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肝血窦内白细胞迁移的数值模拟与分析
Alternative TitleNumerical Simulations and Analyses for Migration of Leukocyte in Liver Sinusoid
陈深宝
Thesis Advisor吕守芹
2020-05-29
Degree Grantor中国科学院大学
Place of Conferral北京
Subtype博士
Degree Discipline一般力学与力学基础
Keyword肝血窦,白细胞迁移,数值模拟,渗透性,细胞粘附
Abstract

肝脏是人体的重要器官,在消化、解毒、造血和免疫等过程中发挥重要作用。肝血窦是肝脏中特殊的毛细血管网络,是肝脏微循环血流灌注和免疫应答的主要发生部位,主要由四种细胞构成,包括肝实质细胞、肝血窦内皮细胞、肝星状细胞和枯否氏细胞。肝血窦具有独特结构,肝血窦内皮细胞上分布的穿透性筛孔结构,以及肝血窦内皮和肝实质细胞之间的狄氏间隙,使得肝血窦形成相互渗透的双层流动结构,间隙内的肝星状细胞赋予肝血窦壁较好的弹性,而肝血窦狭窄的内腔使白细胞常需发生挤压变形才能通过。在肝纤维化或肝硬化等病理条件下,肝血窦内皮细胞上筛孔退化、狄氏间隙中胶原纤维沉积等现象导致肝血窦结构、物理/力学微环境发生显著变化,进而调控其生物学功能。肝血窦内白细胞募集是实现肝脏免疫功能的重要途径,与经典炎症级联反应过程中毛细血管后静脉的白细胞募集存在明显区别,主要表现在:白细胞在肝血窦内的募集没有初期的捕获、滚动过程,而是直接的粘附滞留。而该独特的募集现象的机制尚存在争议。一方面认为是由于肝血窦内皮细胞表面不表达介导细胞滚动粘附的选择素导致的;另一方面也认为该过程是由肝血窦独特的结构及物理/力学微环境所致。因此,受体-配体相互作用介导细胞粘附的生化因素、以及肝血窦物理/力学微环境对肝血窦内独特的白细胞募集过程的各自贡献尚不清楚。在体观测是目前肝血窦免疫研究的主要手段,虽然该方法可以直观获得白细胞在肝血窦内的募集特征,并借助于生物学技术考察特定生物分子的贡献,但是难以孤立不同物理/力学因素的调控作用。随着技术的发展,肝器官芯片在肝脏免疫机制研究中起到越来越重要的作用,并在药物筛选等领域具有广阔应用前景。但是想要完全复现在体肝血窦结构、物理/力学微环境还有技术难度,需要更多的理论基础支撑。
基于此,本论文通过实验测量与数值模拟相结合的方法,开展了如下三方面工作:1)分别采用原子力显微镜与平行平板流动腔技术,测量了肝血窦特异的CD44-HA相互作用反应动力学及其介导的细胞粘附动力学特征,并与毛细血管后静脉特异的CD44-E-/P-选择素相互作用反应动力学及其介导的细胞粘附动力学特征相比较。结果表明:肝血窦特异的CD44-HA相互作用零力负反应率最低,可以介导细胞的稳定粘附;而CD44-E-/P-选择素则零力负反应率相对较高,分别介导细胞的滚动或瞬态粘附。这些分子反应动力学和细胞粘附动力学行为的差异,为考察特定受体-配体相互作用在白细胞肝内滞留过程中的贡献提供基础数据。2)基于浸入式边界模拟方法建立了体外双层肝血窦芯片的模拟体系,并针对目前双层肝血窦芯片缺乏特定流场设计依据的现状,系统考察了芯片几何尺寸及多孔膜渗透性对芯片流场的调控规律。结果表明:随着下层流道高度的增加,其壁面剪切力呈先上升后下降的趋势,芯片长度和多孔膜渗透性虽然可以调控下层流道壁面剪切力的转折点,但并不影响其先上升后下降的趋势。进一步建立了相应理论模型并与数值模拟结果进行了比对,为优化双层肝芯片设计提供支撑。3)在浸入式边界模拟方法基础上,建立了肝血窦内白细胞迁移的力学-化学耦合数值模型,结合体外细胞水平和分子水平的实验,考察了肝血窦几何特征、力学微环境以及细胞-内皮间粘附对白细胞迁移的调控作用。结果表明:在不同的空间受限条件下,受体-配体相互作用介导的细胞粘附是白细胞在肝血窦内滞留的基础,而肝血窦物理/力学微环境是白细胞滞留的重要调控因素。在有渗透条件下,高细胞刚度或狄氏间隙刚度均可有效促进白细胞的粘附与滞留,而低刚度细胞在低狄氏间隙刚度条件下对渗透率不敏感。本文工作为阐明肝血窦内白细胞的募集机制,理清生化与物理条件各自的贡献提供基础,并为未来肝脏炎症相关疾病的治疗提供新思路。

Other Abstract

The liver plays important roles in many processes such as digestion, detoxification, hematopoiesis and immunity. Liver sinusoid, mainly composed of four kinds of cells including hepatocytes, liver sinusoidal endothelial cells (LSEC), hepatic stellate cells (HSC) and Kupffer cells, is a specialized capillary network for blood perfusion and immune response in the liver. The sinusoids present unique structures with penetrating fenestrae distributing in the LSECs and the Disse space separating the endothelium and the parenchymal cells together making a double-layer flow configuration with mutual penetration. HSCs in the Disse space assign the sinusoidal wall a high elasticity, while the narrow lumen of the sinusoid makes the leukocytes squeeze to pass through. Under pathological conditions such as liver fibrosis or cirrhosis, the defenestration of LSECs and the accumulation of extracellular matrix in the Disse space lead to significant changes in anatomical structure and physical / mechanical microenvironment of the sinusoid, and regulate their biological functions. For example, leukocyte recruitment in the sinusoids is important in hepatic immunity and significantly different from that in the post-capillary during classical inflammatory cascade, in which the former lacks the initial capture and rolling. However, the mechanisms of such unique phenomenon are still controversial. On the one hand, it is believed that lack of selectin expression on the surface of LSECs is responsive. On the other hand, it could also be attributed to the unique structure and the physical / mechanical microenvironment of the sinusoid. Thus, it is still hard to isolate the respective contributions of the cell adhesion mediated by receptor-ligand interactions and the physical / mechanical microenvironment of the sinusoid to this unique leukocyte recruitment process. While in vivo observations are mainly applied to intuitively obtain the features of leukocyte recruitment in the sinusoid and elucidate the roles of specific biomolecules, those advanced in vitro techniques such as liver chips play a more and more important role in understanding the liver immunity and extensive applications in drug screening and other fields. Evidently, theoretical modeling is required due to the technical difficulties in replicating the structures and physical / mechanical microenvironment of the liver sinusoid.
To address the above issues, this dissertation has conducted the following three aspects of work: 1) Atomic force microscopy and a parallel plate flow chamber were used to quantify the liver sinusoid-specific CD44-HA binding kinetics and corresponding cell adhesion dynamics, and the post-capillary-specific CD44-E-/P-selectin kinetics and corresponding cell adhesion kinetics. Results showed that CD44-HA interaction had the lowest unstressed dissociation rate and mediated the stable adhesion of leukocytes, while CD44-E-/P-selectin interaction yielded relatively high unstressed dissociation rates and induced the rolling or transient adhesion of leukocytes, respectively. These results provide data sets for the following numerical simulations of leukocyte migration in the sinusoid. 2) Based on Immersed Boundary Method and the fact of lacking specific flow field design for current double-layered liver chips, the impact of chip geometry size and porous membrane permeability on flow field was systematically investigated upon a typical model of a porous-membrane-separated double-layered liver chip. Results showed that, with the increase of the lower channel height, the wall shear stress in the lower channel increases first and then decreases. The chip length and membrane permeability control the turning point of the wall shear stress of the lower channel, but do not affect the biphasic pattern. And a related theoretical model was also established and the predictions were then compared well with the numerical simulations. These analyses provide theoretical bases for optimizing the design of double-layer chip. 3) Also upon the immersion boundary method, a mechanical-chemical coupled numerical model was proposed for elaborating the leukocyte migration in the sinusoid. Combined with the in vitro experiments at the cellular and molecular levels, the effects of geometry, mechanical microenvironment and cell-endothelial adhesion on leukocyte migration were investigated. Results showed that, with varied space constraints, the cell adhesion mediated by receptor-ligand interaction provides the basis of leukocyte retention in the sinusoid, and the physical / mechanical microenvironment is an important regulatory factor for leukocyte retention. With a permeable flow across LSEC monolayer, high cellular stiffness or high Disse space stiffness effectively promotes leukocyte adhesion and retention, while less stiff cells are not sensitive to permeability at low Disse space stiffness. Collectively, this work provides the basis for elucidating the mechanisms of leukocyte recruitment in liver sinusoid and isolating the respective contributions of biochemical and physical conditions, providing new insight into the treatment of liver inflammation-related diseases in the future.

Call NumberPhd2020-004
Language中文
Document Type学位论文
Identifierhttp://dspace.imech.ac.cn/handle/311007/81929
Collection国家微重力实验室
Recommended Citation
GB/T 7714
陈深宝. 肝血窦内白细胞迁移的数值模拟与分析[D]. 北京. 中国科学院大学,2020.
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