CpG-ODN调控PD-L1在自身免疫性心肌炎模型中的作用研究

发布时间：2018-07-27 19:05

【摘要】：研究目的:心肌炎是(myocarditis)由多种病毒感染、药物毒性作用等多种病因引起的心脏急性炎症性疾病,伴有各种炎症细胞浸润与心肌细胞损伤甚至坏死。部分心肌炎可快速发展至扩张型心肌病(Dilated cardiomyopathy,DCM),同时扩张型心肌病患者血清标本中可检测出多种抗心肌的自身抗体,证实了自身免疫性心肌炎(autoimmune myocarditis)在扩张型心肌病发病中起着重要的作用。因此,建立合适的自身免疫性心肌炎动物模型,模拟心肌炎免疫性损伤的发病过程,在心肌炎发病机制、自身免疫损伤的研究,及治疗效果及预后中有着至关重要的意义。本实验拟建立合适的自身免疫性心肌炎小鼠模型,通过PCR及免疫组化等方法研究程序性细胞死亡分子1配体(programmed death ligand 1,PD-L1)在自身免疫性心肌炎小鼠中表达情况,并寻求一种方法可以调控小鼠心脏内皮细胞表达PD-L1,以期为心肌炎发病机制的研究提供一种新的研究方向,并为临床治疗心肌炎提供一种新的治疗措施。研究方法:(1)构建了两种T细胞过继转输诱导自身免疫性心肌炎模型,并通过免疫组化、PCR等方法观察在两种模型中PD-L1的表达水平。(2)先后使用抗PECAM-1抗体和抗ICAM-2抗体的免疫磁珠分离纯化获得小鼠心脏内皮细胞,通过镜下观察和抗CD31抗体和VE-Caherin染色鉴定内皮细胞。(3)探索干扰素对小鼠心脏内皮细胞PD-L1调控。(3)通过在体实验,研究IFN对小鼠心脏内皮细胞PD-L1的调控。(4)通过在体实验,研究含CpG基序的寡聚脱氧核苷酸(Oligodeoxynucleotides containing CpG motifs,CpG-ODN)对小鼠心脏内皮细胞PD-L1的表达。(5)构建自身免疫性心肌炎,通过石蜡包埋切片计算病理积分,观察CpG-ODN对自身免疫性心肌炎治疗作用。(6)采用PCR检测心脏组织中IL-1β、TNF-α的表达水平。研究结果:(1)两种自身免疫性心肌炎中均发现心脏组织mRNA表达PD-L1显著高于对照组;(2)通过免疫组化发现,心脏组织表达PD-L1与CD31呈一致性。(3)采用二次分选法所纯化获得的小鼠心脏微血管内皮细胞生长良好,形态正常,内皮细胞CD31染色阳性率达99.9%,且VE-Caherin表达率达85.1%,并可在体外稳定传代培养,且冻存后复苏仍保持良好活性。(4)Real-time PCR结果显示10U/ml IFN-γ、100U/ml IFN-β刺激小鼠心脏内皮细胞8小时后,小鼠心脏内皮细胞PD-L1均上调,差距有统计学意义;(5)在体实验结果发现,IFN-β、IFN-γ未能上调小鼠心脏PD-L1 mRNA表达,且未能上调小鼠心脏PD-L1蛋白表达;(6)在体实验结果发现,CpG ODN能上调小鼠心脏PD-L1 mRNA表达,且免疫组化同样得以证实;(7)通过HE染色并对心肌炎小鼠进行组织病理学积分量化分析,结果发现相对PBS干预组,预防使用CpG-ODN组可以使自身免疫性心肌炎小鼠心肌炎程度较之减轻。(8)经荧光实时定量RT-PCR方法检测,CpG-ODN组中IL-1β表达量显著低于PBS对照组,IL-1β(0.313±0.022)vs.(1.180±0.148);TNF-α(0.075±0.011)vs.(0.255±0.018)。结论:PD-L1参与了自身免疫性心肌炎的病程,CpG-ODN能上调小鼠心脏内皮细胞表达PD-L1,并通过上调PD-L1降低了小鼠心肌炎炎症程度。在此实验基础上,CpG-ODN很可能成为治疗自身免疫性心肌炎的有效方法。
[Abstract]:Objective: myocarditis is (myocarditis) acute inflammatory disease of the heart caused by a variety of virus infection, drug toxicity and so on, accompanied by various inflammatory cells infiltration and myocardial cell injury or even necrosis. Partial myocarditis can rapidly develop to dilated cardiomyopathy (Dilated cardiomyopathy, DCM), and dilated cardiomyopathy A variety of anti myocardium autoantibodies can be detected in the patients' serum samples, and it is proved that autoimmune myocarditis (autoimmune myocarditis) plays an important role in the pathogenesis of dilated cardiomyopathy. Therefore, a suitable animal model of autoimmune myocarditis is established to simulate the pathogenesis of immune injury of myocarditis and the pathogenesis of myocarditis. The study of autoimmune injury and the therapeutic effect and prognosis are of vital significance. This experiment is to establish a suitable model of autoimmune myocarditis in mice, and to study the expression of programmed cell death molecule 1 ligand (programmed death ligand 1, PD-L1) in autoimmune myocarditis mice by means of PCR and immunohistochemistry. In order to provide a new direction for the study of the pathogenesis of myocarditis and provide a new treatment for the clinical treatment of myocarditis, a method is sought to provide a new way to regulate the expression of PD-L1 in the pathogenesis of cardiac myositis in mice. (1) a model of two kinds of T cell adoptive transfer to induce autoimmune myocarditis was constructed. The expression level of PD-L1 in the two models was observed by immunohistochemistry, PCR and other methods. (2) the mouse cardiac endothelial cells were isolated and purified with anti PECAM-1 antibody and anti ICAM-2 antibody, and the endothelial cells were identified by microscope and CD31 antibody and VE-Caherin staining. (3) to explore the PD of mouse heart endothelial cells by interferon. -L1 regulation. (3) the regulation of PD-L1 in mouse cardiac endothelial cells by IFN was studied in vivo. (4) the expression of Oligodeoxynucleotides containing CpG motifs (CpG-ODN) containing CpG based oligodeoxynucleotides (CpG-ODN) on mouse cardiac endothelial cells PD-L1 was studied in vivo. (5) the construction of autoimmune myocarditis, paraffin embedded and cut through paraffin The therapeutic effect of CpG-ODN on autoimmune myocarditis was observed. (6) the expression of IL-1 beta and TNF- in the cardiac tissue was detected by PCR. The results were as follows: (1) the mRNA expression of mRNA in the cardiac tissue was significantly higher than that of the control group in two kinds of autoimmune myocarditis. (2) the expression of PD-L1 and C in the heart tissue was found by immunohistochemistry. D31 was consistent. (3) the mouse cardiac microvascular endothelial cells obtained by the two separation method had good growth and normal morphology, the positive rate of CD31 staining of endothelial cells was 99.9%, and the expression rate of VE-Caherin was 85.1%, and it could be cultured in vitro, and the resuscitation still maintained good activity. (4) Real-time PCR results showed 10U/ml IF. N- gamma, 100U/ml IFN- beta stimulated the heart endothelial cells of mice for 8 hours, the heart endothelial cells PD-L1 up up, the difference was statistically significant. (5) in the experimental results, it was found that IFN- beta, IFN- gamma failed to increase the expression of PD-L1 mRNA in the heart of mice, and failed to increase the expression of PD-L1 protein in the heart of mice. (6) the experimental results showed that CpG ODN could be up to up. The expression of PD-L1 mRNA in rat heart was also confirmed by immunohistochemistry; (7) quantitative analysis of histopathology in mice with myocarditis was quantified by HE staining and the results were found in the relative PBS intervention group. The prevention of the use of CpG-ODN group could reduce the degree of myocarditis in the autoimmune myocarditis mice. (8) the fluorescence real-time quantitative RT-PCR method was detected. The expression of IL-1 beta in CpG-ODN group was significantly lower than that in PBS control group, IL-1 beta (0.313 + 0.022) vs. (1.180 + 0.148) and TNF- alpha (0.075 + 0.011) vs. (0.255 + 0.018). Conclusion: PD-L1 involved in the course of autoimmune myocarditis, CpG-ODN can increase the expression of PD-L1 in mouse heart endothelial cells and reduce the degree of inflammation in murine myocarditis by increasing PD-L1. Based on this experiment, CpG-ODN is likely to be an effective way to treat autoimmune myocarditis.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.21;R-332

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