肝窦毛细血管化过程差异蛋白质组的研究

发布时间：2018-02-08 20:15

本文关键词： 肝窦内皮细胞肝窦毛细血管化蛋白质组　出处：《北京工业大学》2012年硕士论文　论文类型：学位论文

【摘要】：肝窦内皮细胞(Liver sinusoidal endothelial cell, LSEC)是肝脏非实质主要细胞群，约占肝非实质细胞的总量的50%，它们将肝细胞与血流分隔开，并在肝脏微循环中发挥重要作用．这些细胞缺乏基底膜，形成孔型单层结构，并表达多种清道夫受体，调节肝脏薄壁细胞与血液之间的物质交换，构成机体中最强大的清道夫系统．肝脏在遭到多种病原侵袭时，肝窦内皮细胞窗孔逐渐减少或消失，内皮下基膜形成，产生类似于连续型毛细血管的结构，这一过程称为肝窦毛细血管化．它由多种因素引起，其过程极复杂，而如果肝窦毛细血管化持续发展，将进一步发展为肝纤维化，由于肝纤维化可以起始多种肝病，因此肝窦毛细血管化现象在多种肝病的发病前期阶段均有出现，近年来受到广泛关注，而目前关于肝窦内皮细胞的生理功能及病理机制方面仍少有研究．用亚砷酸钠加入饮水中喂养C57BL/6J小鼠可以成功复制肝窦毛细血管化的动物模型，本研究采用蛋白质组学的技术手段，系统分析了肝窦毛细血管化发生发展过程中肝窦内皮细胞蛋白质组的动态变化，发掘了一批与肝窦毛细血管化密切相关的功能蛋白质组，结合生物信息学手段系统分析差异蛋白的功能和调控网络，从而为深入探讨肝窦毛细血管化发生发展的分子机制奠定了基础．用亚砷酸钠饮水喂养C57/BL6J小鼠5周，肝脏病理表现为肝窦毛细血管化及轻度纤维化．随后，我们利用密度梯度离心结合CD146免疫磁珠分选的方法原位分离正常组和模型组小鼠的肝窦内皮细胞，利用差异凝胶电泳技术(DIGE)分析正常组和模型组小鼠LSEC全蛋白的差异，所得到的差异蛋白质点用MALDI-TOF/TOF-MS质谱仪进行检测，目前共鉴定了8个差异蛋白，其中7个表达量下调，1个表达量上调．在下调的差异蛋白中，Anp32A是G蛋白信号通路相关的细胞因子，可作为S1P的作用靶点，参与多种细胞活动，包括细胞增殖，细胞分化，细胞凋亡，，肿瘤抑制等生理功能；而RhoGDI2是Rho蛋白家族GDP解离抑制因子，可调节Rho家族蛋白GDP与GTP的结合与转换，从而实现Rho蛋白活性的调控，它的下调可以激活NADPH氧化酶亚基Rac1，进而活化NADPH氧化酶；S100a9可与S100a8共同介导NF-κB活性的调控，通过活性氧簇(ROS)的表达调节肝脏恶性肿瘤的发生发展，它们对形成肝窦毛细血管化有着重要的调控作用．上述研究为进一步阐明肝窦毛细血管化发生发展的分子机制，发现多种肝病早期诊断标志物和潜在的药物靶点奠定了基础．
[Abstract]:Liver sinusoidal endothelial cell ( LSEC ) is a non - essential main cell population of the liver , which accounts for 50 % of the total liver non - essential cells , which separates the liver cells from blood flow and plays an important role in liver microcirculation . These cells lack the basement membrane , form the pore - type single - layer structure , express a variety of scavenger receptors , regulate the exchange of substances between the thin - walled cells of the liver and the blood , and form the most powerful scavenger system in the organism . When the liver is invaded by a variety of pathogens , the portal pores of the hepatic sinusoidal endothelial cells are gradually reduced or disappeared , and the subcutaneous basement membrane is formed to produce a structure similar to the continuous capillary vessel , and the process is called the capillary vessel of the hepatic sinus . It is caused by a variety of factors , its process is very complex , and if the capillary of hepatic sinusoid continues to develop , it will be further developed into hepatic fibrosis . In this study , the dynamic changes of hepatic sinusoid endothelial cells were successfully reproduced by adding sodium arsenite to drinking water , and the dynamic changes of the protein groups in the hepatic sinus endothelial cells were analyzed by means of proteomic technique . The function and control network of the differential protein were analyzed by means of bioinformatics , which laid the foundation for further exploring the molecular mechanism of the development of capillary vessel in the liver . The liver and pathology of C57 / BL6J mice were fed with sodium arsenite drinking water for 5 weeks . Subsequently , we used the density gradient centrifugation combined with CD146 immunomagnetic bead sorting method to separate normal and model group mouse liver sinus endothelial cells in situ . The difference of total protein of LSEC in normal group and model group was analyzed by differential gel electrophoresis ( DIGE ) . The difference protein spots were detected by MALDI - TOF / TOF - MS mass spectrometer . Eight differentially expressed proteins were identified , 7 of them were down - regulated and 1 expression was up - regulated . In the down - regulated difference protein , Anp32A is a cytokine related to G protein signaling pathway , and can be used as the target of S1P to participate in various cellular activities , including cell proliferation , cell differentiation , apoptosis , tumor inhibition and other physiological functions . In order to further clarify the molecular mechanism of the development of capillary vessel in hepatic sinus , this study lays a foundation for the early diagnosis of liver diseases and potential drug targets .

【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341

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