【作者】 彭云丽；

【导师】 耿美玉；

【作者基本信息】 中国海洋大学 ， 生药学， 2007， 硕士

【摘要】 内皮细胞作为血管内皮基本的结构和功能单位,它的一个重要功能就是发挥其屏障功能,调节着血管内外的物质交换,维持内环境的稳定,使得邻近或远端的组织器官免受损害。肿瘤血源性转移是一个极为复杂的多步骤过程,每个环节都受到多种因素的影响。肿瘤血源性转移过程主要包括四步,侵袭、内渗、外渗和新的转移灶形成。肿瘤细胞的外渗过程,是肿瘤转移的至关重要的环节,它与炎症反应中白细胞的渗出过程极为相似。白细胞渗出过程极其复杂,经过附壁、粘着、游出和趋化作用等阶段到达炎症灶,在局部发挥重要的防御作用。血管内皮细胞是白细胞由血流渗出的第一道障碍,且文献研究表明,白细胞渗出的同时,其所释放的炎症因子,可以引起内皮细胞表面的糖链变化,并且这种变化与内皮细胞的功能异常密切相关。而肿瘤细胞的外渗过程中,内皮细胞表面糖链是否发生变化,此变化与内皮功能障碍之间有怎样的关系,至今均未见报道。本论文旨在研究肿瘤细胞外渗过程中内皮细胞表面糖链变化,以及该变化所引起的内皮细胞形态和功能的改变。本论文以肿瘤条件培养基刺激内皮细胞为研究手段,观察肿瘤细胞外渗过程中,内皮细胞表面多种糖链的变化情况,以及内皮细胞的形态变化,并初步探讨了致使其功能改变的机制。第一部分:肿瘤条件培养基对内皮细胞表面糖链的影响论文中采用肿瘤条件培养基刺激内皮细胞,观察肿瘤外渗过程中内皮细胞表面的糖链变化。结果发现,肿瘤条件培养基作用下,内皮细胞表面多种糖链均有不同程度的升高,以β1,6分支糖链变化最为明显。进一步以β1,6分支糖链为研究对象,观察了肿瘤条件培养基作用不同时间下,内皮细胞表面β1,6分支糖链的变化情况。结果表明,肿瘤条件培养基作用不同时间下,内皮细胞表明β1,6分支糖链均有不同程度的升高,其中18h时变化最大,且发现β1,6分支糖链表达的升高并不是由负责其催化合成的GnT-V的表达变化所致。第二部分:肿瘤条件培养基对内皮细胞形态功能的影响第一节:肿瘤条件培养基对内皮细胞形态的影响实验发现,肿瘤条件培养基可以明显地引起内皮细胞的皱缩。恢复完全培养基后,皱缩的内皮细胞又可重新伸展开。共聚焦检测细胞骨架相关蛋白发现,tubulin并无明显变化,而内皮细胞内应力纤维明显增多。肿瘤条件培养基使内皮细胞间F-actin明显减少,细胞间隙明显增大。同时,肿瘤条件培养基显著降低内皮细胞与细胞外基质成份Fn的粘附,使内皮细胞易于收缩,致使内皮通透性增大。第二节:肿瘤条件培养基对内皮细胞间粘附分子CD31的影响本节主要研究了肿瘤条件培养基对内皮细胞上的同质粘附分子CD31的影响。首先观察了肿瘤条件培养基对CD31表达的影响,结果表明,肿瘤条件培养基并不影响CD31的蛋白表达。接着检测了肿瘤条件培养基对CD31糖基化和磷酸化的影响,结果表明,肿瘤条件培养基使CD31分子的糖基化和酪氨酸磷酸化水平明显升高。进一步观察细胞迁移相关信号分子RhoA的变化,结果表明肿瘤条件培养基能使RhoA的激活明显增强。本论文研究发现,肿瘤细胞外渗过程中,肿瘤条件培养基使内皮细胞表面多种糖链均有不同程度的升高,β1,6分支糖链变化最明显。肿瘤条件培养基通过使内皮细胞上CD31分子的β1,6分支糖链增加,从而使其磷酸化增强,并使下游信号分子RhoA的活化增强,引起内皮细胞的皱缩,细胞间隙增大,促进肿瘤细胞的外渗。本论文探讨了糖链与肿瘤细胞转移的关系,为糖链在肿瘤转移中的作用增添了新的研究内容,丰富了糖的生物学功能研究内容,也为抗肿瘤药物的研究开发提供新的作用靶标。更多还原

【Abstract】 As an essential structural and functional unit of the blood vessel endothelium, one of the important functions of the blood vessel endothelium is to act as a barrier, regulating the substance exchange across the blood vessels, sustaining the stability of the internal conditions, so that the adjacent or even remote tissues and organs are free from harms.The hematogenous metastasis of the tumour is an extremely sophisticated multi-step process, in which every individual step is affected by various factors. The hematogenousmetastasis process of the tumour consists of four major steps, invasion, intravasation, outward infiltration and the formation of new metastasis focus. The outward infiltration refers to the process in which the tumour cells separate from the original metastasis focus and enter the blood circulation. It is a critical step in tumour metastasis. This process is very similar to the outward infiltration of the leucocyte in inflammation reactionsThe outward infiltration of the leukocytes is a complicated process. It involves the following stages: episporium, adherence, emigration and chemotaxis etc. Through the infiltration, leukocytes are delivered to the inflammation focus and play an important role to protect locally. The endothelial cell is the very first barrier for the leukocytes to infiltrate across the blood vessel. According to the literature review, during the course of leukocyte infiltration, the inflammatory factor released can trigger the change of glycans over the surface of endothelial cell. This chang of glycans is closely related to the abnormal functionality of the endothelial cell. On the other hand, there is no recent research reported on whether there are changes of glycans over the surface of endothelial cell and if so, how is this related to the abnormal functionality of the endothelium, during the course of tumour cell extravasation.This dissertation is to present the research regarding to the change of glycans that expressed on the surface of endothelial cells during the course of tumour cell infiltration, and the impacts of the change of glycans upon the change of conformation and functionality of endothelial cells. This research was carried out by means of stimulating endothelial cells with tumor conditioned medium , observing the changes happened to various glycans over the endothelial cells during the course of tumour cell filtration. The changes in conformation of endothelial cells was also studied. The mechanism of causing the changes in its functionality is discussed.Part 1. The impact of tumor conditioned medium upon glycans which expressed on the surface of endothelial cellIn this thesis we mainly observed the chang of glycans that expressed on the surface of endothelial cell in the process of extravasation by means of tstimulating endothelial cells with tumor conditioned medium. As a reslt, we found that many kinds of glycans on the surface of endothelial cell had different increase, whileβ1,6 branches increased most obviously. Further study focused onβ1,6 branches.β1,6 branches increased in different degreee along with the extention of time, at the point of eighteen hours the change is maximum. Meanwhile, the increase ofβ1,6 branches is not because of the high expression of GnT-V, which is in charge of the biosynth ofβ1,6 branches.Part 2. The impact of tumor conditioned medium upon the conformation and functionality of endothelial cellsSection 1. The impact of tumor conditioned medium upon the conformation of endothelial cells It is observed during the experiments that tumor conditioned medium will apparently cause the crimpling of endothelial cells. Once the complete medium is retrieved, the crimpledendothelial cells will spread again. On the confocal inspection of related proteins of the cell skeleton, there is no obvious reduction in tubulin; however, the stress fibre of the endothelial cell increase enormously. tumor conditioned medium results in great reduction in F-actin linkages among the endothelial cells, and the separation among them are increased. Meanwhile, tumor conditioned medium can markedly reduce the adhesion between endothelial cell and ECM Fn, so that it is easier for the endothelial cell to shed away from the substrate. This will consequently enhance the infiltration across the endothelium.Section 2. The impact of tumor conditioned medium upon the functionality of endothelial cellIn this section the impact of tumor conditioned medium upon the homogeneous adhesion molecule CD31 of the endothelial cell is discussed. First, tumor conditioned medium has no apparent effects observed on CD31 expression. Subsequently the impacts of tumor conditioned medium upon the CD31 glycosylation and phosphorylation have been inspected. According to the experiment results, the tumor conditioned medium can improved the CD31 glycosylation and phosphorylation to a great extent. Further observation has been devoted to cell migration correlated signaling molecule RhoA, whose activation is greatly enhanced by tumor conditioned medium.In this thesis we find that in the process of extravasation tumor cell can induce the increase of so many glycans expressed on endothelial cells.Among these kinds of glycans,β1,6 branches changed most obviously. Tumor conditioned medium increase the CD31 glycosylation, and further increase CD31 phosphorylation and downstream signaling molecule RhoA activation. As a result, endothelial cells become shrinkage and the endothelial junction become larger.All of these can facilitate the extravasation of tumor cell. This thesis mainly study the relationship between glycans and tumor metastasis, which make the roles of glycans in tumor metastasis more clearly and enriching the content of glycobiology. At the same time, this study can provide new target for the development of anti-tumor agents.更多还原