| ObjectiveAcute coronary artery sydrom arising from atherosclerosis has been a common disease that influence the health of midaged people. The disease presents an increasing tendency year by year. However, the prevent and treatment of atherosclerosis is very difficult because of the mechanism has not known. Recently , the idea that atherosclerosis is an inflammatory disease is generally acknowledged. Many immune cells (macrophages, T cells) and cytokines participate the occurrence and development of atherogenesis. Furthermore, the function of CD4~+T cell in atherosclerosis is concerned by many research groups. CD4~+ T cell is a kind of heterogeneous population that could differentiate into different subsets after stimulated with antigen. The subsets include conventional Th1 and Th2, regulatory T cells and new subsets Th17. Nowadays, the function of different CD4~+T cells on the development of atherosclerosis is not clear. There are documents show that Th1 could promote the formation of plaque and the role of Th2 has disputes. Recently, there are two research group think that CD4~+CD25~+Foxp3~+T cells play a protective role in atherosclerosis, but the role of Th17 has not known.Th17 is a CD4~+T cell subsets that mainly secret IL-17 . IL-6 and TGF-βcould promote the Th0 cells differentiating into Th17 subsets when CD4~+T cells were stimulated with antigen. The transcription factors that control the differentiation was RORγt. Many results show that Th17 cells play a crucial role in the induction of autoimmune diseases such as rheumatoid arthritis(RA) and experimental autoimmune encephalomyelitis (EAE). Atherosclerosis is an autoimmune disease occurring in vascular walls. The function of Th17 cells on the formation and development of atherosclerosis was not well known.To determine the relationship between T cell subsets and the occurrence and development of athrosclerosis, and the influence of Th17 cells on the formation of atherosclerotic plaque, we study from four main points:1. The establishment of atherosclerotic model and the process of the atherosclerotic plaque formation.2. The change of different Th subsets in the process of atherosclerotic plaque formation.3. The expression of transcript factors and cytokines in the the process of atherosclerotic plaque formation.4. The confirmed function of Th17/IL-17 in the formation of atherosclerotic plaque.Methods1. The establishment of atherosclerotic mice model(1) ApoE knockout mice were fed with high cholesterol diet from 8 weeks old, and collars were placed around the left common carotid arteries at 10 weeks old. Then, the mice were fed high cholesterol diet to 16 and 24 weeks.(2) Separated the left carotid arteries of different aged mice and prepared continuous frozen sections. Then detected the formation of the atherosclerotic plaque of different aged mice and the lipidoses with HE staining and Oil red O staining.(3) To detect the expression of relative inflammatory cytokines with RT-PCR.2. The change law of Th17, Th1 and Treg cells in the process of atherosclerosis(1) To prepare the splenocyte suspensions liquid from non-atherosclerosis and the early stage and late stage atherosclerosis mice, then labeled with different antibody.(2) To detect the percentage of Th17, Th1 and Treg cells in spleen of mice model with FCM 3. The expression of transcript factors and cytokines in the plaque(1) To prepare carotid arteries and artery from non-atherosclerosis and the early stage and late stage atherosclerosis mice(2) To dectect the expression of T-bet ,RORγt and IFN-γ, IL-17 with RT-PCR4. The influence of cells that secrets IL-17 on the formation of atherosclerotic plaque(1) 20 ApoE knockout mice were divided into 2 group at random and fed with high cholesterol diet from 8 weeks old, and collars were placed around the left common carotid arteries at 10 weeks old. Then, the mice were fed high cholesterol diet.(2) To inject neutralizing anti-mouse IL-17 antibody intraperitoneally to ApoE knockout model mice after surgery 5 days and inject isotype IgG to control.(3) To detect the size of plaque with micro-ultrasound(4) To detect the plaque of carotid arteries with HE staining and Oil red OResults1. The establishment of atherosclerotic model and the process of the atherosclerotic plaque formation.(1) The formation of atherosclerotic plaqueTo determine the function of T cell in the formation of atherosclerotic plaque, we establish quick atherosclerotic model with ApoE-/- mice. The mice were fed with high cholesterol diet from 8 weeks old and the carotid artery was placed with collars at 10 weeks to promote the formation of plaque. To mice 16 weeks old and 24 weeks old, we separate carotid artery and prepare the frozen sections, then staining with HE and oil red O. The results show that no obvious plaque and the vascular wall was only thicken at 8 weeks ApoE-/- mice compared with C57BL/6 mice. There is significant plaque at 16 mice weeks, the fibrous cap was obviously thicken, smooth muscle cells significantly increased and many cells, collagen in lesions . That is the fibrous plaque stage,ie. the early stage of plaque . To 24 weeks, the plaque is no significant thicken compared with 16 weeks, but the fibrous cap obviously become thinning and the content of lipid significantly increased, smooth muscle cells reduced compared with 16 weeks mice. That is the atheromatous plaque, ie. the late stage of plaque. Littermate wild C57BL/6 has no plaque in the whole process.(2) The change of relative inflammatory cytokines in plaqueTo evident the existence of inflammatory response in plaque, we detect the expression of relative adhensive moleculars and inflammatory cytokines in aortic arch and carotid artery that have formed plaque. The results show that the expression of vascular adhensive molecular-1 (VCAM-1), IL-12 and TNF-αwere obviously higher with the formation of plaque.2. The change of different Th subsets in the process of atherosclerotic plaque formation.(1) The change of regulatory T cells in the process of atherosclerotic plaque formationTo research the change law of regulatory T cells with the formation of atherosclerotic plaque, we detect the percentage of Tregs from 8 weeks, 16 weeks and 24 weeks old mice splenocytes with the flow cytometry. The results show that CD4~+Foxp3~+T cells are higher than control from non-atherosclerosis to atherosclerosis. In CD4~+Foxp3~+T cells, CD4~+CD25~-Foxp3~+T cells significantly increase through the whole process, CD4~+CD25~+Foxp3~+T cells has no obvious change. Furthermore, we found whether in ApoE-/- or in C57BL/6 splenocytes CD4~+CD25~+Foxp3~+T cells show a decreasing tendency with the age. However, the proportion of CD4 CD25 Foxp3~+T cells shows an increasing tendency. These data suggests that these two regulatory subsets play different roles in atherosclerosis and aging. The function of them need more research. (The research has acquired the assistance of national natural science funds.)(2) The change of Th1 and Th17 in the formation of atherosclerotic plaqueTo research the function of Th17 in the formation of atherosclerotic plaque and the relationship with Th1, we prepare the splenocyte suspensions liquid from non-atherosclerosis, the early stage and late stage atherosclerosis mice and detect the percentage of Th1 and Th17 cells in spleen of mice model with intracellular immunofluorescent staining for flow cytometry. The results show that the percentage of Th1 and Th17 cells has no obviously change in non-atherosclerotic plaque and significantly increased at 16 and 24 weeks mice compared with control. Furthermore the increasing of Th17 and Th1 is positively related to the size of plaque. This suggests that Th1 and Th17 cells both could promote the development of atherosclerosis3. The expression of transcript factors and cytokines in the plaqueTo determine the existence of Th1 and Th17 in the atherosclerotic plaque, we extract RNA from carotid artery and aortic arch of non-atherosclerotic , early stage and late stage plaque, then we detect the expression of RORγt, T-bet and IFN-γ, IL-17 in plaque with RT-PCR. The result shows that the expression of transcript factors both increase obviously in the early and late stage of the plaque formation. The expression of IFN-γand IL-17 both up-regulates at ApoE-/- mice 16 weeks old, however, they decrease in the late stage of the plaque formation. This proves that Th1 and Th17 both participate the formation of atherosclerosis.4. The confirmed function of Thl7/IL-17 in the formation of atherosclerotic plaqueIL-17 is the mainly effective molecule that Th17 secrets. To determine the influence of Th17 cells in the formation of atherosclerotic plaque, we injected neutralizing anti-mouse IL-17 antibody intraperitoneally to neutralize the function of IL-17. After one weeks of interference, we detect the size of plaque with micro-ultrasound and HE staining. The result shows that carotid artery of all the mice (9/9) in control group receiving isotype control antibody developed larger plaque, whereas only 10% (1/10) of mice treated with anti-IL-17A antibody had minor plaque and other mice has no plaque. This suggests that Th17 cells secreting IL-17 could promote the formation of atherosclerotic plaque and there is significant value to treat atherosclerosis with this.Conclusions1. Different Th subsets both change in the process of the plaque formation(1) CD4~+Foxp3~+T cells were higher than control from non- atherosclerosis to atherosclerosis. In CD4~+Foxp3~+T cells, CD4~+CD25~-Foxp3~+T cells were significantly increased and CD4~+CD25~+Foxp3~+T cells has no obvious change. This present that CD4~+CD25~-Foxp3~+T probably has the role of positive regulation.(2) Th1 and Th17 cells show up-regulatory tendency in the formation of atherosclerotic plaque and the increasing of them is positively related to the size of plaque. Furthermore, the transcript factors and cytokines for Th1 and Th17 both increase. This suggest that Th17 and Th1 both participate the formation of atherosclerosis.2. The plaque was significantly smaller after interference with neutralizing anti-IL-17 antibody than control. This proves that Th17 could promote the formation of atherosclerotic plaque.Innovation and significance1. The innovation of the research is that we observed the change law of T cell subsets with the formation of atherosclerotic plaque. We found that Th1 and Th17 obviously increased in the early stage of atherosclrosis. We firstly found that IL-17 secreting cells-Th17 participate the formation of atherosclerotic plaque in the early stage. This provide a novel view point for the research of the mechanism of the atherosclerosis and provide the first hand information for clinical treatment with anti-IL-17 antibody.2. Furthermore, regulatory T cells has significantly change with the formation of atherosclerotic plaque. We found that CD4~+CD25~-Foxp3~+T cells obviously increased, this raised a new question about the function of regulatory T cells in atherosclerosis.Research limitation1. The plaque of carotid artery is too small with the ApoE-/- mice model, this question limited the research in the local of plaque.
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