【作者】 杨杰；

【导师】 黄岂平；

【作者基本信息】 重庆大学 ， 生物学， 2010， 硕士

【摘要】 肝纤维化(hepatic fibrosis)是指在慢性肝病的影响下,肝脏内弥漫性细胞外基质(特别是胶原)的过度沉积。而慢性肝病的愈伤反应过程中肝星状细胞(hepatic stellate cells, HSC)扮演着十分重要的角色。在正常的肝脏中,肝星状细胞是静止的非肝实质细胞,主要的作用是储存维生素A和保持正常的基底膜成分。但是很多体内外的实验都表明,一旦HSC受到外界信号的刺激,HSC便失去储存的维生素A,变成一个快速增殖,表达Ⅰ型胶原的细胞。本文在参考文献和总结别人经验的基础上,取成年雄性SD大鼠,用链蛋白酶、胶原酶在体门静脉灌注肝脏消化大鼠肝脏细胞,经PERCOLL密度梯度离心分离肝星状细胞。台酚蓝拒染实验鉴定细胞活力,α-SMA蛋白免疫细胞化学染色鉴定细胞纯度。为后续实验的进行打下了基础。本文采用本实验室已经熟练掌握的异源蛋白植入法,将异源蛋白植入1.5Kg新西兰大白兔腹腔中,获得原代成肌纤维细胞。并以高细胞密度种植在盖破片上,对其进行机械损伤,研究损伤对成肌纤维细胞的形态和方向的影响。组织损伤归根结底是细胞损伤,细胞的损伤造成2种病变现象:一是组织功能缺失,二是细胞死亡后形成的间隙会改变组织的空间构象。细胞死亡形成间隙,从力学角度讲即为细胞间预应力的破坏。这会给细胞一个信号并使细胞做出响应。本文从预应力破坏角度出发,力求在体外培养条件下对细胞造成机械损伤,使用生物力学、细胞生物学、免疫组织化学、计算机图像处理等多种研究方法,观察预应力的破坏对细胞的形态和方向的影响。使用SPSS等软件对实验中涉及到的细胞角度等进行分析。主要结论如下:①取成年雄性SD大鼠,用链蛋白酶、胶原酶在体门静脉灌注肝脏消化大鼠肝脏细胞,经PERCOLL密度梯度离心分离肝星状细胞。台酚蓝拒染实验鉴定细胞活力,α-SMA蛋白免疫细胞化学染色鉴定细胞纯度。②将异源蛋白植入1.5Kg新西兰大白兔腹腔中,获得原代成肌纤维细胞。并以高细胞密度种植在盖破片上,进行60小时归一化培养。③将3天、5天、10天的归一化培养的成肌纤维细胞刻划,连续观察细胞的形态和方向的变化,观察极性的改变。④统计分析刻划后细胞角度、长度等,得出刻划对不同天数成肌纤维细胞的影响。⑤通过对成肌纤维细胞与刻划线所成角度的分析,得出细胞间预应力的破坏对细胞有正调控作用。更多还原

【Abstract】 Hepatic fibrosis is the excessively collagen accumulation during the tissue repair response to chronic liver injury due to the novel synthesis. The cells and soluble factors participating in this response in the liver are the hepatic stellate cells. Hepatic stellate cells are non-parenchymal, quiescent cells in normal, whose main functions are to store vitamin A and probably to maintain the normal basement membrane type matrix. However, numerous References indicate that in response to liver injury stellate cells undergo an "activation" process in which they lose vitamin A, become highly proliferative, and synthesize ’fibrotic’ matrix rich in type I collagen.HSCs were isolated by PERCOLL density gradient centrifugation after the hepatocytes obtained from SD adult rats which were digested with pronase and collagenase infused via portal vein. The cell viability was determined by trypan blue exclusion test. The purity of HSCs was identified by detectingα-smooth muscle actin immunohistochemical staining. This is the foundation for the next experiments.Myofibroblast were isolated by using specific approach that foreign protein have been implanted into New Zealand rabbits’cavitas abdominalis for a period of time. The cells are planted in coverslip with high density and studied the form and orientation after scratch.In the final analysis tissue injury is cell injury which might cause by two kinds of pathological phenomena: First, tissues will lose their functions. Second, the gap after cell death will change the structure of the tissue. From the view of cellular mechanics, the gap after cell death can also be called the destruction of cells stressed. Then the cells will get a signal and respond. In this research, we make mechanical damage on cells in vitro and use bio-mechanics, cell biology, immunohistochemistry and computer image processing to study cell’s shape and polarity effects. SPSS was used for statistics analyse.The major founding are as follow:①HSCs were isolated by PERCOLL density gradient centrifugation after the hepatocytes obtained from SD adult rats which were digested with pronase and collagenase infused via portal vein. The cell viability was determined by trypan blue exclusion test. The purity of HSCs was identified by detectingα-smooth muscle actin immunohistochemical staining. ②Static relaxation was exerted on Myofibroblast from processed New Zealand rabbits(1.5kg) using specific approach that foreign protein have been implanted into rabbits’cavitas abdominalis for a period of time. The cells are planted in coverslip with high density and cultivated for 60h.③Scratch 3 days, 5 days, 10 days of the normalized cultured myofibroblast, continuous observation of cell shape and orientation changes, polarity effects.④Statistical analysis of cell characterization angle, length, etc., Get the conclusion of effects by cutting the different number of days.⑤Through the analysis of the angle of MFB with the line of scratching, we get conclusion that there is a positive regulatory effect to MFB by mechanical damage.更多还原