| ObjectiveCardiovascular disease and cerebrovascular disease is the primary disease cause of mortality in many countries,its potential factor is the pathological process of atherosclerosis(AS).AS it is a kind of immune inflammatory disease,and the most important antigen presenting cell is dendritic cells(DC),the dendritic cells through the regulatory T cell responses play a role in the AS.Hower,the mechanism is not clear.AS is also a metabolic disease,research has shown that DC can also regulate blood fat.TGF-beta is a kind of multifunctional cytokine,can be generated by a variety of vascular cells of the immune system to express the main subtypes of TGF-beta 1.A large number of studies have shown that the expression of TGF-beta 1 is associated with the AS form.DC is an expression of TGF-beta 1 important function of cells,this topic research DC expression of TGF-beta1 in AS the role and mechanism of action of beta.For the future provide theoretical basis for the prevention and treatment of atherosclerosis.Method1.Through the cre-flox technology,we successfully prepared dendritic cells(dendritic cells,DC)that is not expressed TGF-beta1 in mice,main method is through a bone marrow transplant will be the chimeric mouse bone marrow in atherosclerosis susceptibility Apoe mice,at the same time,the DC expression of TGF-beta1 bone marrow chimeric in Apoe and mice as a condition for reference.2.DC expression of TGF-beta1 mice CD11 ccre(cre)as contract,induced by high-fat diet CD11 ccre TGF-beta1 fl/fl,cre~+atherosclerosis disease models in mice.3.CD11 ccre TGF-beta1 fl/fl,cre~+mice high-fat after 16 weeks,for cardiac perfusion,embedding the heart,frozen sections.4.Evaluate the oil red O staining in the section of the formation of plaque,the aortic root and plaques area size,the aortic root by software calculation for the whole aorta lipid deposition in dyeing observation,and observed by HE staining and mason dyeing aortic plaques in inflammatory cells infiltration and the content of collagen.5.To immunohistochemical staining and slice observation macrophages,T cells,DCs in atherosclerotic plaque.6.Using biochemical analyzer test in plasma total cholesterol(TC),triglycerides(TG),high-density lipoprotein(HDL),low density lipoprotein(LDL)levels.7.Take the spleen to prepare the single cell suspension.Use mul-ticolor fluorescence labeling and flow cytometry testing,we analyze the changes of Th1.Th17 and Treg.8.Through the Real-time PCR detection of the genes involved in metabolism in the liver and gut,through the Real-time PCR to detect the expression of inflammatory cytokines in artery.Results1.A high-fat diet for 16 weeks later,oil red O staining showed that compared with the control mice,plaque area of the aortic root in experimental mice increased significantly,HE staining results showed that the patchs inflammatory cells infiltration is significantly increased,mason staining of plaque showed that the collagen content increased significantly,that is a more stable plaques.2.Compared with the control mice,in the plasma,total cholesterol(TC)is significantly increased in CD11cCre~+mice but triglyceride(TG)was no significant change,take the analysisof lipoprotein,they found that a significant rise in low density lipoprotein(LDL)but high-density lipoprotein(HDL)and very low density lipoprotein(VLDL)had no significant change.3.The analysis of the composition related to liver lipid metabolism show that although the changes of the enzymes were not significantly associated with cholesterol synthesis,the changes associated with cholesterol intake(LDLRAP1),reverse transportation(ACAT2 LCAT)and secretion of molecules(MTP ApoB100)are significant which is associated with the increased cholesterol.4.By intracellular cytokine staining and flow cytometry analysis showed that compared with control group,experimental group mice showed that no significant difference of Th1.Th17 and Treg cells,CD4~+T cell proliferation increased and a significant increase in the proportion of CD4~+/CD8~+T.5.Through the detection of Real-time PCR in the atherosclerotic aorta,the results showed that compared with the control group,IL-17 and IL-6 were significantly increased,while the TGF-beta significantly reduced,consistent with the deterioration of the plaque.through the detection of chemokine,we found that chemokines changed unsignificantly,through the immunohistochemical study we found that compared with control group,the number of the macrophages(MAC),dendritic cells(CD11c),and T cells(CD3)were significantly increased,this suggested that DC which is not expressed TGF–beta1 can aggravate atherosclerosis plaques by increase immune cells infiltration.Conclusions1.In the process of atherosclerosis disease,DC which is not expressed TGF-beta 1can increase plaque area significantly,inflammatory cells infiltration is also increased sinnificantly in the plaque.DC which is expressed TGF-beta 1 has resistance to AS.2.In the process of atherosclerosis disease,DC which is not expressed TGF-beta1increases total cholesterol and low density lipoprotein cholesterol(hdl-c)in plasma,CD4~+T cell proliferation is also increased,That is to say that the DC which is expressed TGF-betal eventually play a resistant role in atherosclerosis by regulation of the proliferation of CD4+T cellsand cholestrol metabolism. |
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