TGF-β信号通路对口腔癌旁成纤维细胞募集-分化时相特征的调控机制研究

The recruitment and the differentiation of the surrounding fibroblasts in oral cancer, the two important biological behaviors, show essential for the tumor development. The preliminary study of the group leaded by the applicant found that the differentiation of the fibroblasts was inhibited when migrating. The hypotheses are first proposed that the surrounding fibroblasts appear the two-stage differentiation pattern and time phase of initial differentiation, recruitment and complete differentitiation, which might be regulated by the time phase molecular swich of TGF-β. The techniques adopted by the project include three-dimensional culture in vitro model and SCID mouse model of oral cancer, real-time image acquisition, flow cytometric bead analysis, RNA interference, immunoprecipitation and western blotting. The research involves the pattern and time phase of the recruitment and the differentiation of the surrounding fibroblasts in oral cancer, the regulatory pathway and its mechanism of the recruitment and the differentiation by TGF-β, the cross talk between the regulatory pathways of the recruitment and the differentiation and the time phase molecular swich. The project can test the hypothesis of the two-stage differentiation of the fibroblasts by TGF-β, can clarify the molecular mechanism of oral epithelial-stromal interaction, can provide experimental basis and theoretical basis for seeking the new strategies and new targets to block the development of oral cancer and so it possesses important theoretical significance and potential applications.

癌旁成纤维细胞的募集和分化是其促进肿瘤发生发展必不可少的两个重要生物学行为，申请者领导的课题组前期研究发现：成纤维细胞在迁移的同时其分化将受到抑制，并首次提出了癌旁成纤维细胞存在初始分化-募集-完全分化的"两阶段分化"时相特征且受到"TGF-β时相分子开关"调控的假说。本项目通过建立口腔癌体外三维培养模型和SCID小鼠模型，联合应用实时图像采集、激光共聚焦、流式微球分析、RNA干扰、IP-Western等生物学技术研究 （1）口腔癌旁成纤维细胞募集－分化的时相特征;（2）TGF-β刺激口腔癌旁成纤维细胞，募集和分化的信号调控通路及其分子机制；（3）癌旁成纤维细胞募集-分化信号调控通路的交互对话及"时相分子开关"；为验证"TGFβ-成纤维细胞两阶段分化"假说，阐明口腔上皮－间质交互作用的分子机制，寻找阻断口腔癌的新策略和新靶点提供实验依据和理论基础，具有重要的理论意义和广阔的应用前景。

肿瘤微环境在癌上皮细胞的增殖、分化、侵袭、转移过程中扮演着关键角色，癌旁成纤维细胞是上述微环境中最重要的间质细胞成分之一，它通过与上皮细胞间复杂的交互对话发挥了重要的促癌功能。其中，癌旁成纤维细胞的分化和募集迁移是其促进肿瘤发生发展必不可少的两个重要生物学行为。然而，有关上述分化和募集有何特点、二者之间是否存在内在联系或先后时相顺序以及TGF-β信号通路对其的调控机制是尚未解决的科学问题，也正是本课题拟研究的主要内容。.在该基金资助下，本课题组：①首先用了较多的时间和精力来建立一个适用于观察口腔癌旁成纤维细胞迁移分化行为的组织工程化模型，在摸索建立了二维模型、初期三维模型基础上，首次建立并完善了一个较成熟的三维模型；②在该模型上，研究观察了口腔癌旁成纤维细胞分化迁移的特点，明确了TGF-β1对募集、分化的调控作用；③申请人采用系统生物学技术预测得到与口腔癌旁成纤维细胞转分化密切相关的TβRIII，并进一步采用细胞和分子生物学实验证实，干预TβRIII可抑制肿瘤发展，该研究成果发表在国际肿瘤权威期刊Cancer Research上；④系统生物学技术的应用为揭开口腔癌旁成纤维细胞募集和分化的时相关系提供了新的途径，使募集分化的调控机制研究不仅局限于TGF-β相关信号通路，我们还发现：黏着斑激酶FAK和yes相关蛋白YAP对成纤维细胞的迁移特性（如细胞黏附，细胞骨架形成等）也具有一定的调控作用；⑤此外，本课题组发现：白色念珠菌对口腔癌旁成纤维细胞的分化具有刺激作用，且后者还可增强邻近癌上皮细胞的迁移能力加速癌细胞侵袭、转移。上述研究结果为阐明口腔癌旁成纤维细胞的重要生物学功能及调控机制，寻找阻断口腔癌的新策略和新靶点提供了实验依据和理论基础，具有重要的理论意义和广阔的应用前景。