肝脏是哺乳动物体内具有重要生理作用的器官，承担着合成、代谢、消化和免疫等维持机体稳态的重要功能。肝脏还具有强大的再生能力，为肝脏受损后快速恢复功能提供了基础保障，也使得部分肝切除和肝脏移植等肝病临床治疗手段成为可能。肝脏具有十分独特的门静脉和肝动脉双血供系统，其血流动力学是影响肝脏行使复杂生理功能的重要调控因素，表现为典型的力学-生物学耦合特征，对于肝脏维持生理功能和器官特征具有重要意义。部分肝切除后的肝再生涉及细胞增殖、细胞外基质重建以及反馈信号调节等复杂生物学过程，而该过程除生化因子的作用外，还伴随着血流动力学的改变。这一血流动力学的变化在肝血窦层面可以进一步解耦为作用在肝血窦内皮细胞上的力学拉伸和流体剪切应力，这种作用直接、时间迅速且变化显著的力学因素可能作为驱动肝再生的因素之一，与生化因素协同促进肝再生的进程，但目前对于力学因素（尤其基于肝血窦内血流引起的力学拉伸）在肝再生中作用的研究仍不够清晰。在肝脏复杂的细胞组成中，肝血窦内皮细胞能够首先感知肝内环境变化，其对力学拉伸变化的感知、转导及响应的力学-生物学耦合信号转导机制，尚待进一步研究。

本论文基于部分肝切除后的肝再生过程中力学拉伸对肝血窦内皮细胞旁分泌能力的影响及其力学转导机制展开研究。首先，通过肝脏离体灌注模型，发现高灌注流量引起的肝血窦扩张与肝再生因子的表达以及肝细胞的增殖能力呈正相关性。其次，对肝血窦内皮细胞进行体外力学拉伸加载模拟肝血窦的扩张，发现力学拉伸不改变肝血窦内皮细胞的分子表型和窗孔特征，但能够促进肝血窦内皮细胞表达肝再生相关因子，且其表达模式与生理肝再生过程相似，其中肝素结合性表皮生长因子（HB-EGF）的表达和分泌呈现明显的时间和幅值依赖性，对促进肝细胞的增殖具有重要作用。再次，通过收集力学拉伸加载肝血窦内皮细胞后的上清液对肝细胞进行培养，发现力学拉伸后的上清液培养的肝细胞具有更强的增殖潜能以及合成和代谢等功能。最后，通过蛋白质组学及磷酸化蛋白质组学结果分析，结合小分子抑制剂、基因沉默等实验方法，得到力学拉伸调控肝血窦内皮细胞中HB-EGF表达的力学信号转导通路。

肝血窦内皮细胞的力学信号转导对上述生物学过程至关重要。肝血窦内皮细胞通过表面的β1整合素（β1-Integrin）对力学拉伸进行感知，β1-Integrin将力学信号传递给肌动蛋白丝（F-actin），进而促进yes关联蛋白1（YAP）入核以及HB-EGF的表达。力学拉伸促进转录共激活因子YAP进入细胞核后，与细胞核内的转录因子TEA域家族（TEAD）相互作用，直接促进Hbegf基因的转录，进而增加HB-EGF的表达。进一步发现，F-actin通过两方面因素促进YAP进入细胞核：一方面力学拉伸通过F-actin促进细胞核核孔的扩张，被动增加YAP进入细胞核的潜能；另一方面F-actin能够促进BAG家族分子伴侣调节蛋白3（BAG-3）的表达，而增加的BAG-3与YAP结合，主动转运YAP进入细胞核，两种途径共同促进YAP激活并入核以及HB-EGF的表达。力学拉伸在肝再生中的重要作用通过小鼠不同程度肝切除模型得到验证。

综上所述，本论文从肝再生中复杂的力学-生物学耦合背景入手，解耦出力学拉伸在肝再生过程中的重要地位。通过研究力学拉伸在促进肝脏再生中的重要作用，揭示了力学拉伸能够促进肝血窦内皮细胞分泌HB-EGF等肝再生相关因子从而促进肝细胞的增殖潜能，深化了肝再生过程中力学信号与生化信号的协同作用，以及肝血窦内皮细胞对肝细胞的旁分泌作用。最后本文探究出一条力学敏感蛋白YAP介导HB-EGF表达的力学信号转导通路，即β1-Integrin → F-actin → 核孔/BAG-3 → YAP和TEAD → HB-EGF表达。上述结果在深化理解肝再生中力学-生物学耦合规律的同时，也为临床肝病治疗提供了基础数据和新思路。

The liver is an organ with significant physiological roles in mammals, undertaking important functions in maintaining body homeostasis such as synthesis, metabolism, digestion and immunity. It also has a strong regenerative capacity, which provides the basis for rapid recovery of functions after liver injury. The regenerative capacity of the liver also makes it possible for clinical treatments in liver diseases such as partial hepatectomy and liver transplantation. The liver has a unique dual blood supply system of portal vein and hepatic artery, and its hemodynamics is an important regulatory factor affecting the complex physiological functions of the liver, showing typical mechanical-biological coupling characteristics for maintaining its physiological functions and organ characteristics. Liver regeneration after partial hepatectomy involves complex biological processes such as cell proliferation, extracellular matrix reconstruction and feedback signal regulation, which are accompanied by hemodynamic changes in addition to the action of biochemical factors. Hemodynamics can be further decoupled at the hepatic sinusoidal level into mechanical stretch and fluid shear stress acting on the liver sinusoidal endothelial cells, and mechanical factors with direct, immediate and drastic changes may act as one of the driving factors for liver regeneration and promote the process of liver regeneration in concert with biochemical factors. However, the role of mechanical factors (especially based on the mechanical stretch caused by blood flow within the hepatic sinusoids) during liver regeneration is still not clear. Among the complex cellular composition of the liver, liver sinusoidal endothelial cells are the first to sense changes within the intrahepatic environment, and the mechanical-biological coupling signal transduction mechanism of mechanosensing, mechanotransduction and response to mechanical stretch remains to be further investigated.

In this dissertation, the effect of mechanical stretch on the paracrine capacity of liver sinusoidal endothelial cells during liver regeneration after partial hepatectomy and its mechanotransduction mechanism were investigated. Firstly, the dilation of hepatic sinusoids induced at high perfusion flow was positively correlated with the expression of liver regeneration-associated factors and the proliferation of hepatocytes through an ex vivo liver perfusion model. Next, in vitro mechanical stretch loading of liver sinusoidal endothelial cells which simulated the dilation of hepatic sinusoids revealed that mechanical stretch did not change the molecular phenotype and fenestrae characteristics of liver sinusoidal endothelial cells, but could promote the expression of liver regeneration-associated factors in liver sinusoidal endothelial cells, and the expression pattern was similar with the physiological liver regeneration process. The expression and secretion of heparin-binding EGF-like growth factor (HB-EGF) showed a significant time and magnitude dependence, which was important for promoting hepatocyte proliferation. Further, by collecting the supernatant from liver sinusoidal endothelial cells after mechanical stretch, it was found that hepatocytes cultured with supernatant from liver sinusoidal endothelial cells after mechanical stretch had stronger proliferative potential as well as function of synthesis and metabolism. Finally, the analysis of proteomics and phosphoproteomics combined with small molecule inhibitors and gene silencing experiments revealed the signaling transduction pathways of HB-EGF expression in liver sinusoidal endothelial cells regulated by mechanical stretch.

Mechanotransduction of liver sinusoidal endothelial cells is crucial for the above biological process. The mechanosensing of mechanical stretch by liver sinusoidal endothelial cells was carried out through β1-Integrin on the cell surface, which transmitted mechanical signals to F-actin, thereby promoting yes-associated protein 1 (YAP) entry into the nucleus and HB-EGF expression. After entering the nucleus, YAP, a mechanical stretch-promoting transcriptional co-activator, interacted with TEA domain family (TEAD) in the nucleus to directly promote the transcription of Hbegf gene, which in turn increases the expression of HB-EGF. It was further found that F-actin could promote YAP entry into the nucleus through two pathways: on the one hand, mechanical stretch promoted the opening of the nuclear pores through F-actin, which passively increased the potential of YAP entry into the nucleus; on the other hand, F-actin could promote the expression of BAG family molecular chaperone regulator 3 (BAG-3), and the increased BAG-3 binded to YAP enhancing its active transport of YAP into the nucleus. The two pathways worked cooperatively to promote YAP activation and entry into the nucleus. The important role of mechanical stretch in liver regeneration was verified by mouse models with different extents of hepatectomy.

In summary, this dissertation decouples the important position of mechanical stretch from the complex mechanical-biological coupling context during liver regeneration. By investigating the important role of mechanical stretch in promoting liver regeneration, we reveal that mechanical stretch can promote the secretion of HB-EGF and other liver regeneration-associated factors by liver sinusoidal endothelial cells and thus promote the proliferative potential of hepatocytes, deepening the synergistic role between mechanical signal and biochemical signal during liver regeneration, as well as the paracrine effects of liver sinusoidal endothelial cells on hepatocytes. Finally, a mechanical signal transduction pathway of HB-EGF expression mediated by mechanosensor YAP is explored, namely, β1-Integrin → F-actin → nuclear pores/BAG-3 → YAP and TEAD → HB-EGF expression. These results further deepen the understanding of the mechanical-biological coupling in liver regeneration and provide basic data and new ideas for clinical liver disease treatment.