| Currently,coronary heart disease(CHD) has become one of the greatest threatens to human beings.It is particularly important to study on associated risk factors for the pathogenesis of CHD which has not been clearly identified. Generally,traditional risk factors can be divided into genetic factors such as family history of heart disease,gender,age,as well as environmental factors such as smoking,dyslipidemia,hypertension and exercises.Recently,factors including metabolic factors such as hyperhomocysteinemia,infection factors including chlamydia pneumoniae,helicobacter pylori,cytomegalovirus,as well as variety of cells and molecule involved in immune responses which have been found and have an important relationship with CHD.Therefore,it is proposed that inflammatory and immune responses probably have relationship with CHD.Based on the former theories,we investigate relationship between gender factors and immune response to clarify one of the pathogenesis of CHD,thereby enrich the theoretical basis for the treatment of CHD.Atherosclerosis(AS) is the pathological basis of CHD,while inflammatory and immune responses centre on the formation of AS in view of internal and external risk factors respectively.Series of specific immune response caused by T lymphocytes plays an important role in the development of AS.The activation of T lymphocytes depends on interaction between T-cell receptor and antigen presented by MHC molecules of antigen-presenting cells(APC).Dendritic cells(DC) are the most powerful APC and play an important role in immune response.When encountered DC,foreign pathogens will be presented to T-cell which can be activated and would proliferate and secrete cytokines,then activate immune responses.DC have been found in arterial wall and increase in the AS plaque,particularly coexisted with T-lymphocyte in the region of inflammation.It is implied that DC may play an important role in the development of AS because variety of recognized risk factors can promote immature DC into mature DC.Among of those,oxidized low-density lipoprotein(ox-LDL) is an important autoantigen causing immune activation of AS, which can cause vascular endothelial dysfunction,participate in formation of foam cell,promote vascular smooth muscle cell proliferation and induce apoptosis and promote DC differentiation and maturation which derived from human peripheral blood mononuclear cells and thereby triggering immune responses.Effects of ox-LDL on AS depend on its own level in blood and its receptor effect.Scavenger receptor is classic receptor of ox-LDL which can modify the function of uptaking ox-LDL.Recently,a new type of receptor of ox-LDL like condensate of oxidized low-density lipoprotein receptor-1(LOX-1) are considered the specific receptors of ox-LDL which were expressed in vascular endothelial cells mainly,and can bind to, phagocytize and degrade ox-LDL.LOX-1 is expressed on the surface of mature and immature DC,monocytes,macrophages,as well as B cell.So LOX-1 participates in the development of AS in multiple links when activated.But it has not been reported that LOX-1 expression on the surface of DC was induced by ox-LDL.It is well known that there are significant different incidence of cardiovascular disease between men and women.Besides the genetic factor and lifestyle,it is likely that female endogenous estrogen play a role of anti-AS.Estrogen replacement therapy can reduce the dangers of cardiovascular disease.Researchers pay more attention to effects of estrogen on immune system.It is indicated that estrogen may play a regulating role in immune system because estrogen replacement therapy can cure some autoimmune diseases through clinical observations.Some progress has been made on DC influenced by estrogen.But such studies on AS have not been reported.In light of the former evidences,we could assume that estrogen play a protective effect on cardiovascular system through inhibiting the immune response.In order to clarify the protective immunity effect of estrogen on cardiovascular system,our research aim at investigating the relationship between estrogen and immune mechanism of AS,exploring the effect of estrogen on DC maturation induced by ox-LDL.Accordingly,theoretical foundation of preventing of CHD is enriched.Our experiments can be divided into two portions.Firstly,in order to explore the relationship between gender factors and the body's immune response and clarify the possibility of sex hormones playing a protective role on immune response in the development of AS.We employed enzyme-linked immunosorbent assay(ELISA), direct chemiluminescent immunoassay technology and flow cytometric technique to measure hs-CRP,IFN-γ,IL-10,estradiol(E2),subgroup distributions of mDC and pDC in the peripheral blood of postmenopausal women with acute coronary syndrome(ACS) and compared those with chest pain syndrome(CPS) group and the healthy control group.Secondly,we used fluorescence microscope,electron microscope,flow cytometric technique,ELISA,reverse transcription PCR(RT-PCR), and western blotting to observe maturity of DC derived from human PBMC induced by oxidized low-density lipoprotein(ox-LDL),stimulating capacity of T lymphocyte proliferation and secretion of cytokines by DC and expression of mRNA and Protein of LOX-1 on the surface of DC.At last,we observed the protective effects of estrogen on maturity of DC,the proliferation of T cell and the secretion of cytokines and the expression of LOX-1,which might provide a new way in biological therapy of autoimmune diseases.The results are as follows:1 Relevance of ACS of postmenopausal women between E2 levels and changes in DC subsets1.1 Comparison of the general clinical data among different groups showed no significant difference.82 cases were divided into as follow:CHD group[AMI group(20cases),UA group(22cases),SA group(20cases)],CPS group(10cases) and control group (10cases).Comparison of the general clinical data showed no significant difference among different group.1.2 Peripheral blood levels of E2 and ratios of E2/T reduced in patients of ACSLevels of E2 and ratios of E2/T of CHD(SA,UA and AMI group) were lower than the group of healthy control,CPS measured by direct chemiluminescent immunoassay and ACS(UA and AMI) were lower than group of SA.While the levels of T in group of CHD(SA,UA and AMI) were higher than those of groups of healthy control and CPS.1.3 Proportion and actual number of DC subgroups in peripheral blood of different groups.mDC proportion and mDC actual number in CHD group(UA,AMI and SA group) were lower than those of control group.But comparison of pDC among groups showed no significant difference.1.4 Detection of plasma hs-CRP,IFN-γand IL-10 levels in different groupPlasma hs-CRP and IFN-γlevels in cases of ACS group were higher than that of control group,CPS group and SA group.There were no significant differences among them.While ratio of IFN-γ/IL-10 in ACS group were higher than those of control group,CPS group and SA group.They also suggest the existence of inflammatory reactions and immunity activation in ACS cases and the level of IFN-γwas in advantage.1.5 The peripheral blood level of E2 and DC subsets proportion and actual number of ACS patients is related each other.The peripheral blood levels of E2 of ACS patients have positive correlation with DC subsets proportion.While the levels of T have no correlation with DC subsets proportion.2 The roles of ox-LDL and LOX-1 receptor on DC maturation derived from PBMC immune and intervention by estrogen.The test include 5 groups(n=8):(1) Control group:DC+PBS;(2) DC+LDL (25 mg/L) group;(3) DC+ox-LDL(25mg/L) group,(4) DC+ox-LDL(50mg/L) group,(5) DC+ox-LDL(100mg/L) group.The test pretreated by 17β-E2 have 5 groups(n=8):(1) Control group(DC+PBS),(2) DC+ox-LDL(50 mg/L) group,(3) DC+ox-LDL(50mg/L)+17β-E2(10-7mol/L),(4) DC+ox-LDL(50 mg/L)+17β-E2 (10-8 mol/L),(5) DC+ox-LDL(50 mg/L)+17β-E2(10-8 mol/L).2.1 Identification of DC by microscopeCell shapes under inverted phase contrast microscope and electron microscope in different days are reported as following:the cells shapes changed from round to irregular after induced by ox-LDL,some spinule-like structures newly erupted and the cytoplasm were getting thicker and thicker.2.2.Impact on the cell phenotypes,identified by flow cytometry.2.2.1 The upregulation of phenotypes expression on DC by ox-LDLPhenotypes of DC derived from PBMC in peripheral blood were detected by flow cytometry.Phenotypes of DC including CD1a,CD80,CD83,CD86 and HLA-DR.DC stimulated by ox-LDL have high expression of CD1a,CD86 and HLA-DR.ox-LDL can induce DC maturation,and with the increase of concentration DC have higher maturity.2.2.2 17β-E2 decrease the expression of DC phenotypes induced by ox-LDLThe expression of DC phenotypes of CD1a,CD86 and HLA-DR which pretreated by 17β-E2 were lower than that in ox-LDL group in a concentration-dependent manner.This indicate that 17β-E2 may decrease the expression of DC phenotypes induced by ox-LDL.2.3 Impact on allogeneic mixed lymphocyte reaction2.3.1 ox-LDL increase 3H-TDR incorporation efficiency3H-TDR incorporation efficiency of DC stimulated by ox-LDL higher than that of control group and LDL group,and in a concentration-dependent manner.2.3.2 17β-E2 decrease the 3H-TDR incorporation efficiency of DC stimulated by ox-LDLThe 3H-TDR incorporation efficiency of DC stimulated by ox-LDL which pretreated by 17β-E2 lower than those of ox-LDL group,and in a concentration-dependent manner.Indicating that 17β-E2 may reduced the capacity of allergenic mixed lymphocyte reaction of DC stimulated by ox-LDL. 2.4 Impact on levels of IFN-γand IL-10 stimulated by ox-LDL2.4.1 ox-LDL increase the levels of IFN-γof DC stimulated by ox-LDLAfter co-cultured DC stimulated by ox-LDL with T lymphocytes,the levels of IFN-γand the ratios of IFN-γand IL-10 higher than that in PBS and LDL group in a concentration-dependent manner,while ox-LDL have no influence on levels of IL-10. This indicate that ox-LDL may stimulate DC maturation,promote Th1 secreteing IFN-γ,and have capability to promote Th1-type immune response.2.4.2 17β-E2 decreased the levels of IFN-γof DC stimulated by ox-LDL and increase levels of IL-10The levels of IFN-γof DC stimulated by ox-LDL which pretreated by 17β-E2 lower than those of ox-LDL group and in a concentration-dependent manner.This indicate that 17β-E2 may reduce the capacity of allergenic mixed lymphocyte reaction of DC stimulated by ox-LDL and may reduce capability of Th1-type immune response.17β-E2 on concentration higher than physiological concentration may increase levels of IL-10.This indicate 17β-E2 may have the capability to promote Th2-type immune response.2.5 The measurement of LOX-1 mRNA on DCGroup added blocking agent:DC+Polyinosinic acid+Carrageenan(250mg/L+ ox-LDL(50mg/L).2.5.1 The upregulation of mRNA expression on DC by ox-LDLThe levels of LOX-1 mRNA on DC surface in ox-LDL group were much higher than that in PBS group and LDL group in concentration-dependent manner.The blocking agent of LOX-1 can decrease the LOX-1 mRNA expression,which suggest that ox-LDL can induce the mRNA expression on DC and ox-LDL exert the function by binding with its receptor.2.5.2 The downregulation of mRNA expression on DC by 17β-E2The levels of LOX-1 mRNA in the group of adding 17β-E2 and PBS is lower than that in the group of adding ox-LDL,which suggest that 17β-E2 could downregulate expression of LOX-1 mRNA on DC which is induced to maturation by ox-LDL. 2.6 The measurement of protein expression of LOX-1 by Western-blot2.6.1 The upregulation of protein expression on DC by ox-LDLThe expression of LOX-1 protein on DC in ox-LDL group is much higher than that in PBS group and LDL group in concentration-dependent manner.The blocking agent of LOX-1 could decrease expression of LOX-1 protein,which suggest that ox-LDL can induce protein expression on DC and ox-LDL exert the function by binding with its receptor.2.6.2 The downregulation of LOX-1 protein expression on DC by 17β-E2.The levels of LOX-1 protein expression in the group of adding 17β-E2 and PBS is lower than that in the group of adding ox-LDL,which suggest that 17β-E2 could downregulate LOX-1 protein expression on DC which is induced to maturation by ox-LDL.The results are reported as follow:①The level of E2 in post-menopause women decreased and the balance between E2 and T is disturbed.②The subsets and number of DC in post-menopause female patients with ACS have changed.DC in patients with ACS is activated.③The levels of E2 in post-menopause women with ACS correlate with the subsets of DC in peripheral blood.④The levels of hs-CRP and IFN-γin post-menopause female patients with ACS increase and the inflammatory reaction in patients is activated.⑤The level of hs-CRP and IFN-γin peripheral blood vary with the proportion of DC subsets,the change of DC subsets proportion correlate with inflammation,this is concerned with plaque instability.⑥17β-E2 downregulate DC maturity of immune function.⑦ox-LDL induce the maturation of DC by binding with its receptor,and then facilitate the Th1 reaction.⑧The down regulation of ox-LDL by 17β-E2 could induce DC to maturation and the LOX-1 protein to be expressed in dose-dependent manner,this suggest that 17β-E2 possess the function of protective effect on cardiovascular system.
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