The Changes Of Glycans Expressed On Endothelial Cell During The Course Of Tumor Extravasation, And Its Impacts On The Conformation And Functionality Of Endothelial Cell

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.