| Coronary heart disease (CHD) has been the greatest threaten to human beings since the arrival of the 21th century. However, the pathogenesis of CHD has not been clearly identified, so it is a great hindrance to the treatment of CHD. Thus, to study on the pathogenesis of CHD has become a pop trend of cardiology. It is generally accepted that atherosclerosis (AS) is the pathologic basis of cardiac cerebro vascular disease. AS is considered to be a progressing course which including the formation of fatty streak, fatty plaque, fibrous plaque, atherosclerotic plaque, and so on. People's initial recognition on AS is the anabrotic tumor-like proliferation of artery endomembrane in the final stage by clinical autopsy of dead cases. Subsequently, people discovered fatty streak and fatty plaque on arterial vessel walls in young cases which died of any disease but CHD. Finally, people got to know that AS proceeds slowly and gradually by making animal experiments and further clinical observations. Clinical observations in recent years had found that end stage CHD would have an accelerated progress which appears as sudden onset of an event with a high rate of risk, and so on. The mortality and mutilation rate will be surprising high if the salvages and treatments are not available in time. The predominant pathogenic characteristic of the arteries in these cases is the forming of unstable plaques. Plaques in this situation are always fragile so they may disrupt easily and quickly. Eventually, a chute-like thrombogenesis which will worsen the disease is caused. People again realized that end stage CHD may evolve acceleratedly and progress intensively. So we call this kind of end stage CHD acute coronary syndrome (ACS). The concept is emphasizing on a worsen stage in the developmental course of CHD. The clinical symptoms and signs are larvaceous in the initial and advancing stages of CHD, but when it comes to the end stage, the mortality and mutilation rate is getting surprisingly high, making great damage or loss to people' daily life or work.AS experiences a slow and continuous pathological change, but may speed up in the end stage of its natural course. People found that pathological process of this kind has some similarity to that of trauma, tumor, inflammation, or autoimmune diseases, and so on. So different kinds of presumes on the morbility and progressing mechanisms of AS emerged, such as theories of trauma-like disease, tumor-like disease, inflammation-like disease, or autoimmune disease, just name a few. However, no even a literature documents that AS is a process of repair in any kind of trauma for that AS advances to a more serious stage when more repairs are done. No one confirms that AS is a kind of tumor for that more cells will experience apoptosis with more cellular proliferation. No one had even found out an antigen that would generally be accepted if it is proved to be a chronic inflammation or an autoimmune disease. Any of these theory faces a difficulty in explaining the morbility and advancing mechanism of AS. So how to make a breakthrough to find out a more convincing theory to help the explanation of AS pathogenesis is a difficulty to any one of the existed theory. Detections on the activated immunocells and cytokines in vivo in ACS cases and, detections made recently on the activated immunocytes in different stages of AS indicate that not only can we detected a great sum of activated T cell and monocytes, but also we can detected a high level expression of pro-inflammatory factors, in the peripheral blood, this suggests that a intensified inflammatory reaction in the body. With more knowledge on activated T cells are obtained, people pay more attention to the roles of inflammation and immunity in the development of AS than ever before. Studies on T cell subgroup, T cell typing, receptor expression and adhension factor had found that a great sum of activated T cells appeared in the peripheral blood and affected tissues of ACS cases. The difference in subgroups, typing and protein expression can also be noticed. The desire to know what activate T cells makes people pay more and more attention to the existence of dendritic cells (DCs). DC is a main kind of antigen presenting cell (APC) which are capable to induce the activation of T cells. Questions as whether DCs exist in the AS plaques or not, what is the origin of DCs, whethr DCs are in a mature status or not and what are their functioning status, whether DCs phagocytize the antigens and then present the antigen peptide- stimulated signs to incipient T cells so as to induce the proliferation and activation of T cells, have becoming the focus presently.Normally speaking, DCs can hardly be seen in all kinds of body tissues. DCs are less than 1% of all the karyocytes in peripheral blood. It is even harder to find out any DCs in the normal vessel walls. All of these make it difficult to make a further study on DCs. Some scholars obtain DCs in vitro by cultivation of mononuclear cells from peripheral blood. They compared the maturity and immune functions of DCs in different stages of AS. They found that the PBMC derived DCs in ACS cases showed a high expression of costimulatory molecules and adhension molecules such as CD80, CD86, CD83, and so on. What's more, they had found that the DCs appeared to be mature and had a strong ability to activate T cells. All of these suggest that some substances which are capable to induce the activation of DCs and make them appear to be mature, exist in ACS cases. AS-related substances such as oxidated low density lipoprotein, nicotine and heat shock proteins, may induce the maturity of DCs and, give them the immune activity. However, if the study just retents on the in vitro experiments which may only confirm that the AS-induced antigen stimulus play roles in the activation of cells, or only makes a detection on the maturity standards of PBMC derived DCs cultured in vitro in ACS cases, no further progress will be made on the forming and advancing mechanism of AS. So, the questions mentioned above still can't be confirmed.In order to further confirm that DCs certainly play a role in the progress of AS, and that they initiate and activate T cells in the forming of unstable plaques in ACS cases so as to intensify the inflammatory reaction and, on the basis that DCs may experience a developmental process of transforming from bone marrow stem cells to DCs precursor cells in vivo firstly, and then they will deadexis to the antigen-located sites, then the immature DCs may phagocytize and present the antigens so as to become mature cells which will deadexis to the lymph node T cell regions subsequently to activate incipient T cells for their further proliferation, though we can not detect the distribution and mature status of DCs in AS regions, we are capable of analyzing the distribution of DCs in peripheral blood, so it will help to reflect their deadexis to the vessels in a different aspect. If DCs aggregate and be activated by phagocytizing antigens in the plaques in ACS cases, their distribution levels in vessel walls may raise, so a redistribution of DCs in peripheral blood and lymphs can be noticed. We started our study with the presume that detection of DCs proportion and quantity in different stages and different tissues in their genesis and developing courses would help to investigate the roles of DCs in the forming of unstable plaques in AS, so it may help to set up a experimental ground for the establishment of a confirmed immune mechanism of CHD.Our experiments can be divided into three portions. 81 cases were allocated into 4 groups. They were control group (with a normal coronary angiography (CAG)), chest pain syndrome (CPS) group (with chest pain symptoms but a normal CAG), stable angina pectoris (SAP) group and acute coronary syndrome (ACS) group, respectively. Enzyme-linked immunosorbent assay (ELISA) was employed to the analysis of IL-6 and hs-CRP in the peripheral blood of cases. The subgroup distributions of mDC and pDC in peripheral blood were detected by Flow cytometric technique. Then the distributions of DCs and T cells in peripheral blood, spleen and aorta in AS models such as DM rats and DM plus high fat diet rats, were detected by Immunohistochemistry technique and Flow cytometric technique. Bone marrow derived DCs cultured in vitro were fluorescence CFSE marked and reinfused to the caudal veins of employed rats. The distributions of DCs in peripheral blood and aorta were detected by Flow cytometry and observed by fluorescent microscope. Finally, bone marrow derived DCs were stimulated in vitro by AS-related antigens and, chemotatic factor(including MIP-1αand MCP-1) levels in culture supernatant were observed by ELISA. The expression levels of chemotatic factor receptors were detected by Fluorescent quantitative PCR. The results are reported as following.1. mDC subgroup proportion and actual number decrease in the peripheral blood of ACS cases.1.1 Comparison of the general clinical data among different 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 Proportion and actual number of DCs Subgroups in peripheral blood of different groups.Subgroup classification of mDC and pDC was on basis that CD11C and CD123 were positive but PBMC was negative on their cytomembrane. mDC proportion that account for all PBMC in peripheral blood of Control group, CPS group, SAP group, UAP group and AMI group were 12.03±3.9‰, 12.3±3.3‰, 10.96±6.4‰, 4.74±2.63‰and 4.97±2.7‰, respectively. The actual number of mDC in peripheral blood were 84.4±37.3/ul, 81.2±34.8/ul, 73.2±42.2/ul, 41.0±28.3/ul and 45.8±25.2/ul, respectively. Comparison of mDC proportion and mDC actual number in different groups showed a significant difference (P<0.001) . LSD comparison of mDC proportion and mDC actual number in UAP group and AMI group with that of control group, CPS group and SAP group showed a significant difference (all P<0.001) . But comparison of pDC among groups showed no significant difference.1.3 Detection of plasma hs-CRP and IL-6 levels in groupsPlasma pre-inflammatory factor levels (including hs-CRP and IL-6) were detected by ELISA. HsCRP levels in control group, CPS group, SAP group, UAP group and AMI group were 2.1±0.40, 2.2±0.39, 2.2±0.43, 5.95±1.22 and 22.2±5.72ng/ml respectively. IL-6 levels were 4.47±1.02, 4.5±0.94, 4.7±0.98, 6.17±1.27 and 20.1±4.75pg/ml respectively. Hs-CRP and IL-6 levels in five groups differed significantly from each other (P<0.001). LSD Comparison of Hs-CRP and IL-6 raised levels in UAP group and AMI group with that of control group, CPS group and SAP group showed a significant difference (all P<0.001). These comparisons indicate that plasma hs-CRP and IL-6 levels in cases of ACS group were higher than that of control group, CPS group and SAP group. They also suggest the existence of inflammatory reactions and immunity activation in ACS cases.1.4 DCs subgroup proportion and quantity in the first day of hospitalization and 1 week after PCI, and detections of plasma hs-CRP and IL-6 of in AMI cases.For the fact that both DCs subgroup proportion and quantity raised significantly in peripheral blood of AMI cases, we re-checked the subgroup proportion and quantity of DCs and, detected the plasma hs-CRP and IL-6 levels in some of the AMI cases 1 week after PCI. We found that mDC and pDC in peripheral blood account for 9.24±2.64‰and 1.33±0.32‰, respectively, of all PBMC number. mDC proportion post-PCI differed significantly from that of pre-PCI(p<0.05). Plasma hsCRP and IL-6 levels post-PCI were 14.37±2.50ng/ml and 15.83±3.02pg/ml, obviously lower than that of pre-PCI (p<0.05). The details can be seen in figure 1-3. mDCs subgroup proportions and quantity and pre-inflammatory factors levels in the peripheral blood of AMI cases decreased significantly 1 week after treatments. These suggest that mDCs subgroup proportions are highly correlated to the variation of inflammatory factors.1.5 Detection of DCs subgroup proportions and quantity, and the plasma hsCRP, IL-6 levels in peripheral blood immediately before and after PCI.PCI may do some injuries to the involved regions. In order to avoid the influences of operation on plasma inflammatory factors and DCs subgroups, we detected DCs subgroup proportions and quantity and plasma hsCRP, IL-6 levels in peripheral blood 12 hours after operation in some of the PCI cases. The results showed that in PCI cases before operation, mDC and pDC accounted for (0.38% -2.47%) and (0.12-0.34%) of all PBMC number, plasma hsCRP and IL-6 levels were (1.8-29.2ng/ml) and (4-25 pg/ml), respectively. 12 hours after PCI, mDC and pDC accounted for (0.38%-1.49%) and (0.11-0.21 %) of all PBMC number, plasma hsCRP and IL-6 level were (1.5-30.6ng/ml ) and (3.4-23.8pg/ml ), respectively. Comparisons of DCs subgroup proportions and plasma hsCRP and IL-6 levels in peripheral blood between post-PCI and pre-PCI showed no significant difference (p>0.05). This suggests that PCI have no obvious effects on plasma inflammatory factor levels and the distribution of DCs subgroups.2.The distribution level of DCs decreased in peripheral blood in DM -rats, and marked-DCs tracing suggests that DCs deadexised to aorta walls accerelatedly.2.1 The modeling of DM-rats and comparison of the general data among groups.Blood sugar exceeded 16. 7mmol/L in all 20 rats 3 days after an intraperitonealinjection of STZ. The achievement ratio of DM-rats modeling was 100%. 2 months after intraperitoneal injection of STZ, body weight in control group raised a big more when compared with that of DM group but with no significant difference among groups. Blood sugar in DM group was higher than that of control group when the experiment was finished. The cholesterol levels in 3 groups differ significantly from each other. LSD comparison indicate that cholesterol level in high fat diet group was significantly higher than that of DM group and control group (P<0.001). Triglyceride levels in 3 groups differed significantly from each other. LSD comparison find that Triglyceride level in high fat diet group was significantly higher than that of DM group and control group (P<0.001), while DM group was higher than that of control group (P<0.001).2.2 Comparison of aortic tunica intima proliferation among groups.Aorta morphology of the cases in 2 general groups was compared by Light microscope after 4 weeks' feeding. Tunica media and tunica intima in DM group thickened significantly with much of proliferated smooth muscle cells which circularly lined up in order. Much of extracellular matrix formed in the newly grew tunica intima and, obvious stenosis could be observed in the lumens. Comparison of different parameters such as total encircle area of endo-elastic membrane, newly grew tunica intima area, lumen area and tunica intima proliferation percentage in 3 groups showed a significant difference. LSD comparison indicate that the newly grew tunica intima areas in high fat diet group and DM group were larger than that of control group (P<0.05). The lumen areas diminished obviously (P<0.001) and the tunica intima proliferation percentages increased obviously in high fat diet group and DM group when compared with that of control group (P<0.001). All these suggest that the forming of new intima in aorta tunica intima in rats. 2.3 Comparison of DCs proportion in the peripheral blood, spleen and aorta in all 3 groups.The detection of DCs was on the basis of a positive expression of OX-62 and MHCⅡon the cytomembrane of cell mass. Results analysis by Flow cytometry showed that DCs distribution percentage in peripheral blood in control group, DM group and DM combined with high fat diet group were 1.18±0.79%, 0.31±0.17% and 0.32±0.19%, respectively. A significant difference can be seen in 3 groups (P<0.01). LSD comparison showed that peripheral blood DCs distribution percentage in high fat diet group and DM group were lower than that of control group. DCs distribution percentage in spleens in control group, DM group and DM combined with high fat diet group were 1.74±0.65%, 1.80±0.25% and 1.86±0.31%, respectively. No significant difference cab be seen among 3 groups. Aorta DCs distribution percentage in control group, DM group and DM combined with high fat diet group were 0.44±0.23%, 1.66±0.24% and 1.57±0.32%, respectively. A significant difference can be seen among 3 groups(P<0.01). LSD comparison indicate that DCs distribution percentage in aortas in DM group and DM combined with high fat diet group were higher than that of control group.2.4 DCs were CFSE fluorescent-labeled for tracing uses. The tracing of CFSE fluorescent -labeled DCs in peripheral blood within 1-7 was reported as following.CFSE fluorescent-labeled DCs distribution percentages in peripheral blood from rat vena caudails were detected by Flow cytometry in the 1st, 3nd, 5th and 7th day after a pellet-like venous injection of 0.5ml DCs tracing solution. They were 11.73±1.68%, 5.53±1.24%, 6.9±1.21%, 3.92±0.52% and 1.32±0.6% respectively in control group , 10.59±2.82%, 4.34±0.9%, 3.74±0.79%, 1.94±0.58% and 0.67±0.15% respectively in DM group , and 10.9±2.65%, 3.99±1.07%, 3.03±0.5%, 1.74±0.28% and 0.43±0.24% respectively in DM combined with high fat diet group. These suggest that CFSE fluorescent -labeled DCs distribution percentages in peripheral blood in DM group and DM combined with high fat diet group were obviously lower than that of control(P<0.001).2.5 The tracing of CFSE fluorescent -labeled DCs in aorta.The rats were executed and their aorta cells were isolated 7 days after the tracing. DCs distribution proportions were detected by Flow cytometry. They were 0.73±0.49%, 2.01±0.32% and 1.59±0.52% in control group, DM group and DM combined with high fat diet group, respectively. A significant difference can be seen among 3 groups. LSD comparison showed that DCs percentage in aortas of high fat diet group and DM group were higher than that of control group (P<0.01).3. ox-LDL may stimulate bone marrow derived DCs to excrete MCP-1 and express a high level of CCR-7 receptors.3.1 Ox-LDL and bone marrow derived DCs were co-cultured and the MIP-1αand MCP-1 levels were detected afterward.Supernatant MIP-1αand MCP-1 levels in different time stages were detected by ELISA. Supernatant MCP-1 level raised gradually along with time-lapse and the raising of oxLDL concentration. This differed significantly from that of control group (P<0.01). Comparison of the raised MIP1γlevels in both groups showed no significant difference.3.2 Expressions of chemotatic factor (CCR5 and CCR7) receptors were detected by Fluorescent quantitative PCR.Detection results of CCR5 and CCR7 gene expression by Fluorescent quantitative PCR also suggest that CCR5 gene expression levels in both experiment group and control group do not differ significantly from each other. While CCR7 gene expression level in experiment group was higher than that of control group.According to our experiments, some conclusions can be drawn as following.①Variation of mDCs subgroup proportion and actual number in peripheral blood of ACS cases can be observed and, DCs in vivo of ACS cases are on a status of activation.②Excretion of cytokines such as IL-6 and hs-CRP in the peripheral blood of ACS cases increase and ACS cases are being in a inflammation activated state.③mDCs subgroup percentages in the peripheral blood of ACS cases are varying with the changes of plasma IL-6 and hs-CRP levels. Variation of mDCs subgroup proportion is highly correlated to inflammations. This may have some matters with the instability of the plaques.④DCs distribution in peripheral blood in both DM group and DM combined high fat diet group decreased and, DCs proportion in the aortas of both groups can be seen a raise. So it is indicated that peripheral blood DCs in DM rats may speed up to deadexis to the arteries.⑤Bone marrow derived DCs were CFSE fluorescent-labeled in vitro, and then reinfused them to experiment cases for a tracing. Observations suggest that marked-DCs concentration in peripheral blood decreased obviously with time -lapse. Marked-DCs proportions raised in the aortas of DM group. This suggests the speeding up deadexis of DCs to arteries walls is highly correlated to the genesis of AS.⑥ox-LDL was co-cultured with bone marrow derived DCs. MCP-1 level in the culture supernatant fluid raised gradually with time-lapse and the raising of oxLDL concentration. While expressions of CCR7 receptors in experiment groups were obviously higher than that of control group. DCs may be stimulated by oxLDL to become mature and then express mature chmotatic factor receptors.
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