肿瘤微环境诱导内皮祖细胞内皮间质转化及机理研究

By a process called Endothelial-to-Mesenchymal Transition (EndoMT) endothelial cells (ECs) or endothelial progenitor cells (EPCs) convert to a more mesenchymal cell type, participate embryonic development, fibrosis, wound healing,or tumor progression. Interestingly, EndoMT contributes to the generation of cancer associated fibroblasts that are known to influence the tumor-microenvironment favorable for the tumor cells. Our and others' investigations showed that EPCs play critical roles during tumor progression, metastasis, and recurrence, and are crucial roles in tumor microenvironment. Our further investigations showed that GFP labelled EPCs trans-differentiated into fibroblasts in HCC models. EndoMT related genes or phenotypes of EPCs were up-regulated when EPCs were co-cultured with HCC cells co-culture. Some factors relevent to EndoMT, such as PDGF, TGFβ, were up-regulated in the tumore tissue and adjacent non-tumor tissue. Given the close interaction between EPCs and cells or factors in the tumor environment, we asked whether EPCs convert to mesenchyal cells under tumor microenvironment or not, which promote tumor progression. The followings will be investigated in vitro and in vivo: 1, the surface makers of EndoMT and biological characters of EPCs, co-cultured with HCC cells, CAFs, or under hypoxia, will be detected. 2, the key proteins and their signal pathways of EPCs during EndoMT, will be validated.3，EndoMT of EPCs in vivo with HCC will be confirmed; 4, The clinical correlationship between EndoMT relevent indices and the biological characters of HCC, will be analyzed. We are the first to testify that EPCs convert to mesenchymal cells under tumor microenvironment, which promote HCC progression. It will be useful to filter the molecular target for HCC therapies.

内皮间质转化（EndoMT）是内皮细胞或内皮祖细胞在特定条件下分化为具有间质表型细胞的分化方式，参与血管发育、纤维化、肿瘤等病理生理过程。EndoMT是内皮细胞参与肿瘤微环境构建的重要方式。我们和其他研究证实：内皮祖细胞（EPCs）参与肿瘤发展、转移或复发，是肿瘤微环境的重要成分。我们进一步研究提示：肝癌模型内EPCs分化为成纤维细胞；与肿瘤细胞共培养，EPCs的EndoMT相关基因表达上调；肝癌及其癌旁肝组织高表达PDGF、TGF-β等EndoMT相关因子。故我们假设：在肿瘤微环境作用下，诱导EPCs发生EndoMT，转化为间质样细胞，促进肿瘤生长和转移。故我们预备明确：促进EPCs发生EndoMT的微环境因素、具体细胞因子及具体信号通路；EPCs发生EndoMT的肿瘤模型在体研究；靶向抑制EndoMT治疗肝癌小鼠肿瘤。该研究将进一步阐明EPCs参与肿瘤的作用方式和机制。

内皮间质转化（EndoMT）是内皮细胞或内皮祖细胞在特定条件下分化为具有间质表型细胞的分化方式，参与血管发育、纤维化、肿瘤等病理生理过程。EndoMT 是内皮细胞参与肿瘤微环境构建的重要方式。本课题使用Nunu裸小鼠作为载体， MHCC97-H细胞株作为肿瘤来源。我们通过文献调研及网络检索，选取了人EMT PCR Array进行分析EPCs与肝癌细胞共培养时EMT相关基因的表达谱变化情况，并在此基础上通过相关体外实验基本明确了肿瘤细胞诱导EPCs 发生EndoMT 的关键蛋白为TGFβ3、MMP3等细胞因子，并研究了TGFβ3、MMP3相关信号通路上下游蛋白的表达变化，我们进一步的研究提示TGFβ、MMP3可能通过TGFβ/BMP及Estrogen receptor信号通路参与EPCs的EndoMT。此外，我们构建原位肝癌裸小鼠模型，并将GFP-EPCs由尾静脉注入，检测EPCs 的分化和EndoMT 相关指标，研究发现EPCs可以归巢于肝癌癌巢，肿瘤边缘可检测到发生EndoMT的EPCs细胞，而肿瘤内部及正常组织内罕见EndoMT的EPCs细胞，且肝内转移灶中亦可观察到发生EndoMT的EPCs细胞。肿瘤边缘TGFβ3、MMP3的表达较内部更强、比例更高，与发生EndoMT的EPCs比例呈正相关。但EPCs 发生EndoMT与肿瘤大小、肿瘤增殖等没有显著的相关性。随后我们经尾静脉继续静脉注射TGFβ3、MMP3拮抗剂，发现实验组裸鼠在建模12周左右肿瘤的转移明显少于对照组。综上所述，我们的研究结果提示肿瘤细胞可以通过分泌TGFβ3、MMP3等细胞因子诱导EPCs 发生EndoMT转化为间质样细胞，进一步促进肿瘤生长和转移，而通过拮抗TGFβ3、MMP3可以减少肝癌小鼠肿瘤转移的发生。本研究为肝癌的抗转移复发提供又一思路和理论依据。