构建基质硬度与生长因子可控动态模型研究肝癌发展中的细胞行为和分子机制

The development of hepatocellular carcinoma (HCC) involves a series of changes in cell behaviors and functions, which are mediated by diverse factors in the extracellular matrix. However, current biological methods are difficult to present those factors (stiffness, growth factor, etc.) simultaneously and dynamically with a tunable manner. In the proposal, we will fabricate a controllable and dynamic model based on the layer-by-layer self-assembly technique, and adjust matrix stiffness by using a specific crosslinker containing the matrix metalloproteinase (MMP) sensitive peptide, and then deliver growth factor in a matrix-bound manner with tunable amounts. The early behaviors of HCC cells in the mimicking microenvironment (adhesion, morphology, migration, etc.) will be studied, to reveal the synergistic effect of different factors to influence cell performances. Matrix stiffness and growth factor binding will gradually change during cell incubation, due to crosslinking destruction by MMP releasing from HCC cells. We will also investigate the long-time behaviors of HCC cells (proliferation, epithelial-mesenchymal transition drug responses, etc.) in the dynamically changing process of microenvironment. Furthermore, hidden molecular mechanisms of cell behaviors will be discovered by studying involved surface receptors and signaling pathways. Our project will open new insights for providing a powerful platform to mimic tumor microenvironment and to build in vitro disease models, which would accelerate the studies of interactions between cancer cells and their microenvironment, as well as screening the appropriate periods for clinical therapy.

肝癌的发展是受微环境中多因素协同动态作用而产生的一系列细胞行为和功能变化的结果，但传统生物学研究方法难以实现微环境中基质硬度和生长因子等因素的精准同步呈现和动态变化。在本项目中，申请人计划利用层层自组装膜构建可控动态模型来克服这一技术瓶颈，以含基质金属蛋白酶（MMP）敏感多肽的交联剂调控基质硬度，然后携载生长因子并调控其结合量，研究两种因素协同影响肝癌细胞在模拟微环境中的早期行为（黏附、形态、迁移等）。利用肝癌细胞在培养过程中释放MMP改变基质硬度和生长因子结合量，探索微环境性质重塑过程中肝癌细胞长期行为（细胞增殖、上皮间质转化和药物响应性等）的变化。进一步明确感应微环境中不同因素刺激的表面受体介导胞内信号通路活化的分子机制，揭示微环境中多因素协同影响肝癌发展的规律。本项目成果将为模拟肿瘤微环境和构建体外疾病模型奠定基础，促进癌细胞与微环境之间相互作用的研究，为临床治疗筛选合适的干预信息。

肿瘤微环境中的基质硬度和生长因子等因素会对肝癌的发展起到重要的调控作用，但传统的生物学研究方法难以实现多因素的同步可控呈现。在本项目中，我们成功制备出硬度可控的多层生物膜，实现从肝硬化到肝癌晚期基质硬度的有效模拟。在此基础上使用化学结合法进行TGF-β1的负载和调控，生长因子的使用效率可达90%。为研究复杂微环境多因素对癌症发展的作用规律和分子机理提供了有效的技术手段。首次提出了肝癌细胞响应基质硬度的三种机械反馈方式：逐步式（适用于细胞伸展、迁移和奥沙利铂给药后的早期凋亡），阶梯式（适用于细胞黏附、增殖、黏着斑形成和耐药性），特定方式（适用于EMT过程）。硬基质+结合态TGF-β1（i-TGF）被证明是促进肝癌间质表型的最佳条件。分子机制研究显示i-TGF可以增加其特异性识别受体表达并激活部分PI3K通路，受体-配体相互作用促进癌细胞黏附，扩大细胞与基质之间的接触面积以便更好地感受基质硬度，从而导致integrinβ1表达量增加，然后通过integrinβ1/vinculin/p-FAK的途径增强力学信号转导，促进更多的PI3K磷酸化并加快EMT进程。首次提出了生物因素通过影响肝癌细胞感应微环境中的力学因素而发挥作用的分子机制，为复杂微环境中不同来源的因素如何发挥协同作用影响癌症发展提供了新的研究思路。

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