内皮祖细胞诱导肝细胞肝癌上皮间质转化及相关分子机制研究

Endothelial progenitor cells (EPCs) play critical roles during tumor progression, metastasis, and recurrence, which support the idea that tumor biology is introduced directly or indirectly by EPCs. Our previous investigations showed that endothelial progenitor cells were mobilized and recruited into microenvironment in hepatocellular carcinoma (HCC) during tumor progression and metastasis, and were relevant to early recurrence of HCC. However, the exact mechanism remains unclear. The tumor microenvironment plays crucial role in facilitating cancer metastasis and may induce the occurrence of EMT in tumor cells. Given the close interaction between EPCs and cancer cells in the tumor environment, we asked whether EPCs might play critical roles in EMT in tumor cells or not. Our further investigations showed: 1, some cytokines relevant to EMT are secreted from EPCs; 2, the mobility of HCC cells is promoted and mesenchyme surfaces are up-regulated when HCC cells are co-cultured with EPCs. It is assumed that EPCs induce EMT of HCC cells, and promote HCC metastasis. The followings will be investigated in vitro and in vivo: 1, the surface markers and their relevant pathways of EMT and biological characters of HCC cells, co-cultured with EPCs, will be detected in vitro and in vivo. 2, EMT in HCC cells, co-cultured with EPCs under tumor microenvironment, will be evaluated. 3, the key proteins and their signal pathways of HCC cells during EMT induced by EPCs, will be validated in vivo. We are the first to testify that EPCs induce EMT in HCC cells, and promote HCC metastasis, which will be useful to filter the molecular targets for HCC therapies.

内皮祖细胞（EPCs）在肿瘤转移与复发过程中扮演重要角色，说明EPCs直接或间接影响肿瘤生物学行为。我们先后证实EPCs参与构建肝癌微环境，促进肝癌发展与转移，且与其早期复发相关，但确切机理不明。在肿瘤进程中，肿瘤微环境诱导肿瘤细胞上皮间质转化（EMT），促进肿瘤转移。因此，作为肿瘤微环境重要成员，EPCs对肝癌EMT的作用值得研究。我们前期实验显示：EPCs分泌EMT相关因子，促进肝癌细胞迁移，并调节其EMT表型改变。我们假设：在肿瘤微环境中，EPCs通过诱导肝癌细胞EMT促进其转移。本课题通过体外和体内实验研究EPCs对肝癌细胞EMT及生物学行为的影响；体外模拟肿瘤微环境下EPCs对肝癌细胞EMT的作用；筛选EPCs诱导肝癌细胞EMT关键蛋白，并体外验证拮抗该类蛋白抑制肝癌转移。本研究首次阐明EPCs在肝癌细胞EMT过程中的作用及相关分子机制，为开发靶向肝癌关键分子的治疗手段打下基础。

内皮祖细胞(EPCs)在肿瘤转移与复发过程中扮演重要角色,说明EPCs直接或间接影响肿瘤生物学行为。我们先后证实EPCs参与构建肝癌微环境,促进肝癌发展与转移,且与其早期复发相关,但确切机理不明。在肿瘤进程中,肿瘤微环境诱导肿瘤细胞上皮间质转化(EMT),促进肿瘤转移。因此,作为肿瘤微环境的重要成员,EPCs对肝癌EMT的作用值得研究。我们前期实验显示:EPCs分泌EMT相关因子,促进肝癌细胞迁移,并调节其EMT表型改变。本课题试图证实：在肿瘤微环境中,EPCs通过诱导肝癌细胞EMT促进其转移。实验结果如下：.1、.体外实验结果证实EPCs可以促进肝癌细胞的增殖和迁移。流式细胞学结果提示：随着肝癌的进展，外周血单个核细胞中EPCs的比例逐渐升高。.2、.动物试验显示在肝癌小鼠肝内转移灶中EPCs整合入转移微环境。同肝叶EPCs与肝癌细胞共移植组小鼠原发灶大小与单独移植组未见明显差异，而各共移植组转移灶数均明显多于单独移植组（21天时转移灶数量4.8±1.9 vs 4.2±1.1 vs3.1±0.3 vs 0.9±0.3，共移植组两两比较p>0.05，但与单独移植组比较p值均小于0.01），小鼠的长期生存时间的结果与转移灶数目具有相关性。而异肝叶共移植各组EPCs移植处均未见HCC定向转移，提示EPCs对HCC的转移没有趋化作用，但对肿瘤的大小和趋化转移方面未见明显影响。.3、.Transwell共培养细胞爬片免疫组织化学结果提示EPCs可以诱导人MHCC97-H肝癌细胞发生上皮/间质表型的转变。裸鼠原位肝癌模型提示，相比肝癌细胞单独移植组，共移植组HCC组织内Vimentin+细胞数显著升高，并遍及肿瘤实质，E-cadherin+细胞则明显减少，并集中HCC组织内部，而EMT关键因子TGFβ的表达则无显著性差异，均高表达于HCC内。.4、.筛选EPCs诱导肝癌EMT的关键蛋白，我门采用EMT PCR Array检测肝癌细胞，提示共培养组肝癌细胞间质表型相关基因表达明显上调，同时上皮表型相关基因下调，通过信号通路分析，EPCs可能主要通过TGFβ/BMP、Estrogen信号通路诱导肝癌发生EMT。.本研究首次阐明EPCs在肝癌细胞EMT过程中的作用及相关分子机制,为开发靶向肝癌关键分子的治疗手段打下基础。