肝细胞是肝脏内细胞数目占比和体积占比最多的细胞，也是肝脏执行诸多生物学功能的主力军。肝病的治疗、研究等都需要大量具有成熟表型的肝细胞，但是肝细胞在体外培养时会丧失增殖的能力，分化表型也会逐渐消失。因此，在体外探究如何启动肝细胞的增殖以及调控肝细胞特异性的功能具有重要的应用价值。生理情况下，肝脏内几乎所有的肝细胞处于无增殖能力的休眠状态，但是进行2/3部分肝切除（Partial hepatectomy, PHx）手术后，肝脏展示出强大的再生能力。肝细胞是肝再生过程中首先进入细胞周期的细胞，其再生速率与剩余肝脏单位质量的血流量密切相关，但是血流引起的剪切应力能否触发肝细胞增殖仍不清楚。除了细胞增殖，肝细胞特异性功能的力学调控也十分重要。肝细胞处在复杂的力学微环境中，除了感受到血流剪切外，还可以感受到周围组织硬度的变化，尤其在肝脏发生纤维化时，但是目前尚缺乏合适的肝纤维化模型可以同步探究剪切应力和基质硬度对肝细胞特异性功能的耦合调控作用。基于上述问题，本文分别以PHx后肝再生和肝纤维化为背景，探究剪切应力及基质硬度对肝细胞功能的力学调控作用及其潜在的分子机制，主要包括以下两个方面：

1. 剪切应力启动肝细胞的增殖。本文以不同的流量经门静脉向小鼠的肝脏进行灌注，并从组织、蛋白和基因水平检测增殖标志物的表达，结果发现高流量灌注可以促进肝细胞由休眠期进入细胞周期。借助荧光粒子扩散和肝脏活体成像技术，观测到灌流液可以穿过内皮层渗流到肝细胞区域，而且间隙流随着灌注流量的增大而增大，提示间隙流在肝细胞增殖中可能存在潜在的作用。在体外的肝血窦流动剪切模型中，对分离提取的小鼠原代肝细胞施加0~5 dyn/cm²的剪切应力，加载时间为0~24 h，结果证实流体剪切确实可以启动肝细胞增殖，并且呈现幅值和时间依赖性，其最优力学加载参数为0.05 dyn/cm²、6 h。进一步收集力学加载后的肝细胞进行测序分析，筛选得到Yes相关蛋白（Yes-associated protein, YAP）信号通路在剪切应力加载后有差异性激活，并且抑制或激活YAP可以削弱或增强剪切应力下肝细胞的增殖。通过对分子机制的探究，发现剪切应力可以激活细胞膜上的β1整合素，将力学信号传递至胞质内的局部黏着斑激酶，然后通过负调控Hippo信号通路、从而正调控YAP在细胞核内的应答，最终促进肝细胞的增殖。此外，剪切应力可以促进肝细胞成熟表型的维持或提升，复现肝再生过程中细胞增殖能力和代谢活性同步提高的生理现象。上述结果提示，间隙流剪切应力可能是PHx后肝再生的触发机制之一，为体外实现肝细胞的大量扩增提供了新的思路。

2. 剪切应力和基质硬度调控肝细胞特异性功能。本文借助微流控芯片技术在构建了肝纤维化的体外模型，该模型分为上下两层，以多孔膜间隔，模拟肝血窦内皮细胞（Liver sinusoidal endothelial cells, LSEC）上的窗孔结构。下层流道接种肝细胞，接种前与不同浓度的胶原水凝胶（1~7.5 mg/mL）预混，模拟肝纤维化不同阶段的情况；上层流道接种LSEC，并通过注射泵施加不同大小的流体剪切（0~1 dyn/cm²），模拟血流的作用，从而实现剪切应力和基质硬度的同步、可控加载。结果表明，细胞可在所构建的肝纤维化模型中维持很好的存活率和分化状态。随着基质硬度的增加，肝细胞的白蛋白合成和细胞色素P450还原酶的表达不断降低。随着剪切应力的增加，肝细胞特异性的功能呈现阈值现象，即低剪切应力可以增强肝细胞的合成代谢功能，而高剪切应力会导致肝细胞表型的丢失。同时剪切应力下肝细胞对基质硬度的响应差异会增大，提示多种力学加载可以放大细胞的响应。肝细胞对两种力学因素的响应规律不受LSEC和胶原支架的影响，但和流道的尺寸有关。进一步的探究发现，基质硬度对肝细胞特异性功能的影响可能受肝细胞核因子4α的调控。上述工作基于体外肝纤维化模型揭示了剪切应力和基质硬度对肝细胞特异性功能的单独及耦合调控作用，可为深入认识肝纤维化过程中的肝细胞功能紊乱提供基础。

综上，本文诠释了力学刺激对肝细胞增殖和特异性功能的生物力学调控规律及其潜在的分子机制，对从生物力学角度深入理解肝再生的触发机制和肝纤维化的发病机理具有重要的意义。

As the key cell type with the largest number and highest volume ratio in the liver, hepatocyte serves as the backbone of liver to perform various biological functions. Although numerous hepatocytes with mature phenotype are in great demand for the therapy and research of liver disease, it is noted that hepatocyte can not proliferate and will lose differentiated phenotype gradually in in vitro culture. Therefore, it is significant of practical value to explore how to initiate hepatocyte proliferation and regulate hepatocyte-specific functions in vitro. Physiologically, almost all the hepatocytes are kept in quiescent state without proliferative ability. In contrast, liver exhibits powerful regenerative potential after 2/3 partial hepatectomy (PHx). Hepatocyte is the first one to enter cell cycle during liver regeneration, and its regenerative rate is closely related to the blood flow per unit mass of the remnant. However, whether the blood flow-induced shear stress is able to trigger hepatocyte proliferation remains unclear. Besides cell proliferation, hepatocyte-specific functions regulated by mechanics is also very important. Hepatocytes are exposed to a complex mechanical microenvironment, consisting of blood flow-induced shear stress, and surrounding tissues-imposed stiffness, especially during liver fibrosis. To date, there is no suitable fibrotic liver model to study the coupling effects of shear stress and matrix stiffness on hepatocyte-specific functions simultaneously. Based on the scientific issues above, the present dissertation aims to investigate the roles of shear stress and matrix stiffness play in regulating hepatocyte functions and their underlying molecular mechanisms in the context of PHx-induced liver regeneration and liver fibrosis, respectively. The major works are summarized into two parts as follows:

1. Direct shear stress exposure initiated hepatocyte proliferation. In this study, mouse liver was perfused at various rates via portal vein, followed by proliferative markers measurement at tissue, protein, and gene levels. Results indicated that high perfusion rate was able to promote hepatocytes to step into cell cycle from quiescent state. By means of fluorescence particle diffusion and in vivo liver imaging, it was observed that the perfusion fluid reached hepatocyte region through endothelial layers, and interstitial flow was enhanced with the increasing perfusion rate, implying a potential effect of interstitial flow on regulating hepatocyte proliferation. In the in vitro flow-exposed hepatic sinusoid model, isolated mouse primary hepatocytes were exposed to shear stress of 0~5 dyn/cm² for 0~24 h. It was confirmed that flow shear indeed initiated hepatocyte proliferation, in a shear amplitude- and duration-dependent manner with an optimized set at 0.05 dyn/cm² for 6 h. Further sequencing analysis of collected hepatocytes after mechanical stimuli identified differentially activated Yes-associated protein (YAP) signaling pathway, and its inhibition or activation weakened or enhanced shear-induced hepatocyte proliferation. Mechanotransductive pathway exploration indicated that shear stress enabled β1 integrin activation on the membrane before transferring signals toward Focal adhesion kinase in the cytoplasm, and thus positively regulated YAP response within nucleus through negative Hippo signaling pathway, leading to hepatocyte proliferation finally. In addition, the mature phenotype of hepatocytes was promoted, or maintained at least, by shear stress, which resembled the physiological phenomenon of simultaneous improvement in cell proliferation and metabolic capacity during liver regeneration. These results suggested that interstitial flow-induced shear stress might be one of the trigger mechanisms responsible for liver regeneration after PHx, providing a new idea for accomplishing massive expansion of hepatocytes in vitro.

2. Shear stress and matrix stiffness regulated hepatocyte-specific functions. An in vitro fibrotic liver model was constructed here based on microfluidic chip technology. The model consisted of two layers separated by a porous membrane to mimic the fenestrae of liver sinusoidal endothelial cells (LSEC). The hepatocytes premixed with collagen I at various concentrations (1~7.5 mg/mL) were inoculated into the lower channel, representing different stages of liver fibrosis. The upper channel lined with LSEC and was accessible to various shear stress (0~1 dyn/cm²) via injection pump to mimic blood flow. Therefore, both shear stress and matrix stiffness were applied to this model simultaneously and controllably. Characterization of the fibrotic liver model demonstrated high survival rate and well differentiated phenotype of seeded cells. Hepatocytes cultured on stiffer matrix produced less albumin and had decreased cytochrome P450 reductase expression. In contrast, shear stress showed biphasic effects on hepatocytes, where low shear stress elevated the synthetic and metabolic functions of hepatocytes while high shear stress resulted in loss of hepatocellular phenotype. Moreover, shear stress increased the responsive differences of hepatocytes to matrix stiffness, suggesting that multiple mechanical loading was able to amplify cell response. This regulating pattern was neither affected by LSEC nor collagen scaffolds but related to channel size. Further exploration suggested that hepatocyte-specific functions regulated by matrix stiffness were likely attributed to hepatocyte nuclear factor 4 alpha. These results enabled us to elucidate the separated or coupled effects of shear stress and matrix stiffness on hepatocyte-specific functions depending on our fibrotic liver model, providing a basis to understand hepatocyte dysfunction during liver fibrosis.

In summary, the present dissertation deciphered the roles of mechanical stimuli in regulating hepatocyte proliferation and liver-specific functions, and unraveled underlying molecular mechanisms. This work shed light on understanding the triggering mechanisms of liver regeneration and pathogenesis of liver fibrosis from a biomechanical viewpoint.