【摘要】：研究目的:心肌炎是(myocarditis)由多種病毒感染、藥物毒性作用等多種病因引起的心臟急性炎癥性疾病,伴有各種炎癥細胞浸潤與心肌細胞損傷甚至壞死。部分心肌炎可快速發(fā)展至擴張型心肌病(Dilated cardiomyopathy,DCM),同時擴張型心肌病患者血清標本中可檢測出多種抗心肌的自身抗體,證實了自身免疫性心肌炎(autoimmune myocarditis)在擴張型心肌病發(fā)病中起著重要的作用。因此,建立合適的自身免疫性心肌炎動物模型,模擬心肌炎免疫性損傷的發(fā)病過程,在心肌炎發(fā)病機制、自身免疫損傷的研究,及治療效果及預后中有著至關(guān)重要的意義。本實驗擬建立合適的自身免疫性心肌炎小鼠模型,通過PCR及免疫組化等方法研究程序性細胞死亡分子1配體(programmed death ligand 1,PD-L1)在自身免疫性心肌炎小鼠中表達情況,并尋求一種方法可以調(diào)控小鼠心臟內(nèi)皮細胞表達PD-L1,以期為心肌炎發(fā)病機制的研究提供一種新的研究方向,并為臨床治療心肌炎提供一種新的治療措施。研究方法:(1)構(gòu)建了兩種T細胞過繼轉(zhuǎn)輸誘導自身免疫性心肌炎模型,并通過免疫組化、PCR等方法觀察在兩種模型中PD-L1的表達水平。(2)先后使用抗PECAM-1抗體和抗ICAM-2抗體的免疫磁珠分離純化獲得小鼠心臟內(nèi)皮細胞,通過鏡下觀察和抗CD31抗體和VE-Caherin染色鑒定內(nèi)皮細胞。(3)探索干擾素對小鼠心臟內(nèi)皮細胞PD-L1調(diào)控。(3)通過在體實驗,研究IFN對小鼠心臟內(nèi)皮細胞PD-L1的調(diào)控。(4)通過在體實驗,研究含CpG基序的寡聚脫氧核苷酸(Oligodeoxynucleotides containing CpG motifs,CpG-ODN)對小鼠心臟內(nèi)皮細胞PD-L1的表達。(5)構(gòu)建自身免疫性心肌炎,通過石蠟包埋切片計算病理積分,觀察CpG-ODN對自身免疫性心肌炎治療作用。(6)采用PCR檢測心臟組織中IL-1β、TNF-α的表達水平。研究結(jié)果:(1)兩種自身免疫性心肌炎中均發(fā)現(xiàn)心臟組織mRNA表達PD-L1顯著高于對照組;(2)通過免疫組化發(fā)現(xiàn),心臟組織表達PD-L1與CD31呈一致性。(3)采用二次分選法所純化獲得的小鼠心臟微血管內(nèi)皮細胞生長良好,形態(tài)正常,內(nèi)皮細胞CD31染色陽性率達99.9%,且VE-Caherin表達率達85.1%,并可在體外穩(wěn)定傳代培養(yǎng),且凍存后復蘇仍保持良好活性。(4)Real-time PCR結(jié)果顯示10U/ml IFN-γ、100U/ml IFN-β刺激小鼠心臟內(nèi)皮細胞8小時后,小鼠心臟內(nèi)皮細胞PD-L1均上調(diào),差距有統(tǒng)計學意義;(5)在體實驗結(jié)果發(fā)現(xiàn),IFN-β、IFN-γ未能上調(diào)小鼠心臟PD-L1 mRNA表達,且未能上調(diào)小鼠心臟PD-L1蛋白表達;(6)在體實驗結(jié)果發(fā)現(xiàn),CpG ODN能上調(diào)小鼠心臟PD-L1 mRNA表達,且免疫組化同樣得以證實;(7)通過HE染色并對心肌炎小鼠進行組織病理學積分量化分析,結(jié)果發(fā)現(xiàn)相對PBS干預組,預防使用CpG-ODN組可以使自身免疫性心肌炎小鼠心肌炎程度較之減輕。(8)經(jīng)熒光實時定量RT-PCR方法檢測,CpG-ODN組中IL-1β表達量顯著低于PBS對照組,IL-1β(0.313±0.022)vs.(1.180±0.148);TNF-α(0.075±0.011)vs.(0.255±0.018)。結(jié)論:PD-L1參與了自身免疫性心肌炎的病程,CpG-ODN能上調(diào)小鼠心臟內(nèi)皮細胞表達PD-L1,并通過上調(diào)PD-L1降低了小鼠心肌炎炎癥程度。在此實驗基礎(chǔ)上,CpG-ODN很可能成為治療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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R542.21;R-332

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