Study On The Relationship Between The Gene Expression Of Dendritic Cells And Unstable Plaque In Acute Coronary Syndrome

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(DCs)are the most powerful specific antigen presenting cells that play a role of presenting antigens in the immunologic process.Minor DCs may intensively activate T cells,which may initiate the specific cellular immunity reaction. Their capacities of T cells activation are as 100-1000 folds as that of macrophages. They play an important role in the induction and regulation of immunologic response. It is confirmed by recent studies that arterial wall may offer a good microenviroment to the immunological reactions.A Vascular-associated lymphoid tissue(VALT)that similar to mucous membrane-associated lymphoid tissue in respiratory tract and gastrointestinal tract,exist in the normal endarterium.Some cell masses which are comprised of immunological competent cells and antigen presenting cells distribute diffusedly in these tissues.They may monitor and screen the potential endogeneous or exogenous harmful antigens in the vascular tissues.Vascular dendritic cells(VDCs) can be found in VALT.Normally speaking,only a very small number of VDCs which in an immature form,can be found in the vessel tunica intima and tunica adventitia. Aggregation of VDCs in the affected regions increase and,they always aggregate with T cells and macrophages in the inflammatory infilitrated regions.People presume that DCs may play a role in initiating inflammatory and immunological reactions in the genesis and development of AS for that DCs co-emerge with T cells in the weak affected regions.Recent studies showed that oxidated LDL and nicotine may promote the advancement of AS by activating DCs-mediated acquired immunological reactions.This suggest that DCs may play a role in the pathogenesis of AS and that they may play an important role in the regulation of the triggering and amplification of inflammatory and immunological reactions in AS.However,viewed from the discovery and study history of DCs,we consider that DC is a new kind of immunocyte.Mature status,functional status and typing of DCs differ significantly not only in different populations and different individualities,but they also differ obviously in different developmental stages and different tissue environments in the same individuality.According to hemopoietic stem cell lineage,it is generally accepted that DC can be classified as marrow-like DC and lymphoid-like DC.They are corresponding to DC1 and DC2,respectively.Phenotypes of DC1 subgroups are CD1a+,CD1b+,CD1c+,CD1d low,CD4 low,CD11b+,C D11c+, CD13+,CD 33+,CD 45RA-,CD 45RO+,GM-CSFRa+ and IL-3Ra low.They express high levels of MHC-1,Ⅱ,CD40 and B7,which may induce the differentiation of Th0 to Th1.They also induce Th1-like immunological response. Phenotypes of DC2 subgroups are CD1a-,CD1b-,CD1c-,CD1d-,CD4-,CD11b-,C D11c low,CD13 low,CD 33 low,CD 45RA+,GM-CSFRa low and IL-3Ra+. Relatively,they express low levels of MHC-1,Ⅱ,CD40 and B7 which may induce the differentiation of Th0 to Th1.They also induce Th2-like immunological response. DC2 distribute mainly to Peyer's lymph nodes under the mucous membrane in small intestine,lung,thymus and the liver.DCs Functional statuses differ not only in different cell subgroups but also in different mature statuses.Expression levels of MHC-1,Ⅱ,costimulating factor and adhension molecule in immature DCs are low so they can not activate T cells effectively.But they have great capacity to capture and process the antigens.After their capturing of the antigens,they may become mature. Then an increased expression of MHC-1,Ⅱ,costimulating factors and adhension molecules can be observed.Their capacities to capture antigens decrease but capacities to process and present antigens and activate T cells increase.DCs' transformation of this type is closely correlated to upper regulation of the expression of immunological regulatory molecules such as CD40,CD80 and CD83.If the activation of immunological system and the intervention of inflammation are the pathogenesis of AS,then some questions need to be further confirmed.For examples,what are the functional statuses of DCs which have antigen presenting capacity,whether the excretion of functional protein and immunological regulatory molecules in health people are different from that of CHD cases or not and,whether the gene expression of these proteins change or not.To study on the gene variations of DCs is the key of all the questions mentioned above.Our studies can mainly be divided into 3 portions.Peripheral blood mononuclear cell (PBMC)and serum abstracted from clinical cases are the objectives of our study. The starting point of our study is that whether gene expressions and functional changes of PBMC derived DCs play roles in the genesis and development of CHD or not.The relationship of the Mature status,immunological function and gene expression difference of PBMC derived DCs in peripheral blood with the genesis and development of CHD were observed emphasizely by means of Immunofluorescence,Flow cytometry,Gene array,RT-PCR and ELISA,and so on. The roles of the functional changes of antigen presenting cells in different stages of AS development are further investigated.Its purpose is to offer the experimental evidences and immunotherapeutic means to the immunity-mediated theory of the genesis and development of AS.It may provide new theoretical platforms and technical means to the prevention and treatment of cardiac cerebro vascular diseases. So they will bring long-term economical values and social significance to us eventually.The results are reported as following.1.PBMC derived DCs in ACS cases are in a mature status and,they have capacities to induce the proliferation of T cells.1.1 Comparison of the general clinical data among groups showed no significant difference.81 cases were allocated to AMI group(20cases),UAP group(20cases),SAP group(20cases),CPS group(11cases)and control group(10cases),respectively. Comparison of the general clinical data such as age and gender showed no significant difference among different groups(P＞0.05).Comparison of the main CHD risks such as smoking,hypertension,diabetes mellitus,total cholesterol, triglyceride,HDL cholesterol,and LDL cholesterol among different groups showed no significant difference(P＞0.05).1.2 PBMC derived DCs were induced in vitro and their cell phenotypes were identified by Flow cytometry.Cell shapes under Inverted phase contrast microscope in different days are reported as following.In the 3th day of cultivation,the cells were adhering to the cellular wall for a further growth and their shapes changed from round to irregular, some spinule-like structures newly erupted and,the cytoplasm were getting thicker and thicer.In the 5th day,dendritic-like structures appeared more obvious,cytoplasm got even thicker,DC cells were gradually floating or semi-adhering.On the 7th day of cultivation,the cell shapes made no more changes but the cells started aggregating to from cell masses.Most of the cells were in a state of floating or semi-adherencing, few were adhering.Trypan blue staining indicated that the cellular vigor was higher than 96%.Phenotypes of PBMC derived DCs in peripheral blood were detected by Flow cytometry.They were CD1α+,CD80+,CD831ow,CD86+ and HLA-DR+.1.3 Detection results of IL-12 in DCs and self plasma co-culture solution supernatant.Expression levels of IL-12 in the 12th hour in sample supernatants in control group,CPS group,SAP group,UAP group and AMI group were 19.24±6.24, 27.24±6.65,22.5±6.81,55.23±31.79 and 62.18±32.18 pg/ml,respectively.In the 24th hour they were 45.23±13.14,50.82±14.25,48.77±13.48,127.32±58.16 and 130.26±60.35 pg/ml,respectively.In 48th hours they were 50.21±13.04, 59.25±14.48,51.67±13.82,145.24±64.55 and 151.45±67.47 pg/ml,respectively. IL-12 levels in all 5 groups differed significantly from each other(all P＜0.001).LSD comparison suggest that IL-12 levels in DCs cultured solution supematant of UAP group and AMI group were obvious higher than that of control group,CPS group and SAP group in the 12th,24th and 48th hours.1.4 FACS analysis of the Changes of DCs phenotypes after DCs co-cultured with self plasma.Expression proportions of CD1αon the surface of PBMC derived DCs in control group,CPS group,SAP group,UAP group and AMI group were 11.08±3.82 %,12.28±3.91%,13.71±5.53%,20.09±4.9%and 20.57±4.75%,respectively. CD80 proportions were 46.78±16.86%,48.51±14.75%,46.81±15.92%, 61.39±11.49%and 60.91±17.77%,respectively.CD83 proportions were 47.57±12.34%,46.59±10.22%,45.76±14.11%,54.17±16.41%and 51.31±18.8%, respectively.CD86 proportions were 46.83±7.45%,46.08±10.25%,45.76±17.75%, 62.27±14.01%and 67.98±17.03%,respectively.HLA-DR proportions were 79.01±11.05%,73.77±13.55%,72.16±17.11%,74.13±12.27%and 79.23±9.79%, respectively.CD1α,CD80 and CD86 in all 5 groups differ significantly from each other(P＜0.001).LSD comparison of the increased CD1α,CD80and CD86 phenotypes in UAP group and AMI with that of control group,CPS group and SAP group showed a significant difference.1.5 Allogeneic mixed lymphocyte reaction in all group cases.PBMC derived DCs in control group,CPS group,SAP group,UAP group and AMI group were co-cultured with allogeneic T cells.3H-TDR incorporation efficiency were 1035.8±280.9,1054±247.39,1112.4±319.9,2633.6±426.1 and 2660.4±316.02 cpm/2000cells respectively when the cell number ratio of DCs to T cells was 1:50.3H-TDR incorporation efficiency were 2043.8±303.5,2169.55±311.6, 2139±295.1,5122.3±640.4 and 5069.1±748.9 cpm/2000cells repectively when the cell number ratio of DCs to T cells was 1:20.3H-TDR incorporation efficiency in 5 groups differ significantly from each other(P＜0.001).LSD analysis indicate that the increase of 3H-TDR incorporation efficiency in UAP group and AMI group were significantly higher than that of control group,CPS group and SAP group(P＜0.001). But 3H-TDR incorporation efficiency in control group,CPS group and SAP group did not differ significantly from each other(P＞0.05).3H-TDR incorporation efficiency increased obviously in both UAP group and AMI group when DCs were mixed with T cells in a cell ratio of 1:50.2.Different gene expressions of PBMC derived DCs in peripheral blood were detected by Gene array.Detection by Gene array showed that 5 functional protein expression genes, including G1P2,G1P3,IFIT4,IL-1βand MX1,increased obviously in ACS cases. All they are the highly correlated interferon-induced protein genes.17 genes, including ACPP,AIM2,ATM,CCR1,CCR5,CD1C,FCGR3A,FI16,IL-16,IL-18, LY75,MAP4K3,TAP1,TAP2,TLR1,TNFRSF6 and VCL,expression levels decreased obviously.They belong to antigen recognition receptors,cell chemokine receptors,cytokines and intracellular signal transducting system,respectively.Just as what we described before,the antigen recognition and capture capacity of mature DCs decreased but obtained an increased antigen presenting capacity.The expression levels of the subcellular structure proteins which are related to antigen processing can be seen a decrease,while the expressions levels of antigen presenting related proteins increased.Our studies indicated that the expression of antigen recognition receptors such as TLR1 and FCGR3A decreased in PBMC derived DCs in peripheral blood of ACS cases.Protein genes which in charge of the transmission of specific antigen peptide which will bind with the MHC molecules,including TAP1 and TAP2, seen a decreased expression level.Chemokine receptors,including CCR1 and CCR5, which function to attract DCs to make a deadexis to the antigen-located regions,can be seen a decreased expression when DCs became mature.Decreased expressions of the intracellular signal transducting system-highly related proteins such as MAP4K3 and some cytokines such as IL-16 and IL-18 genes may be correlated to the functional changes of self cell signaling and intracellular cell signaling in DCs.The increased expression genes mostly are INF-induced proteins.Studies on their structures and functions are still on the initial stage.No reports can be read on the relationship between proteins such as IFIT and mature DCs.3.Results by Fluorescent quantitative PCR detective Gene array indicated that Gene array detection may offer a good degree of confidence.The up-regulated expression genes MX1 and IL-1βand the down-regulated expression genes FIF16 were detected by Fluorescent quantitative PCR detective Gene array.The detection results by Fluorescent quantitative PCR detective Gene array indicated that the increased extent of MX1 and IL-1βgene expression levels in ACS group when compared with that of control group are similar to that of the detection results by Gene array.Detection results by Fluorescent quantitative PCR detective Gene array of the decreased FIF16 gene expression level and extent in ACS group and control group are similar to that of the detection results by gene array detection.According to our studies,some conclusions can be drawn as following.①Cell phenotypic expressions of PBMC derived DCs in the peripheral blood of ACS cases are in a status of maturity.DCs in ACS cases are in a state of maturity and activation.②IL-12 excretion levels of PBMC derived DCs in peripheral blood in ACS group are obvious higher than that of control group.DCs in ACS group are capable to induce the proliferation of homogeneous T cells.③Functional protein expression genes,including G1P2,G1P3,IFIT4,IL-1βand MX1,can be seen an increased expression in ACS cases.All they are interferon-induced protein genes which are highly correlated with each other.17 genes,including ACPP,AIM2,ATM, CCR1,CCR5,CD1C,FCGR3A,FI16,IL-16,IL-18,LY75,MAP4K3,TAP1,TAP2, TLR1,TNFRSF6 and VCL,showed obvious decreased expression levels.They belong to antigen recognition receptors,cell chemokine receptors,cytokines and intracellular signal transducting system,respectively.④The up-regulated expression genes MX1 and IL-1βand the down-regulated expression genes FIF 16 were detected by Fluorescent quantitative PCR detective Gene array.So the detection results by Gene array were further confirmed.