基于微流控平台的细胞共培养及生物微环境模拟的研究

发布时间：2018-12-15 04:31

【摘要】：微流控芯片技术因其具有的一系列优点,如样品试剂消耗少、结构功能多样化、集成化程度高、与细胞尺度接近等优点,近来广泛应用于细胞相关研究领域。通过结合不同的分析检测方法以及集成不同的功能结构单元,取得了显著的研究进展。其中相对传统方法,微流控芯片最大的优势是其集成化的功能,能够将多种不同的细胞或组织有序地整合为一个体系,而这也是当前细胞相关研究的一个重要发展方向,将体内状态下相关的多种细胞共培养并进行相互作用,可以更好地保持细胞的功能及生物学特性,这对于建立更完善的体外生物模型有至关重要的意义。在本论文中,我们围绕微流控芯片上细胞共培养开展了一系列研究。首先,我们对微流控技术的发展现状及在细胞研究中的应用进行了综述,其中着重介绍了在细胞共培养及生物微环境模拟中的的工作,分析论证了微流控芯片上进行细胞共培养的优势和意义。二、在微流控芯片上共培养了肝癌细胞和乳腺癌细胞,建立了一种微流控芯片上的肝-肿瘤模型,成功实现了前体药物卡培他滨的代谢与作用,并与质谱联用对原药及中间代谢产物进行了检测。使用这一平台,不仅可以对药物作用过程中的细胞状态进行活性检测、增殖检测及显微镜直接观察,同时可以使用质谱对系统中的信号分子或中间产物进行检测与鉴定。三、发展了一种使用inkjet在微流控芯片内对不同细胞进行微量精准定位共培养的方法,成功地将肝癌细胞和胶质瘤细胞同时打印并整合至微流控芯片内,用于前体药物的代谢及扩散实验。这一方法可以大幅提高微流控芯片内多种细胞精确定位共培养的效率,具有方便高效、易于自动化等优点。四、通过在微流控芯片中共培养胶质瘤细胞、内皮细胞及巨噬细胞,对胶质瘤微环境进行了一定程度的模拟,并对微环境中细胞间相互作用及细胞形态变化、表型变化、迁移情况进行同时监测,观察到了肿瘤细胞在微环境中与体内状况相一致的上皮-间充质转化的现象,同时也检测到了细胞的迁移运动、及通过细胞因子变化对微环境中巨噬细胞表型变化进行分析。
[Abstract]:Microfluidic chip technology has been widely used in cell related research recently because of its advantages such as less consumption of sample reagent, diversity of structure and function, high degree of integration and close to cell scale. Significant research progress has been made by combining different analysis and detection methods and integrating different functional structure units. The biggest advantage of microfluidic chip is its integrated function, which can integrate many different cells or tissues into one system in an orderly way, which is also an important development direction of cell related research. Co-culture and interaction of several related cells in vivo can better maintain the function and biological characteristics of the cells, which is of great significance for the establishment of a more perfect biological model in vitro. In this thesis, we have carried out a series of studies on cell co-culture on microfluidic chips. First of all, we review the development of microfluidic technology and its application in cell research, especially in cell co-culture and biological microenvironment simulation. The advantages and significance of cell co-culture on microfluidic chip were analyzed and demonstrated. Secondly, hepatoma cells and breast cancer cells were co-cultured on microfluidic chip, and a liver-tumor model on microfluidic chip was established. The metabolism and action of capecitabine, a precursor drug, was successfully realized. The raw drug and its intermediate metabolites were detected by mass spectrometry. Using this platform, not only can the activity of cell state, proliferation detection and direct observation by microscope, but also the signal molecules or intermediate products in the system can be detected and identified by mass spectrometry. Thirdly, a method of using inkjet to co-culture different cells in microfluidic chip was developed, which successfully printed and integrated the hepatoma cells and glioma cells into the microfluidic chip at the same time. It is used for metabolism and diffusion of precursor drugs. This method can greatly improve the efficiency of accurate location and co-culture of many cells in microfluidic chips, and has the advantages of convenience, efficiency, automation and so on. Fourthly, glioma cells, endothelial cells and macrophages were co-cultured on microfluidic chips. The microenvironment of glioma was simulated to a certain extent, and the interaction between cells, the morphological changes and phenotypic changes of glioma cells in microenvironment were also studied. At the same time, we observed the epithelium-mesenchymal transformation of tumor cells in microenvironment, and also detected the migration of tumor cells. The phenotypic changes of macrophages in microenvironment were analyzed by cytokine changes.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q813.1

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