| Hepatic fibrosis (HF) is a keypoint pathophysiological process form chronic liver diseases to cirrhosis. It has now been confirmed that HF is reversible under certain conditions. As one of hepatic non-parenchymal cells, sinusoidal endothelial cells (SECs) constitute the wall of hepatic sinusoid, receive the blood flow mechanical stimulation directly and maintain the normal hepatic microcirculation. If the mechanical properties or the mechanics environment of SECs changes, capillarization of sinusoids will be formed, that is, disappearance of the unique structure of fenestrae and formation of the basement membrane. That will be the pathological basis of HF, and then ultimately lead to HF. So SECs play a very important role in the process of HF. Traditional Chinese medicine believes that the basic pathogenesis of HF characterized by the syndrome of qi deficiency and blood stasis. As one of qi-invigorating herbs, Astragalus has been confirmed for its good anti-hepatic fibrosis from clinical and experimental studies. Astragalus polysaccharides (APS) is the most and effective monomer component of Astragalus. Based on SECs cultured in vitro, we not only observed the cell ultrastructure, but also made a preliminary exploration on micro-mechanics response of SECs to APS at the single cell level, which might reach the final purpose of reversing HF.This thesis is divided into two parts:experimental studies and literature researches.Part One:Experimental StudiesI Primary culture, identification and nano-imagings of SECs.Objective As the body of high-resolution in situ imaging techniques, atomic force microscopy (AFM) and low vacuum environmental scanning electron microscope (SEM) were carried out on nano-imagings of SECs cultured in vitro. Methods SECs were isolated by the combination of collagenase perfusion, percoll gradient isopycnic centrifugation and selective adherence, and then the expression of surface antigens was measured by CD14. After the dehydration, desiccation and fixation, the morphology and ultrastructure of SECs was observed under SEM. The AFM, laser scanning confocal fluorescence microscope (LSCFM) and total internal reflection fluorescence microscope (TIRFM) consisted the joint imaging system, which gained the high-resolution and three-dimensional imagings. Results The isolated SECs were positive for CD14. Growth periods and spreading state of SECs were recorded under light microscope. High resolution imagings under SEM showed the typical morphology including the distribution of fenestrae, microfilaments and connections between the cells. AFM took the real-time dynamic and three-dimensional imaging of the mural SECs. Conclusions SECs isolated by this method provided a cell population not only with high purity and viability, but also with typical morphology, that is fenestrae and surface molecules. Nano-imagings of SECs were obtained by using AFM and SEM.II Micro-mechanics response of SECs to APS at the single cell level.Objective Based on SECs cultured in vitro, we use AFM to investigate micro-mechanics response of SECs to APS at the single cell level, which made the experimental foundation of the Biomachanopharmacology study on HF with the model of SECs damage. Methods Young’s modulus which reflected the cellular flexibility was obtained from force curves gained by AFM. With the intervention of a single concentration of APS, Young’s modulus was compared between the intervention group and the control group. Through the above, the APS with SECs experimental system was established and micro-mechanics response of SECs to APS at single cell level was carried out accordingly. Results When the probe was in the depth of300nm, the median Young’s modulus of the SEC from the intervention group was0.644107KPa, while the control group was0.448870KPa. When the probe was in the depth of500nm, the intervention group was0.700691KPa and the control group was0.540682KPa. The force curves given by AFM showed much lower elasticity of SECs in the intervention group than those in the control group, the differences were statistically significant (P<0.01). Conclusions AFM was used to elasticity of SECs gained by loading force on partial single SECs and portraying force curves. Young’s modulus of SECs had a significant increase under the action of APS with the concentration of50μg/ml, that is to say. APS with this concentration could make SECs hard. As a preliminary consideration, the hardening made by APS may reduce the energy dissipation from blood flow to hepatic sinusoid, which can improve hepatic microcirculation.Part Two:Literature ResearchesThis section reviewed advances on present research of SECs and HF, general situation of the treatments on HF in TCM and research development of Astragalus in curing HF. |
