基于PTMC 3D纳米纤维支架的仿生微环境构建肝癌肿瘤干细胞的体外模型的研究

Liver cancer stem cells (LCSC) have been demonstrated to retain tumor-initiating potential and self-renewal capability which are responsible for tumorigenesis and resistances to chemotherapy and radiotherapy of malignant liver tumors. In this study, we aim to engineer cancer microenvironments for in vitro three-dimensional (3D) LCSC models using poly (trimethylene carbonate) (PTMC) 3D nanofibrous scaffolds, and to investigate the influence of microenvironments on the functions of LCSC in vitro. First, for the first time, 2D nanofiber mesh by electrospinning of PTMC is rolled up to form a stable 3D nanofibrous structure through UV crosslinking. In contrast to traditional nanofiber mesh, the fabricated 3D nanofibrous scaffolds have large pore size allowing cell infiltration within scaffolds. LSCS, isolated from a human liver cancer tissue, will be seeded in 3D nanofibrous scaffolds. To form in vitro 3D LCSC models, we will mimic the native liver cancer microenvironments including hypoxia, co-culture with MSCs, and the physical properties of scaffolds. Finally, the tumorigenicity of LCSC from in vitro 3D culture will be evaluated by using immunodeficient laboratory mice. This study will not only potentially provide a new avenue to fabricate 3D nanofibrous scaffolds but also uncover the underlying mechanism of microenvironments for engineering LCSC models in vitro. Furthermore, our approach can be extended to cancer stem cells from other tumor tissues, and can help researchers better understand CSC biology and thus develop more effective therapeutic agents to treat cancer.

肝癌肿瘤干细胞被认为是导致肝肿瘤耐药性、耐化疗性和复发转移的根源。本项目拟通过仿生肿瘤干细胞微环境来构建肝癌肿瘤干细胞的体外模型，并阐明肿瘤干细胞微环境对维持肝癌肿瘤干细胞干性的作用机制。首先，我们创新性地利用光交联方法将聚三亚甲基碳酸酯2D纳米纤维“卷”成一个结构稳定且尺寸大小可调控的3D纳米纤维支架。与传统纳米纤维支架相比，所制备的3D纳米纤维支架有可调控的大孔结构，有利于细胞向支架内部迁移。接着，我们将肝癌肿瘤干细胞种植在3D纳米纤维支架上，从支架材料的理化特性、与间充质干细胞共培养以及低氧微环境等方面来仿生构建肝癌肿瘤干细胞的体外模型。最后，利用免疫缺陷小鼠模型来评价肝癌肿瘤干细胞的致瘤性。本项目的成功不仅将为制备新型3D纳米纤维支架提供一个新的思路，而且为构建其它癌组织肿瘤干细胞的体外模型奠定基础，从而为研究肿瘤干细胞的靶向治疗策略和治疗剂提供模型样本和新的研究模式。

肝癌肿瘤干细胞被认为是导致肝肿瘤耐药性、耐化疗性和复发转移的根源。本项目拟通过仿生肿瘤干细胞微环境来构建肝癌肿瘤干细胞的体外3D培养模型，并阐明肿瘤干细胞微环境对肝癌肿瘤干细胞干性维持的作用机制。我们成功从病人肝癌组织中提取出2株肿瘤干细胞。所提取的肿瘤干细胞ALDH阳性率达到93%左右，表达多种肿瘤干细胞标志物，且具有高度致瘤性（将10个肿瘤干细胞皮下注射到免疫缺陷小鼠上进行成瘤实验，免疫缺陷小鼠就会长出肿瘤）。我们发现vitronectin可作为包被底物用于肿瘤干细胞的2D细胞培养，在连续传代3代以内，肿瘤干细胞的干性能够得到较好维持。我们进一步将肿瘤干细胞种植在明胶/透明质酸的酶交联水凝胶中，通过仿生微环境构建肿瘤干细胞的体外3D培养模型。研究表明，细胞种植密度影响肿瘤干细胞在3D培养条件下的干性维持；水凝胶的软硬度对肿瘤干细胞的增殖、干性维持、形态等有较大影响。使用明胶/透明质酸（w/w=1:1）水凝胶培养肿瘤干细胞，经过4天培养，ALDH阳性率仍然维持在88.1%。继续培养至7天，ALDH阳性率为80.6%，而使用传统成球悬浮培养方法，ALDH阳性率仅为64%。综上所述，我们建立了以明胶/透明质酸酶交联水凝胶为基质材料的肝癌肿瘤干细胞体外3D细胞培养模型。该3D培养模型可以为针对肿瘤干细胞的靶向治疗和药物筛选的研究提供一个较好的疾病模型。