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Title:
肝脏疾病中肝细胞损伤的基底刚度调控和中性粒细胞的迁移动力学
Alternative Title: Substrate stiffness regulation of hepatocyte damage and neutrophil migration dynamics in liver disease
Author: 杨浩
Abstract:

肝脏是人体重要的多功能器官,其功能结构单元——肝血窦主要包含肝细胞(hepatocyte, HC)、星形细胞(hepatic stellate cell, HSC)、肝血窦内皮细胞(liver sinusoidal endothelial cell, LSEC)和枯否氏细胞(Kupffer cell, KC)等,并与血液中流动的中性粒细胞(neutrophil, PMN)等免疫细胞发生复杂的多细胞相互作用。酒精性肝病、非酒精性脂肪性肝炎(non-alcoholic steatohepatitis, NASH)、病毒性肝炎等肝脏疾病发生和发展过程中,肝细胞持续损伤会释放胞内物质和炎症信号、引起炎症反应,同时激活星形细胞分泌大量细胞外基质,使肝组织刚度显著增加,造成肝脏纤维化甚至硬化。随着炎症反应的发生,血流剪切下募集到肝血窦的大量中性粒细胞也可能造成进一步肝损伤,形成正反馈途径,使肝纤维化加重、肝脏疾病病程恶化。本文围绕肝脏疾病过程中组织刚度变化与肝细胞损伤的调控关系及血流剪切下中性粒细胞在肝血窦内的募集动力学,开展了如下研究:

1)基底刚度对脂肪酸引起的肝细胞损伤的调控作用。组织刚度变化是肝纤维化和肝硬化的典型特征,已有研究表明,刚度相对较小的基底有利于肝细胞功能的维持,但缺乏基底刚度调控病理情况下肝细胞损伤的研究。本文制作了模拟正常肝组织和纤维化肝组织刚度的聚丙烯酰胺水凝胶基底、用于体外培养肝细胞系,利用棕榈酸刺激模拟非酒精性脂肪性肝炎,考察了基底刚度对棕榈酸引起的肝细胞损伤的调控作用。发现随着基底刚度的增加,体外培养的肝细胞细胞骨架微丝蛋白(actin)重组,应力纤维增多,细胞铺展面积增大。刚度大于1 kPa的基底上,棕榈酸刺激的肝细胞死亡比例显著上调。另外,棕榈酸刺激肝细胞损伤过程中,也会造成IL-1β等炎症因子的释放增多,促进炎症反应。该部分研究阐释了基底刚度对肝细胞损伤的影响,可为调控肝纤维化病程中的肝损伤提供新思路。

2)炎症反应中中性粒细胞在肝血窦内的募集机制。几乎所有的肝脏疾病发展过程中,均发现大量中性粒细胞募集的炎症反应。已经有研究表明,中性粒细胞在肝血窦内的募集机制与传统炎症级联反应不同,在特定的炎症刺激下受不同粘附分子调控,但其募集过程中粘附、爬行和跨膜迁移的精细过程和分子调控机制尚不明确。本文通过原代分离小鼠肝血窦内皮细胞和枯否氏细胞,构建了一个体外二维的肝血窦流动腔实验系统,能够维持较好的肝血窦表面筛孔结构。利用此实验系统,研究了生理血流剪切应力下,fMLP激活的中性粒细胞在TNF-α刺激的肝血窦内皮细胞上的粘附、爬行和跨膜迁移动态过程及其分子机制。发现β2整合素中的LFA-1(lymphocyte function-associated antigen 1)主导了中性粒细胞在肝血窦内皮上的粘附,并提供主要的粘附力抵抗高流体剪切;β2整合素中的Mac-1(macrophage-1 antigen)限制了中性粒细胞在肝血窦内的爬行速度和沿流体方向爬行的倾向性;枯否氏细胞共培养增加了中性粒细胞爬行的随机性,但不影响其粘附和爬行速度。该部分研究深化了对流体剪切下中性粒细胞在肝血窦内募集过程及其分子机制的认识。

3)气体驱动的微管吸吮技术中细胞运动和接触分析(与课题组佟春芳博士合作)。为进一步从单细胞层面和表面受体-配体分子二维反应动力学的角度阐明中性粒细胞在肝血窦内的运动和粘附机制,本文利用课题组建立的气体驱动的微管吸吮技术,分析了中性粒细胞在微管内的运动过程和其与另一侧的肝血窦内皮细胞或乳腺癌细胞的碰撞接触过程。发现单次实验中,细胞运动速度、接触面积、接触时间等参数在连续的循环测试中保持独立;不同的循环周期设定下,细胞运动速度、接触面积趋于稳定;接触时间与循环周期成正比;在一定范围内,气体流量和溶液粘度等不影响细胞之间的粘附频率。本文证明了该实验方法能稳定、可靠地用于细胞运动与接触研究,并量化两个有核细胞表面的受体-配体反应动力学,为后续精细研究单细胞层面中性粒细白在肝血窦内粘附和分子反应动力学机制提供基础。

 

English Abstract:

The liver is an important multi-functional organ of the human body. The hepatic sinusoid, as its functional unit, is mainly composed of hepatocytes (HCs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs). Complex multi-cell interactions between hepatic cells and flowing blood immune cells (including neutrophils) occur under physiological or pathological condition. During the development of alcoholic liver disease, non-alcoholic steatohepatitis (NASH), viral hepatitis and other liver diseases, hepatocytes are continuously impaired to release intracellular substances and inflammatory signals, which can cause inflammation and HSC activation. The activated HSCs secrete a large amount of extracellular matrices, inducing fibrosis and even cirrhosis with dramatical increase of tissue stiffness. Under inflammation condition of blood flow, a large number of neutrophils recruitment in the liver sinusoids also mediate further liver damage, forming a positive feedback pathway and aggravating the development of liver fibrosis. Focusing on the impact of tissue stiffness on hepatocyte injury in liver disease and the recruitment of neutrophils in liver sinusoid under shear flow, the following scientific issues were addressed in the current study:

 

1) Effect of substrate stiffness on hepatocyte damage induced by fatty acid. The tissue stiffness increases with the development of liver fibrosis and cirrhosis. Recent studies have indicated that soft substrate is beneficial to the maintenance of hepatocyte function, but little is known about the effect of substrate stiffness on pathogen induced hepatocyte damage. In this study, we used palmitic acid stimulation to induce an in vitro NASH model, and made up the polyacrylamide hydrogel with different stiffnesses to mimic the normal or fibrosis liver tissue. The effect of substrate stiffness on palmitic acid-induced hepatocyte damage was studied. It was found that, with the increase of substrate stiffness, the cytoskeleton of hepatocytes was reorganized to form more stress fibers, and the cell spreading was incrased. Finally, palmitic acid induced hepatocyte death was raised when the substrate stiffness was increased. In addition, the release of inflammatory cytokines such as IL-1β was increased after palmitic acid stimulation, promoting the inflammatory response. These results indicate the effect of substrate stiffness on hepatocyte damage, and provide an insight into recovering the tissue damage in the liver fibrogenesis.

 

2) Neutrophil recruitment mechanism in liver sinusoids during inflammation. Almost in all kinds of liver diseases, neutrophil infiltration into hepatic tissue is widely observed. Recent studies have shown that the recruitment of neutrophils in the liver sinusoids is unlike the classical inflammatory cascade and is regulated by different adhesion molecules under different inflammatory stimuli. But the elaborative processes and molecular mechanisms of adhesion, crawling and transmigration during neutrophil infiltration are still not very clear. Here we constructed an in vitro two-dimensional (2D) liver sinusoid flow chamber system using primary LSECs and KCs, which can maintain the fenestrae feature of liver sinusoids and decipher the multistep processes including adhesion, crawling and transmigration of fMLP-activated neutrophil recruitment on TNF-α-stimulated LSEC monolayer under physiological flow. Our results indicated that one β2-integrin member, LFA-1 (lymphocyte function-associated antigen 1), is positively dominant in neutrophil adhesion and provides the main adhesion strength to resist higher shear flow. Another β2 integrin member, Mac-1 (macrophage-1 antigen), is negatively dominant in neutrophil crawling velocity and directionality on LSECs. Moreover, KC presence only enhances randomized crawling but is not associated with the cell adhesion or crawling dynamics. These findings further the understandings of neutrophil recruitment under shear flow in liver sinusoids.

 

3) Analyses of cell movement and contact using a gas-driven micropipette aspiration technique (GDMAT) (collaborated with Dr. Tong Chunfang). In order to clarify the movement and adhesion mechanism of neutrophils in liver sinusoids at single cell level and quantify the 2D binding kinetics of liver specific adhesion molecules to their ligands, the movement of a neutrophil in a micropipette and its contact with a LSEC or a breast cancer cell on the other side were analyzed using the GDMAT. Our results demonstrated that the cell movement speed, contact area, and contact duration in the pipette are independent of the repeated test cycles. The measured cell movement speed and the contact area are stable with different cycle periods. The contact duration is linearly correlated with the cycle period. In the experimental settings of the current study, the cell adhesion is not affected by the gas flow and solution viscosity. These results proved that the GDMAT assay is robust to quantify the dynamics of cell movement and contact in a micropipette and determin the receptor-ligand kinetics between two nucleated cells. These findings also provide a platform to investigate precisely the neutrophil adhesion in the liver sinusoids and adhesion molecule kinetics at single cell level.

 

Degree Level: 博士
Issued Date: 2018-05-28
Degree Grantor: 中国科学院大学
Place of Degree Grantor: 北京
Supervisor: 龙勉
Keyword: 基底刚度 ; 肝细胞损伤 ; 血流剪切 ; 肝血窦 ; 中性粒细胞募集
Major: 一般力学与力学基础
Language: 中文
Other responsible: 中国科学院力学研究所
Content Type: 学位论文
URI: http://dspace.imech.ac.cn/handle/311007/73170
Appears in Collections:国家微重力实验室_学位论文

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Recommended Citation:
杨浩. 肝脏疾病中肝细胞损伤的基底刚度调控和中性粒细胞的迁移动力学[D]. 北京. 中国科学院大学. 2018-05-28.
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