|英文题名||Substrate stiffness regulation of hepatocyte damage and neutrophil migration dynamics in liver disease|
|关键词||基底刚度 肝细胞损伤 血流剪切 肝血窦 中性粒细胞募集|
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：
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。
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.
|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.|