| Atherosclerosis is the leading cause of death in the developed world and nearly the leading cause in the developing world,so the more and more researches were focus on the pathological process of AS.Epidemiological studies have revealed that the high level plasma lipoprotein,especial low density lipoprotein would facilitate the AS lesion initiation.Atherosclerosis remains a geometrically focal disease,preferentially affecting the outer edges of vessel bifurcations.In these predisposed areas,hemodynamic shear stress is weaker than in protected regions and the flow patterns are disturbed.As an interface between the blood and the vessel wall,the endothelium occupies a unique location directly exposed to the hemodynamic forces imposed by the flow of blood. Pathological studies have revealed the primary initiating event in AS is the accumulation of LDL in the subendothelial matrix.Under normal physiological condition,LDL should be internalized into endothelial cells through integration between LDL-LDLR and digested by lysosome. But why LDL will accumulate in subendothelial matrix at AS preferential site of the artery and cause the AS by a series of pathological change? Furthermore,how about the degradation process of the internalized LDL in this special flow area? Many studies has been concerned about these questions intensively,but most of them are based on lamina flow or low shear stress condition and the result can not completely replace the exact biological effect caused by blood flow kinetics near arterial branches and bends.It will be established that there are two kinds of pathway for the vascular endothelium uptake LDL:receptor mediated endocytosis and permeability between cells.In this thesis,we try to mimic the disturbed shear flow in AS preferential area of the artery and then with cultured endothelial cells,some potential protein that affect the uptaking,digestion and accumulation of LDL,such as caveolin,eNOS,LDLR and lysosome will be studied to address the process LDL accumulating in subendothelial matrix.Based on these studies, we hope to provide the direct evidence for the high risk of AS near bifurcation and crook area of artery.Our studies include:①The vertical-step flow chamber system was set up and the PDMS chamber was made by soft lithography.Based on fluid mechanics,we have defined flowing type and shear stress distribution with finite element analysis to provide the theory principal and detail parameter for the disturbed flow area under the step in flow chamber(modeling software:gambit2.0,Data analysis software:FLUENT5.5).According to the dimension of the chamber and fluid nature,the entrance flow rate was defined as v=2 cm·s-1,which is equal to the flow area in vivo.②With the flow channel we established in step(1),Real-time PCR,ELISA and immunofluorescence were used to explore the response of the CAV-1 to the stimulation of the disturbed flow including the expression on mRNA and protein level and the distribution in the cell.We found that in disturbed flow area,CAV-1 distribution shifted to the membrane from cytoplasm to enhance the interaction between endothelial cells and LDL.In early stage of the stimulation,the expression of the CAV-1 decreased both on mRNA and protein level enhancing permeability among cells.All the responses may increase the lipid level in blood especially the accumulation of the LDL under endomembrane in AS early stage resulting in a series of pathological change.③eNOS is an important adjusting factor for the artery stability.The major catalytic site of the eNOS is distributed on the membrane of the caveolae connecting with CAV-1 scaffolding.After the stimulation of the disturbed shear stress,we found that the synthesis of the NO changed in the time course of the stimulation.At the primary stimulation stage,the synthesis of the NO increased enhancing the permeability among cells and this process will profit for the accumulation of the LDL under endomembrane in AS early stage.After a long term of stimulation,the synthesis of the NO decreased followed by the enhancement of the adhesion between leucocyte and thrombocyte to the endothelial cells to elevate the chance for the AS development.④With the flow chamber we established in step(1),Real-time PCR and ELISA were used to explore the response of the mRNA and protein expression of LDLR on endothelial cells to the stimulation of the disturbed flow.After stimulation,we found that the LDLR expression decreased intensively.Because this process may diminish the amount of the LDL absorbed by endothelial cells conversely increase the accumulation of the LDL in blood plasma,more LDL can be detained around AS predilection area that may intensify the LDL concentration gradient between exterior and interior of the cells enhancing the permeation of the LDL into the cells.Because of the attenuation of the receptor mediated endocytosis for the LDL,LDL accumulation will increase in the endothelial cells and then raise the accumulation under the endomembrane in the early stage of the AS.⑤Under normal condition,LDL will be absorbed into the cells through receptor mediated endocytosis and digested by lysosome.In this digestion process,acid ACP is a critical enzyme for the digestion of LDL.So we try to figure it out that if the expression of ACP also influenced by the shear stimulation.After stimulation,we found that the activity of the ACP decreased diminishing the digestion of internalized LDL thus increase the LDL accumulation in cells.It's consistent with our finding in former steps.⑥In order to obtain better integration and comparison between experiment data and catch the cell response more promptly,this thesis also contains some studies in further research:With the same method of the PDMS flow chamber processing,we tried to make micro-pattern stamp with PDMS on silicon micro-pattern model and then converted the endothelial cells on the bottom of the flow chamber into image model by micro-pattern stamp analysis.Based on these processing,every single cell in different site can be identified and it's a kind foundation for us to integrate the information in different experiment lot into a time course for signal protein expression.We have amplified the gift plasmid of LDLR-EGFP and then transfected into CRL-1730(cultured human umbilical endothelial cells) by transfect reagent FuGENE 6. Screened by G418,we have obtained the stable cell clone CRL-1730/LDLR-EGFP and the identification was processed with immunofluorescence and western blotting to check the plasmid and fusion protein expression.This will be a good preparation for us to do the real time study with the endothelial cells about the influence of the disturbed flow on LDLR expression and other protein adjusted by shear stress.
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