Study On The Effect Of Oxidized High Density Lipoprotein On Maturation And Secretion Of MCP-1 Of Rabbit Dendritic Cells

Coronary heart disease(CHD) has been the main threaten to human health since the arrival of 21th century.The effective prevention and treatment of CHD has being the emphasis and challenge of the whole society of human beings.However, the pathogenesis of CHD has not been clearly identified so it is a great hindrance to the clinical treatment of CHD.Atherosclerosis(AS) is the pathologic basis of cardiac-cerebro vascular disease.In the past,there were mainly 3 theories,including lipidose deposition,injury repair and thrombogenesis,to explain the forming of AS. On basis of these theories,factors such as aging,hyperlipemia,hypertension, hyperglycaemia,hypercysteinemia and high blood uric acid are termed as high risks of CHD because they may induce blood vessel endothelium injury and lipidose deposition.Some progresses have been made in prevention of the genesis and advancement of AS by getting rid of or controlling of these high risks.However, people are beginning to question the effects of the preventions and treatments on these high risks for that the morbility age of CHD are younger than ever before,and the advancement of AS speeds up.With the terming of ACS on acute advancing stages of CHD,People re-recognized the pathologic and pathophysiologic characteristics of CHD in their developmental stages.Studies on AS found that unstable plaques are the pathological basis of ACS.The interventions of the inflammatory cells and immunocells to unstable plaques are key factors that leading to the instability and easily rupture of AS plaques.It is considered that systemic activation of the inflammatory factors and inflammatory reactions exist in ACS cases.What's more,people found that immunocells involve in all the AS developmental stages.They had proposed a new point of view that CHD is a process of chronic inflammation and a disease of autoimmunity.It is considered that inflammatory reactions and immune reactions induced by endogenous and exogenous risks are the pathological basis of AS.Dendritic cells have been considered as the most powerful specific antigen presenting cells that play a role in presenting antigens in the immunologic process since discovered by Steinman and Cohn in 1973.Minor DCs may intensively activate T cells,which may initiate the specific cellular immunity reaction.They play an important role in the induction and regulation of immunologic response.It is confirmed by recent studies that DCs may play a role in initiating inflammatory and immunological reactions in the generation and development of AS for that DCs co-emerge with T cells in the weak affected regions.Statins may play a role in immune regulation and therapy by inhibiting the immune activity of DCs.So in the further research,the function of DCs should be investigating in AS animal model.Rabbit AS model is one of the most widely used in AS research,as well as rat.The source of dendritic cells mainly derived from the monocytes of the blood,bone marrow,cord blood and mouse bone marrow in current research. Virginia sucessful took the human granulocyte macrophage colony stimulating factor and human interleukin-4 to stimulate and induce the cultured DCs maturation, thus provide a new choose for cultured DCs. The change of blood lipid play a important role in the generation of AS.High density lipoproteins have the function of anti-atheroslerosis,low levels of high density lipoproteins is one of the risk factors for CHD.In current opinion,the anti-atherosclerosis function of HDL mainly closely related to its function in reverse cholesterol transport.HDL reversing cholesterol transport mediate by apolipoprotein AI binding to the cell membrane of the ATP binding cassette transporter A1.Recent studies have found that HDL can be oxidative modified by endothelial cells,macrophages and smooth muscle cell in the body,and the structural of oxidized HDL changed,leading to bind and the ability to transport cholesterol through the ABCA1 decline,but also have cytotoxicity,thus promote the generation of AS.Chemokines are small molecule cytokine which make cells chemotactic move.The structure and function is similar between chemokine,molecular weight of chemokine many between 8-12KD,and have chemotactic effection on neutrophils, lymphocytes,monocytes,etc.In recent years,chemokines and their receptors are more and more attention in the study,and have proven to play an important physiological and pathological effects in body,and participate in inflammation, cancer,autoimmune diseases,allergy,AIDS and other diseases.The reorganization of chemokine,chemokine and antibody antagonists have been entered into clinical research,and become the new hot spots of biological treatment.As known as the most powerful professional antigen-presenting cells,whether DCs swallow and capture ox-HDL and presenting antigen to T cells,as well as the effection of ox-HDL on the mature of DCs and the secretion of monocyte chemotactic protein-1 and macrophage inflammatory protein-1αor not,in present study these questions still unknown.The subject is divided into two parts,the first part of research is cultured the DCs with hGM-CSF+hIL-4 in vitro.Induction of rabbit peripheral blood mononuclear cells(PBMC) and bone marrow mononuclear cells(BMMC) into DCs, take LPS induce the maturation of DCs,and lay the foundation for the tissue engineering and clinical cell diagnosis,treatment of the AS study.In the second part, the process DCs capturing ox-HDL and the situation of ox-HDL inducing DCs maturation should be observed by using fluorescent dyes DiI to mark ox-HDL in vitro,and the effection of ox-HDL on the secretion of chemokine MIP-1αand MCP-1 of DCs should be observed by using ELISA in vitro,and thus the foundation for further study the mechanism of antigen-presenting and atherosclerosis generation are lay.The results are as follows:1 Sthdy on cultured the rabbit PBMC and BMMC-derived DCs in vitroThrough the method of density gradient centrifugation,the number of DCs which collected from 20mL peripheral blood of single rabbit is about 10-20×10~6, and the number of DCs which collected from 10mL bone marrow liquid of single rabbit is about 5-10×10~6.1.1 The result of cell morphologyIn cultured 2 day,cells adhesion to the bottom of polystyrene bottle in the control group,part cells are suspended growth in medium in DCs group,smaller, and no significant difference shape.In cultured 6 day,cells adhesion to the culture bottle bottom with round or oval in shape in control group;cells,which in DCs group showing suspended growth in medium,part of them aggregation in clusters, dendritic-like protrusion on cell surface in different quatity and shape under light microscopy,and irregular shape,rough surface,different protrusion on cell body under scanning electron microscopy.1.2 The result of cell phenotype by flow cytometry analysisIn cultured 6 day,MHCⅡ,CD86 are upregulated expression both in PBMC and BMMC-derived DCs,but CD14 is downregulated expression.There have significant difference in MHCⅡ,CD86,CD14 between PBMC derived DCs group and PBMC derived control group(P＜0.001),and between BMMC derived DCs group and BMMC derived control group(P＜0.001).There have no significant difference in MHCⅡ,CD86,CD14 between PBMC derived DCs group and BMMC derived DCs group(P＞0.05),and between PBMC derived control group and BMMC derived control group(P＞0.05).There have no significant difference in cutured cell among different specimen source,but have significant difference in different stimulated method to cultured cells(P＜0.001).1.3 Detection phagocytosisIn matured DCs,the phagocytosis is declined but the antigen-presenting function is enhanced,so the abilitry to uptake FITC-dextran of macrophages and mature DCs should be compare under the condition of 37℃by flow cytometry. There have no significant difference in cutured cell among different specimen source,but have significant difference in different stimulated method to cultured cells(P＜0.001).The phagocytosis function is lower in PBMC DCs group and BMMC DCs group,but the phagocytosis function is higher in two control groups. There have no significant difference in cutured cell between specimen derived DCs and macrophages,but have significant difference beween DCs and macrophages in each source(P＜0.001).2 Effection of ox-HDL on the maturation of rabbit derived DCs and the secretion of the chemokines2.1 Culture and observation the DCsUnder the inverted phase contrast microscope:in cultured 2 day,cells suspended growth in medium,round,smaller in each group of cells small.In cultered 5 day,cells suspended growth,some cells aggregated into clusters,with irregular shape,different quantity and pattern of the dendritic-like protrusion on the surface, and rich in the cytoplasm.After add 50μg/mL HDL,ox-HDL no apparent changes in cell morphology,a small number of cells suspended or semi-paste on the wall.2.2 DCs processing and antigen-presenting ox-HDL labeled by DiIDCs mixed-incubat with ox-HDL labeled by DiI for 2 hours,fluorescence microscopy showed that the original no color of the DCs,after uptake of ox-HDL labeled by DiI,the cytoplasm also changed into red,cultured for 2 days,this showed more obvious than before.DiI showed good DCs antigen uptake and ox-HDL maturation procession.At the beginning,DCs as a smaller and naive cells,can clearly see the procession of antigen uptake,in which the cell gradual development, riched cytoplasm,transform into many types of irregular and have more extended pseudopodia shapes after uptake antigen.2.3 The effection of ox-HDL on the phenotype of DCsThe suspended cells after intervention for 48h are collected and analysed by FACS,and the result show that,compared with the PBS negative control group,the expression of DCs surface molecules CD86 and HLA-DR which treated with the HDL and ox-HDL are increased,but the expression of CD14 are reduced.There have significant diference in HLA-DR,CD86,CD14 among groups(P＜0.001),and ox-HDL group and PBS group,ox-HDL group and HDL group,but no significant difference in HLA-DR,CD86,CD14 between HDL group and PBS group.2.4 The result of detection chemokine MIP-1αand MCP-1 in the supernatant of DCs which co-cultured with ox-HDLDetection the chemokine MIP-1αin supernatant by ELISA and the results suggest that:the time factor has impact on the expression of MIP-1αin the supernatant(F=34.520,P＜0.001),sub-groups factors(interference factor in training environment) has no impact on the expression of MIP-1αin the supernatant (F=0.722,P＞0.05).The expression of MIP-1αare different vary in different periods,the expression of MIP-1αin HDL group with the exception of at 24 hours and 48 hours have no significant difference(P=0.102),the rest of the time period and compared between each other at other periods have significant differences(P＜0.05),but different group,that is,intervention by different conditions on the expression of MIP-1αin the supernatant at the each time have no significant difference(P＞0.05).The results of detection chemokine MCP-1 in supernatant suggest that:the time factor have an inpact on the expression of MCP-1 in supernatant(F=31.481,P＜0.001),as well as the sub-groups factors(F=47.931,P＜0.001).Different time have different MCP-1 expression,the expression of MCP-lin PBS group with the exception of at 12 hours and 24 hours have no significant difference(P=0.383),the rest the time period and compared between each other at other periods have significant differences(P＜0.001),but different group,that is, intervention by different conditions on the expression of MCP-1 in the supernatant at the each time have significant difference(P＜0.001),and the resultof multiple comparisons showed that the effects of ox-HDL alone compared with other factors at each time have significant differences(P＜0.05).From above two parts experiment,we can conclude that:(1) Rabbit PBMC and BMMC can be induced to differentiate into DCs under the effection of hGM-CSF+hIL-4 for cuturing 5 days,but the positive rate phenotype of expression of PBMC- derived DCs is higher than that in BMMC-derived DCs.DCs from these two sources can be converted into matured dendritic cells under the effection of LPS;(2) In line with the characteristics of matured DCs,the expression of CD86, HLA-DR,are upregulated in rabbit PBMC derived DCs after the intervention of ox-HDL in vitro,but the expression of CD14 are downregulated.(3) The procession of DCs uptaking ox-HDL and maturation is showed by the morphology of DCs uptaking ox-HDL which labeled by diI fluorescent dye,further confirmed that ox-HDL can be identified by DCs,and through receptor uptake, ingestion within the cytoplasm by DCs.(4) After intervention with ox-HDL,rabbit DCs can increase the secretion of MCP-1.(5) The effection of ox-HDL on the chemotactic factor of DCs makes DCs migrate,may be involved in the generation of atherosclerosis.