| With the social development and population aging, cardiovascular diseases such as coronary heart disease and stroke have become the major threats to human health. Atherosclerosis(AS), having the common pathological basis for cardiovascular diseases,is the key factor in the research of cardiovascular disease prevention and treatment. AS is not caused by only one factor,but by the interaction of several factors. However, the causes of AS are still unknown. One of the recent theories is that AS may be an inflammatory disease resulting from several factors.The increasing of the endothelium permeability and the plasma lipoprotein levels(mostly low density lipoprotein, LDL)makes it easy for lipoprotein to permeate underneath endothelium. This is one of the major features during the process of AS developing. The most direct cause of AS is regarded as oxidized low density lipoprotein, Ox-LDL. The prevailing theory is that LDL in plasma enters into artery walls through endothelium barrier,accumulates under endothelium,and be modified as all kinds of modified LDL. Ox-LDL stimulates adjacent endothelial cells to producing inflammation signal,and induce all kinds of chemical stimulations and cytokines,such as VEGF,M-CSF and MCP-1. Also, Ox-LDL induces circular monocytes to enter artery walls,differentiate into macrophage with high expression of scavenger receptor(SR),internalize modified Ox-LDL through SR unlimitedly, result the formation of peroxide lipid, and transform macrophages into monocyte/macrophage-derived foam cells. All these factors further activate smooth muscle cells(SMC), make it become extracellular matrix,induce the migration and proliferation of SMC, and the formation of fibrous tissues. This process repeats until the formation of AS lesion. Modified LDL obtained from AS lesion internally is similar to oxidized LDL by Copper ion oxidization in vitro. Thus, we use Ox-LDL oxidized by Copper ion to stimulate the coculture system of monocyte/macrophage- endothelial cell so as to research its effects on the function of endothelial cell, and its signal transduction process.Vascular endothelial growth factor (VEGF) is the only growth factor found up to now which elicits mitosis of vascular endothelial cell, and is the only growth factor which is able to increase the vascular permeability. Williams B. believes that VEGF affects endothelial cell through paracrine,increases permeability of vascular endothelial cell, and elicits the formation of AS. Some research indicate,the signal transduction pathway of Tyrosine kinases of the Src family is part of the cause of VEGF angiogenesis. Thus,the further research on the molecular level about the relationship between Src,VEGF and the formation of AS is very important for the clarification of the causes of AS. For in depth research on the factors of AS formation, this project designed a monocyte-endothelial cell coculture system so as to better intimate the physiology situation in human body. This pharmacological model, together with the GEArrayTM Q series Human Endothelial Cell Biology Gene Arrays, is used to study the functional change of endothelial cell and the differentiation of mRNA expression under Ox-LDL stimulation, detect of Tyrosine kinases of the Src family signal transduction pathway, and observe the affects of this pathway related to the increase of Ox-LDL permeability due to VEGF induction for further clarification of VEGF action on the formation of AS.Part I Changes of endothelial cells functions following Ox-LDL stimulation of monocyte-endothelial cell coculture systemUse Trizol reagent to extract total RNA from singly cultured human umbilical vein endothelial cells(HUVEC)and monocyte(U937)-endothelial cell coculture system that have been stimulated by Ox-LDL(40 mg/L) over 24hr.Take 5ulRNA solution,dilute 100 times,and measure its absorbance at 260nm and 280nm,so as to calculate 260/280 ratio and solution concentration. Take total RNA 5μg,use GEArrayTM Q series Human Endothelial Cell Biology Gene Arrays made by SupurArray corporation to...
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