基于微流控芯片的中性粒细胞运动及趋化性研究

发布时间：2018-02-15 13:20

本文关键词： 免疫细胞中性粒细胞趋化性微流控芯片　出处：《中国科学技术大学》2017年博士论文　论文类型：学位论文

【摘要】：免疫细胞趋化性在免疫反应中发挥着重要作用,进一步开展免疫细胞趋化性研究具有十分重要的意义。微流控芯片可以生成精确可控的浓度梯度,是一种理想的免疫细胞趋化性研究工具。然而,在缺乏专业设备和技术人员的情况下使用微流控芯片非常困难,阻碍了微流控芯片在生物和医学研究领域中推广。本论文针对细胞趋化性研究领域所面临的一系列问题,开发了相应的微流控芯片,旨在为免疫细胞趋化性研究提供易于使用和实用的解决方案。首先,开发了一种集成细胞全血分离和细胞预富集功能的微流控芯片,可快速地从全血中筛选中性粒细胞,有利于提高实验效率和分析精度。通过检测中性粒细胞对趋化因子(N-formylmethionyl-leucyl-phenyl-alanine,fMLP)和慢性阻塞性肺病病人(Chronic obstructive pulmonary disease,COPD)痰液样本的趋化性验证了基于微流控芯片的全血分离方法。在此基础上,进一步设计了集成多区细胞校准和梯度生成功能的微流控芯片(Dual-Docking Device),使其具备中性粒细胞趋化性和细胞趋化性记忆效应实验研究的双重功能。研究结果支持在简单二维平面上使用偏向随机运动(bias-random-walk)描绘中性粒细胞趋化性,且证明了趋化性记忆效应不仅与趋化物化学浓度相关,而且与细胞"过去和现在"所经历的梯度环境有关。为进一步提高实验效率,我们开发了具有三个平行梯度通道的微流控芯片,该芯片允许在单一显微镜视野(10X物镜)中同时开展三组不同物理条件下的细胞趋化性实验,有利于节省时间成本、降低劳动强度,并保证了实验的真实性与可靠性。使用三通道微流控芯片研究了与慢性肾病相关的生物标志物成纤维生长因子 23(Fibroblast growth factor 23,FGF23),并首次阐述了 FGF23 影响中性粒细胞趋化性的过程,揭示了研究FGF23介导的中性粒细胞趋化性对研究疾病发病机理和制定相关治疗方案的重要意义。最后,针对当前细胞趋化性研究需要依托昂贵的显微镜且时间和实验成本较高等缺点,开发了基于微流控芯片和智能手机的细胞迁移运动研究专用实时成像系统。该系统集成了微流控芯片、活细胞成像、实验环境控制和数据分析功能,可快速、实时采集实验数据并实现自动化的分析,极大提高了细胞迁移实验的效率。总之,本论文研究成果为基于微流控芯片技术的细胞趋化性研究设备的开发提供了设计思想和实验依据,所开发的微流控芯片有望被广泛用于细胞趋化性实验研究。
[Abstract]:Immunocyte chemotaxis plays an important role in immune response. It is of great significance to further study the chemotaxis of immune cells. Microfluidic chips can generate precise and controllable concentration gradient. Is an ideal tool for chemotaxis of immune cells. However, using microfluidic chips is very difficult in the absence of professional equipment and technicians. In this paper, a series of problems in the field of cell chemotaxis are discussed, and the corresponding microfluidic chips are developed. The aim of this paper is to provide an easy-to-use and practical solution for the study of immunocyte chemotaxis. Firstly, a microfluidic chip which integrates the functions of whole blood separation and cell preconcentration is developed, which can quickly screen neutrophils from the whole blood. The chemotaxis of neutrophils to the samples of chemokine N-formylmethionyl-leucyl-phenyl-phenyl-alanine (MLP) and chronic obstructive pulmonary disease (COPD) sputum proved the whole blood separation method based on microfluidic chip. A microfluidic chip, Dual-Docking device, which integrates the functions of multizone cell calibration and gradient generation, is further designed to provide the dual function of neutrophil chemotaxis and chemoattractant memory effects. The results of the study support the experimental study of neutrophil chemotaxis and chemotactic memory effects. In a two-dimensional plane, neutrophil chemotaxis is depicted using bias-random walks, It is proved that chemoattractant memory effect is not only related to chemoattractant chemical concentration, but also to the gradient environment experienced by cells "past and present". We have developed a microfluidic chip with three parallel gradient channels, which allows three sets of chemotaxis experiments under different physical conditions to be carried out simultaneously in a single microscope field of vision 10X objective, which can save time and cost. Reduce labor intensity, The reliability and authenticity of the experiment were ensured. Using a three-channel microfluidic chip, the fibroblast growth factor 23 FGF23, a biomarker associated with chronic nephropathy, was studied, and the process of FGF23 affecting neutrophil chemotaxis was first described. The significance of the study of neutrophil chemotaxis mediated by FGF23 in the study of disease pathogenesis and the development of related treatment protocols is revealed. In view of the disadvantages of the current study of chemotaxis, such as expensive microscope and high time and experimental cost, A real-time cell migration imaging system based on microfluidic chip and smart phone is developed. The system integrates microfluidic chip, living cell imaging, experimental environment control and data analysis functions. Collecting experimental data in real time and realizing automatic analysis greatly improve the efficiency of cell migration experiment. The results of this paper provide a design idea and experimental basis for the development of chemotaxis research equipment based on microfluidic chip technology. The developed microfluidic chip is expected to be widely used in the study of chemotaxis.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q2-33

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