| Part1The expression and correlation analysis of CCL20and Th17cells from peripheral blood of patients with acute viral myocarditisBackground:Acute viral myocarditis is a common infectious disease in Cardiovascular Medicine. Infiltration of inflammatory cells is the main pathological change of myocarditis. Several kinds of innate immune cells and acquired immune cells, such as lymphocytes and macrophages, are involved in the pathological process. The cytokines and anti-myocardial antibodies secreted by them can introduce autoimmune reaction, and then cause the myocardial necrosis and heart disfunction. Th17cell, which derived from CD4+T cells, is a kind of helper T cells. Th17cells are characterized by secreting IL-17cytokine, which is involved in the process of rheumatoid arthritis, systemic lupus erythematosus, asthma, etc. CCL20, also known as macrophage inflammatory protein (MIP3a), can be secreted by a variety of immune cells. CCL20takes part in the inflammatory response of psoriasis, rheumatoid arthritis, inflammatory bowel disease, cancer and other immune diseases. Thus, we make a study on the CCL20and Th17cell expression in peripheral blood of patients with acute viral myocarditis to find the possible effect of CCL20on Th17cell and their roles in myocarditis.Methods:From2010.06to2013.06,30hospitalized patients with acute viral myocarditis (AVMC) were enrolled in this program as the experiment group, while a group of30healthy volunteers with the similar age and gender ratio was recruited as control group. We used enzyme-linked immunosorbent assay method to detect the concentration of CCL20and IL-17in peripheral blood plasma of the patients and healthy volunteers. Second, we leveraged the density gradient centrifugation to separate mononuclear cells from peripheral blood. Finally, we detected the proportion of Th17cellls in CD4+T cells by flow cytometry and made an analysis on the correlation of them.Results:Compared with healthy volunteers, the CCL20levels from AVMC patients was significantly elevated. CCL20plasma concentration is (11.76±3.61) ug/L for AVMC patients and (2.78±0.64) ug/L for healthy volunteers with P<0.05. In the peripheral blood of AVMC patients, IL-17level and the proportion of Th17cell were higher than healthy volunteers:AVMC patients’ plasma IL-17concentration was (95.6±19.8) ng/L, relatively, healthy volunteers’plasma IL-17was (9.7±4.7) ng/L, P<0.05; The proportion of Thl7cells in peripheral blood CD4+T cells for AVMC patients and healthy volunteers were (1.52±0.86)%and (0.24±0.17)%, P<0.05.Conclusion:For patients with acute viral myocarditis, the CCL20and IL-17concentration in peripheral blood was significantly higher than normal. Similarly, the proportion of Thl7cells in peripheral blood CD4+T cells also increased in AVMC patients. Part2Effect of CCL20on Th17cell chemotaxis in acute viral myocarditisBackground:Innate immune cells, such as macrophages, can secrete CCL20. Th17cells constitutively express CCR6, which is the specific receptor of CCL20. However, the effect that CCL20has on Th17cells is still unclear. SiRNA is a double-stranded RNA with small molecules that can specifically bind to the target gene mRNA, block the translation of mRNA and silence the target gene expression. The goal of our experiment was set to investigate the role of CCL20on Th17cells and the related mechanisms. Fisrt, we separated macrophages and Th17cells from the peripheral blood of AVMC. Second, we leveraged siRNA technology to inhibit the synthesis of CCL20in macrophages. Finally, we explored the chemotaxis effect of CCL20on Th17cells by using transwell chamber to co-cultivate CCL20and Thl7cells.Methods:We seperated macrophages from peripheral blood of AVMC patients. The cells were seeded in a12-well plate and divided into three groups:(1)Test group-transfected with CCL20siRNA,(2) negative control group-transfected with scramble siRNA,(3) blank control group-nontransfected. We detect the concentration of CCL20in the culture supernatant by enzyme-linked immunosorbent assay method. The CD4+T cell isolation kit and Human IL-17secretion assay-cell enrichment and detection kit were used to separate CD4+T cells and Th17cells from the peripheral blood of AVMC patients. First, We put the transwell chambers upon the cultured macrophages, then added CD4+T cells into the upper chamber, co-culture for8h in the cell incubator, and collected the recruited cells in the lower chamber. Then we test the proportion of Th17cells and CCR6+IL-17+cells in the lower chamber by flow cytometry. Secondly, Th17cells were labeled by anti-human FITC-CD4monoclonal antibody and added in the transwell upper chamber. After co-cultured for8h in the cell incubator,we took away the transwell upper chamber, observed the number of Th17cells in the lower chamber under a fluorescence microscope. Last, we added Th17cells in the transwell upper chamber, co-cultured with macrophages for24h in the cell incubator, then the CCR6mRNA expression of recruited cells in the lower chamber was detected by real-time quantitative PCR.Results:The CCL20sccreted by CCL20siRNA transfected macrophages was significantly less than that of the scramble siRNA transfected group and non-transfected group. Under the fluorescence microscope, the number of Th17cells of test group was significantly lower than negative control group and blank control group, while there was no significant difference between two control groups. Moreover, both the proportion of Th17cells and CCR6+IL-17+cells showed an decrease in the test group compared with the control groups. The CCR6mRNA expression of the recruited cells in the lower chamber of test group was significantly lower than the other two groups, p<0.05; while between the two control groups, the cell CCR6mRNA expression showed no significant difference.Conlusion:CCL20siRNA can inhibit macrophages express CCL20. In AVMC, CCL20can recruit Th17cells and promote the expression of CCR6on Th17cells. |
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