血瘀相关因素在肝纤维化形成中的作用与机制

发布时间：2018-05-15 04:04

本文选题：血瘀 + 肝纤维化　；参考：《中国人民解放军医学院》2014年博士论文

【摘要】：背景及目的：肝硬化是一种严重危害人类健康的疾病，由不可逆性肝纤维化发展而来，预后差。中医从血瘀病机出发常用活血化瘀药物治疗肝纤维化、肝硬化，临床疗效显著。中医血瘀与西医淤血微循环障碍有病理学交叉之处，近年来研究发现肝脏微循环障碍为肝纤维化主要发病机制和病理基础之一，但基于中医血瘀的肝纤维化形成机制研究还不够清晰，，因此我们拟建立一种能够去除其他致病因素干扰，独立体现血瘀致肝纤维化这一病理过程的动物模型，尝试观察血瘀致肝纤维化的形成特点，分析可能的发病机理，单纯探索血瘀与肝纤维化之间的关系，寻找活血化瘀中药防治肝纤维化作用的关键靶点，为防治肝纤维化、肝硬化中药新药物筛选提供实验室依据及参考。方法：（1）采用经肝后段下腔静脉部分结扎术（partial ligation of thesuprahepatic inferior vena cava，pIVCL）制作慢性瘀血性肝纤维化小鼠模型，部分小鼠无特殊处理措施常规喂养，部分小鼠给予抗凝剂华法林口服，部分采用组织旁路因子抑制剂(Tissue factor pathway inhibitor,TFPI)转基因小鼠制作模型。于术后6周处死小鼠。（2）记录小鼠体重及肝脾重量，门静脉压力等一般情况，收集血清、肝脏或细胞样本，分别进行肝功能检测、组织学检查（苏木精-伊红染色、天狼猩红染色），羟脯氨酸含量检测、免疫荧光染色、RT-PCR、Western Blot、ELISA以及脱氧胆酸溶解性分析实验等方法检测，观察评估血瘀致肝纤维化后，肝纤维化相关指标平滑肌肌动蛋白（Smoothmuscle acetin α，α-SMA）、胶原、纤维连接蛋白、纤维蛋白、羟脯氨酸等变化，行磁共振扫描评估肝硬变程度及其与门静脉压力、肝内羟脯氨酸含量关系分析。（3）以人或小鼠肝星形细胞（Hepatic stellate cell，HSC）为目标细胞进行体外实验，评估细胞受到周期性机械拉伸、细胞周围纤维蛋白含量变化、应用β1整合素受体或肌动蛋白抑制剂对HSC分泌纤维连接蛋白及溶解性的影响。结果：①本研究采用肝后段下腔静脉部分结扎术（pIVCL）成功建立了瘀血性肝纤维化小鼠实验模型，描述了模型小鼠肝/体重量比、脾/体重量比、门静脉压力、肝功等一般情况。②体内试验：pIVCL手术组6周时胶原沉积、肝内羟脯氨酸含量、α-SMA较假手术组均显著增加；术后口服抗血凝剂华法林6周或采用TFPI转基因小鼠进行pIVCL术后，胶原沉积、肝内羟脯氨酸含量、α-SMA表达较野生型小鼠pIVCL术后增加幅度显著减少； pIVCL手术后小鼠肝脏MRE提示肝硬度增加，与门静脉压力升高及肝内羟脯氨酸含量增加成正比。③体外实验：HSC受到周期性机械拉伸、予纤维蛋白刺激，则纤维连接蛋白（Fibronectin，FN）产量增加，不可溶性FN增加；给予β1整合素受体或肌动蛋白抑制剂刺激则使HSC分泌FN减少，不可溶性FN含量减少；在纤维蛋白胶内孵育HSC，MRE检测结果示纤维蛋白胶硬度增加。结论：①通过瘀血性肝纤维化小鼠模型的建立，证实血瘀可以作为独立致病因素导致肝纤维化产生；②该小鼠模型搭建了传统中医血瘀与现代西医淤血之间联系桥梁，为中医血瘀在肝纤维化中作用的认识找到现代生物学依据提供了实验室数据；③一些抗凝处理措施抑制肝纤维化形成的结果，反证了血瘀可以导致肝纤维化形成这一结论，并为活血化瘀类中药抗肝纤维化作用病理基础研究找到了关键靶点，为下一步筛选抗肝纤维化、肝硬化新药物提供了实验室参考。
[Abstract]:Background and objective: liver cirrhosis is a serious disease which endangers human health. It is developed from irreversible liver fibrosis, and the prognosis is poor. Traditional Chinese medicine is used to treat liver fibrosis and cirrhosis with significant clinical effect. It is found that the liver microcirculation disorder is one of the main pathogenesis and pathological basis of liver fibrosis, but the mechanism of the formation of liver fibrosis based on blood stasis of traditional Chinese medicine is not clear. Therefore, we intend to establish an animal model which can remove the interference of other pathogenic factors and reflect the pathological process of liver fibrosis independently, and try to observe it. The characteristics of the formation of liver fibrosis caused by blood stasis, analysis of possible pathogenesis, simple exploration of the relationship between blood stasis and liver fibrosis, looking for the key targets for the prevention and treatment of liver fibrosis by activating blood and removing stasis, providing laboratory basis and reference for the prevention and control of liver fibrosis and the screening of new drugs of traditional Chinese medicine.
Methods: (1) a mouse model of chronic stagnant liver fibrosis was made by partial ligation of the posterior inferior vena cava (partial ligation of thesuprahepatic inferior vena cava, pIVCL). Some mice were fed without special treatment, and some mice were given the anticoagulant to warfarin, and some of the tissue bypass factor inhibitors (Tissu) were used. E factor pathway inhibitor, TFPI) transgenic mice were made in mice. 6 weeks after the operation, mice were killed. (2) the weight of the mice and the liver and spleen weight, the pressure of the portal vein were recorded, the serum, liver or cell samples were collected, the liver function test, histology examination (hematoxylin eosin staining, Sirius scarlet stain), and hydroxyproline content examination Test, immunofluorescence staining, RT-PCR, Western Blot, ELISA and deoxycholic acid dissolution test and other methods, observe and evaluate the changes of smooth muscle actin (Smoothmuscle acetin alpha, alpha -SMA), collagen, fibrin protein, hydroxyproline and other changes of hepatic fibrosis related indexes after blood stasis induced liver fibrosis. Analysis of the relationship between the degree of liver cirrhosis and the pressure of portal vein and the content of hydroxyproline in the liver. (3) in vitro experiments were conducted on human or mouse Hepatic stellate cell (HSC) for target cells, and the cells were subjected to periodic mechanical tension, the changes of fibrin content around the cells, and the application of beta 1 integrin receptor or actin inhibitor Effects of HSC on fibronectin secretion and solubility.
Results: (1) a mouse model of blood stasis induced liver fibrosis was successfully established by partial ligation of the posterior inferior vena cava (pIVCL). The liver / body weight ratio, spleen / weight ratio, portal pressure and liver function were described. (2) in vivo test: collagen deposition, intrahepatic hydroxyproline content and alpha -S in the pIVCL operation group at 6 weeks. MA was significantly higher than that in the sham operation group; after 6 weeks of oral antiblood coagulant and TFPI transgenic mice, the collagen deposition, the content of hydroxyproline in the liver and the increase in the expression of alpha -SMA were significantly lower than that of the wild type mice after pIVCL; the MRE of the liver of mice after pIVCL operation showed the increase of the liver hardness and the pressure of the portal vein. High and intrahepatic hydroxyproline content increased in proportion. (3) in vitro experiment: HSC was subjected to periodic mechanical tension and stimulated by fibrin protein, the yield of fibronectin (Fibronectin, FN) increased, and the insoluble FN increased; the secretion of HSC by beta 1 integrin receptor or actin inhibitor resulted in the decrease of HSC secretion and the decrease of the insoluble FN content. HSC was incubated in fibrin glue, and the hardness of fibrin glue was increased by MRE.
Conclusion: (1) through the establishment of a mouse model of blood stasis of liver fibrosis, it is proved that blood stasis can lead to liver fibrosis as an independent pathogenic factor. 2. The model set up a bridge between traditional Chinese medicine blood stasis and modern western medicine, which provides a modern biological basis for the understanding of blood stasis in liver fibrosis. Some measures of anticoagulant treatment inhibit the formation of liver fibrosis and prove that blood stasis can lead to the formation of liver fibrosis, and the key target is found for the basic research on the pathological basis of liver fibrosis, which provides a laboratory reference for the next screening of liver fibrosis and new drugs for liver cirrhosis. Exam.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575.2

【参考文献】