通路抑制剂进行验证；活体中通过建立裸鼠移植瘤模型，尾静脉回输绿色荧光标记的EPCs，探讨调控EphA1/EphrinA1通路对EPCs归巢肝癌新生血管的影响。旨在通过本研究明确EPCs归巢肝癌新生血管的分子机制及可能涉及的信号轴，寻找EPCs归巢肝癌新生血管的关键调控位点，有望为肝癌的抗血管生成治疗提供新靶点。

项目背景. 肝癌为典型的多血管型肿瘤，故抗血管生成治疗具有重要意义。近来的很多研究表明循环内皮祖细胞参与了肝癌新生血管的形成，然而调控其归巢肿瘤新生血管的机制尚未明确。.主要研究内容. 本课题利用EphA1SiRNA建立不同EphA1表达水平的内皮祖细胞系。调控EphA1/EphrinA1信号轴后检测对内皮祖细胞迁移、粘附及体外血管生成能力的影响，活体部分构建不同EphA1表达水平的裸鼠皮下移植瘤，经尾静脉输注荧光素酶标记的内皮祖细胞，观察调控调控EphA1/EphrinA1信号轴对内皮祖细胞向肝癌新生血管归巢的作用，并利用基因芯片技术分析其中可能涉及的下游传导通路蛋白。.重要结果、关键数据. 本研究首先成功构建了受强力霉素调控的高表达低背景的EPCsTet-On-EphA1SiRNA细胞株；强力霉素可诱导EPCsTet-On-EphA1SiRNA细胞株中EphA1mRNA表达下调。体外实验中用10ug/ml强力霉素能最大程度抑制EphA1/EphrinA1信号通路活性；10ug/ml强力霉素调控时能最有效的抑制内皮祖细胞运动及侵袭能力；活体实验显示调控内皮祖细胞中EphA1/EphrinA1通路的活性将有效抑制肝癌的血管生成及移植瘤的生长。. 另一方面，我们也发现，肝癌细胞中EphA1基因的高表达可通过旁分泌途径促进内皮祖细胞的运动、侵袭及成血管能力；EphA1在肝癌细胞中的高表达能促进趋化因子SDF-1蛋白在肿瘤微环境中的高表达，进而通过与内皮祖细胞表面的CXCR4受体结合，促进其运动、侵袭及成血管能力；进而发现PI3K和mTOR通路是EphA1促进SDF-1表达的关键信号通路；活体内肝癌细胞中EphA1/EphrinA1通路的活性是调控循环内皮祖细胞归巢肝癌新生血管的关键因素。进而通过全基因组测序及TACG数据库验证我们发现RPS15A基因是EphA1/EphrinA1通路关键的作用位点，其在肝癌细胞中的高表达与其血管生成及预后密切相关。.科学意义.本研究结果显示抑制肝癌细胞及内皮祖细胞中EphA1/EphrinA1通路将有效抑制内皮祖细胞向肝癌新生血管的的归巢并抑制肿瘤的血管生成及生长，EphA1/EphrinA1/SDF-1通路及核糖体中的RPS15A基因作为调控内皮祖细胞归巢的重要调控位点，有望为肝癌的抗血管生成治疗提供新的靶点。