【作者】 刘欣；

【导师】 谷秀；

【作者基本信息】 中国医科大学 ， 内科学， 2024， 硕士

【摘要】 目的:肺癌作为常见的恶性肿瘤,大部分体外研究基于简单的二维模型,不能模拟体内包括细胞外基质在内的肿瘤复杂微环境。三维(Three-dimensional,3D)模型在细胞增殖,基因表达等方面更接近人体内肿瘤,并能实现不同细胞的共培养。本文旨在利用微流控技术,结合水凝胶特性,调控3D结构,制备并调控水凝胶纤维及微珠,使其接近人体肺内形态,以其为支架,构建基于肿瘤细胞和巨噬细胞共培养体系的肺癌体外模型。研究方法:搭建同轴毛细管微流控装置,采用微流控技术,利用液体层流制备甲基丙烯酸酯明胶(Methacrylated gelatin,GelMA)/海藻酸钙复合多层纤维;利用剪切作用制备海藻酸钙气泡微珠;结合材料特性制备GelMA/海藻酸钙复合项链状纤维。使用体式显微镜、扫描电镜、荧光显微镜进行结构表征,观察溶液浓度、各相流速比以及温度对水凝胶形态、结构的影响。通过CCK-8实验验证水凝胶的细胞相容性,通过划痕实验验证材料对细胞迁移能力的影响。在水凝胶内包封A549细胞或RAW264.7细胞进行培养,使用AMPI染色验证细胞存活。结果:通过自搭建的毛细管微流控装置,制备GelMA@海藻酸钙双层中空纤维,获得了溶液浓度、流速等条件对纤维形态的影响规律。海藻酸钙具有良好的生物相容性,A549细胞在海藻酸钙中空纤维内存活率良好,可长期培养。制备海藻酸钙气泡微珠,溶液浓度是影响微珠内部形态的主要因素,海藻酸钠浓度越高,微珠内气泡数量越多,体积越小。在海藻酸钙内负载A549细胞,细胞存活良好,最长可培养28天。制造GelMA@海藻酸钙项链状纤维,探究了溶液浓度、温度和流速对纤维形态的影响,发现温度越高,内部GelMA卷曲越多;常温时,内相流速增加,GelMA层直径增加;高温时,内相流速降低,GelMA层卷曲增多。一定浓度的海藻酸钙和GelMA水凝胶对RAW264.7细胞和A549细胞表现出良好的细胞相容性,肺癌细胞和巨噬细胞在纤维内共培养,可稳定存活,细胞存活率大于80%。结论:海藻酸钙中空纤维是良好的细胞生长支架,可用于A549细胞长期3D培养,构建基础肺癌体外模型。海藻酸钙气泡微珠内部形态可调控,截面与肺泡结构类似,负载A549细胞培养可构建模拟体内结构的优化肺癌体外模型。GelMA@海藻酸钙项链状纤维的结构特点和材料特性协同作用,诱导海藻酸钙内部GelMA纤维规律折叠卷曲,项链状纤维可作为A549细胞和RAW264.7细胞长期3D分区共培养的支架,构建肿瘤细胞和巨噬细胞共培养体系,模拟肺癌的多细胞微环境。更多还原

【Abstract】 Objective: Lung cancer is a common malignant tumor,and most in vitro studies are based on 2-dimensional models that cannot simulate the complex microenvironment of tumors in vivo,including the extracellular matrix.The 3-dimensional(3D)model is closer to the in vivo tumor in terms of cell proliferation and gene expression and enables the co-culture of different cells.The aim of this thesis is to use microfluidic technology,combined with hydrogel properties,to fabricate and regulate hydrogel fibers and microbeads so that they are similar to the morphology of human lungs.Using them as scaffolds,we constructed in vitro models of lung cancer,and co-culture systems of tumor cells and macrophages.The feasibility of culturing A549 cells and RAW cells was also evaluated.Methods: Constructed coaxial capillary microfluidic devices and prepared gelatin methacrylate(GelMA)/calcium alginate composite multilayered fibers by laminar flow of liquids,calcium alginate bubbling beads by shear,and GelMA/calcium alginate composite necklace fiber by combining the material properties.It was characterized using the stereomicroscope,scanning electron microscope,and fluorescence microscope.The effect of solution concentration,flow rate ratio,and temperature on the morphology and structure of the hydrogels was observed.The cytocompatibility of the hydrogels was demonstrated by the CCK-8 assay,and the effect of the material on cell migration ability was verified by cell scratch assay.A549 cells or RAW264.7 cells were encapsulated within hydrogels for culture and cell survival was verified using AMPI staining.Results: GelMA@calcium alginate bilayer hollow fibers were prepared by a self-built capillary microfluidic device.The pattern of change of fiber morphology by the change of conditions such as flow rate and concentration was found.Calcium alginate showed low toxicity,and 549 cells survived well in the calcium alginate hollow fibers and could be cultured for a long time.Calcium alginate bubbling microbeads were prepared.The higher the concentration of sodium alginate,the higher the number of bubbles inside the beads and the smaller the volume.A549 cells were loaded inside calcium alginate and survived well for up to 28 days.GelMA@calcium alginate necklace fibers were prepared.The effects of solution concentration,temperature,and flow rate on fiber morphology were explored,it was found that the higher the temperature,the more the internal GelMA curls.At room temperature,the flow rate of the internal phase increases,and the diameter of the GelMA layer increases.At high temperatures,the flow rate of the internal phase decreases,and the curling of the GelMA layer increases.Certain concentrations of calcium alginate and GelMA hydrogels showed good cytocompatibility for RAW264.7 and A549 cells,and lung cancer cells and macrophages stably survived and proliferated within the fibers with more than 80% cell viability.Conclusions: Sodium alginate hollow fibers are good scaffolds for cell growth and can be used for long-term 3D culture of A549 cells to construct an in vitro basal model of lung cancer.The internal morphology of sodium alginate bubble microbeads is tunable and the cross-section is similar to the alveolar structure.Embedding A549 cells inside the microbeads for culture can construct an optimized in vitro model of lung cancer that mimics the in vivo structure.The structural features and material properties of GelMA@calcium alginate necklace-like fibers act synergistically to induce regular folding and curling of GelMA fibers within calcium alginate.The necklace-like fibers can be used as a scaffold for long-term 3D partitioned co-culture of A549 cells and RAW264.7 cells,to construct a co-culture system of tumor cells and macrophages,and to simulate the multicellular microenvironment of lung cancer.更多还原