CD40siRNA對(duì)自身免疫性心肌炎大鼠Th17細(xì)胞及IL-17與IL-23的作用
發(fā)布時(shí)間：2019-06-13 18:45
【摘要】：目的病毒性心肌炎是兒童的常見疾病之一,可導(dǎo)致心律失常、擴(kuò)張型心肌病、心力衰竭、心源性休克甚至猝死等嚴(yán)重后果。其發(fā)病機(jī)制尚不明確,但越來越多的研究資料證明自身免疫性損傷是造成病毒性心肌炎后期心肌損害的重要原因。因此建立自身免疫性心肌炎的動(dòng)物模型,模擬病毒性心肌炎后期免疫性損傷的發(fā)病過程,是研究病毒性心肌炎的自身免疫損傷過程、探討病毒性心肌炎發(fā)病機(jī)制的較好的方法。Thl7細(xì)胞是近年來新發(fā)現(xiàn)的CD4+T細(xì)胞的一個(gè)亞群,其分化的特異性轉(zhuǎn)錄因子是維甲酸相關(guān)孤兒受體。Th17細(xì)胞以主要分泌細(xì)胞因子IL-17為特點(diǎn),參與了許多自身免疫性疾病的發(fā)病過程,比如系統(tǒng)性紅斑狼瘡、多發(fā)性硬化、病毒性肝炎的后期等。有研究發(fā)現(xiàn),在病毒性心肌炎和自身免疫性心肌炎中Thl7細(xì)胞亞群及外周血IL-17、IL-23濃度有明顯變化,表明Th17細(xì)胞及其細(xì)胞因子IL-17、IL-23在病毒性心肌炎及免疫性心肌炎炎性反應(yīng)及免疫損傷中發(fā)揮了重要作用。小干擾RNA (small interfering RNA, siRNA)也被稱為短干擾RNA (short interfering RNA)或沉默RNA (silencing RNA),一般含有20-25個(gè)核苷酸,主要參與RNA干擾現(xiàn)象,專一性的調(diào)節(jié)基因的表達(dá)。使用小干擾RNA (siRNA)特異性的敲除分子靶點(diǎn)是一種新興的基因沉默的方法。因此使用小干擾RNA治療自身免疫性疾病也許是一種極有前景的治療方法。目前小干擾RNA的干擾效果已經(jīng)在越來越多的細(xì)胞實(shí)驗(yàn)中被驗(yàn)證,但是,關(guān)于小干擾RNA在動(dòng)物實(shí)驗(yàn)中的干擾效果的研究還比較少見,使用小于擾RNA治療大鼠自身免疫性心肌炎尚未有人研究報(bào)道。本研究第一次應(yīng)用CD40小干擾RNA(CD40siRNA)慢病毒表達(dá)載體對(duì)豬心肌球蛋白免疫誘導(dǎo)的實(shí)驗(yàn)性自身免疫性心肌炎(experimental autoimmune myocarditis, EAM)大鼠進(jìn)行免疫治療,并觀察CD40siRNA慢病毒表達(dá)載體對(duì)EAM大鼠心肌組織病理積分、心肌組織RORC mRNA表達(dá)水平、外周血中IL-17及IL-23濃度的影響,分析其潛在的作用機(jī)制,以便幫助醫(yī)務(wù)工作者找到更有效的治療病毒性心肌炎的方法。 方法選取40只6-8周齡健康雄性Lewis大鼠,大鼠均為無特殊病原體的SPF級(jí)實(shí)驗(yàn)動(dòng)物,體質(zhì)量在185-210g之間,平均體質(zhì)量為197.25±5.82g。40只大鼠被隨機(jī)分為EAM模型組、正常對(duì)照組、CD40siRNA治療組及siRNA對(duì)照組四組,每組10只大鼠。EAM模型組、CD40siRNA治療組及siRNA對(duì)照組大鼠以豬心肌球蛋白與完全弗氏佐劑的混合液0.2m1于大鼠雙后足足墊區(qū)皮下注射,正常對(duì)照組大鼠僅在雙后足足墊區(qū)皮下注射PBS緩沖液0.2ml/只。CD40siRNA治療組大鼠與siRNA對(duì)照組大鼠于免疫后第8天分別尾靜脈注射CD40siRNA慢病毒表達(dá)載體、siRNA慢病毒表達(dá)載體。免疫后第21天處死全部大鼠,光鏡下觀察大鼠心肌組織病理變化并計(jì)算心肌組織病理積分；實(shí)時(shí)定量PCR法(RQ-PCR)檢測心肌組織Th17細(xì)胞分化的特異性轉(zhuǎn)錄因子RORC mRNA表達(dá)水平；酶聯(lián)免疫吸附法(ELISA)檢測大鼠外周血中細(xì)胞因子IL-17與IL-23濃度。 結(jié)果 1. CD40siRNA治療組心肌組織病理積分比EAM模型組心肌組織病理積分明顯下降(2.34±0.60vs.3.40±0.35,p0.05),siRNA對(duì)照組心肌組織病理積分與EAM模型組心肌組織病理積分相比無差異(3.56±0.21vs.3.40±0.35,p0.05); 2. CD40siRNA治療組心肌組織RORC mRNA表達(dá)比EAM模型組心肌組織RORC mRNA表達(dá)明顯下降(2.13±0.28vs.2.93±0.36, p0.05), siRNA對(duì)照組心肌組織RORC mRNA與EAM模型組心肌組織RORC mRNA相比無差異(3.05±0.16vs.2.93±0.36,p0.05)； 3. CD40siRNA治療組外周血IL-17濃度較EAM模型組外周血IL-17濃度明顯下降(114.38±8.29vs.148.70±5.04, p0.05), siRNA對(duì)照組外周血IL-17濃度與EAM模型組外周血IL-17濃度相比無差異(144.15±5.82vs.148.70±5.04.p0.05)； 4.CD40siRNA治療組外周血IL-23濃度較EAM模型組外周血IL-23濃度明顯下降(107.00±7.69vs.136.98±23.16, p0.05), siRNA對(duì)照組外周血IL-23濃度與EAM模型組外周血IL-23濃度相比差異無統(tǒng)計(jì)學(xué)意義(142.11±15.87vs.136.98±23.16,p0.05)。 結(jié)論 1.CD40siRNA可減輕實(shí)驗(yàn)性自身免疫性心肌炎大鼠的心肌炎癥反應(yīng)。 2.CD40siRNA可以下調(diào)實(shí)驗(yàn)性自身免疫性心肌炎大鼠RORC mRNA的表達(dá),減少外周血IL-17及IL-23的分泌水平,具體機(jī)制可能與其干擾共刺激分子CD40的生成,阻斷CD40-CD40L共刺激信號(hào)通路有關(guān)。
[Abstract]:Objective Viral myocarditis is one of the common diseases of children, which can lead to serious consequences such as arrhythmia, dilated cardiomyopathy, heart failure, cardiogenic shock and sudden death. The pathogenesis of viral myocarditis is not clear, but more and more research data prove that the autoimmune damage is an important cause of the late-stage myocardial damage of viral myocarditis. Therefore, the animal model of the autoimmune myocarditis is established, the pathogenesis of the late-stage immune injury of the viral myocarditis is simulated, the self-immune injury process of the viral myocarditis is studied, and the better method of the pathogenesis of the viral myocarditis is discussed. Thl7 cells are a subset of newly discovered CD4 + T cells in recent years, and the specific transcription factor of its differentiation is a retinoic acid-related orphan receptor. Th17 cells are characterized by mainly secreted cytokines IL-17, and are involved in the pathogenesis of many autoimmune diseases, such as systemic lupus erythematosus, multiple sclerosis, late stage of viral hepatitis, and the like. It was found that Th17 cells and their cytokines IL-17 and IL-23 played an important role in the inflammatory reaction and immune injury of viral myocarditis and immune myocarditis. Small interfering RNA (siRNA) is also referred to as short interfering RNA or silent RNA, generally containing 20-25 nucleotides, mainly involved in the expression of RNA interference and specific regulatory genes. The use of a small interfering RNA (siRNA)-specific knockout molecule target is a new method of gene silencing. The use of small interfering RNA in the treatment of autoimmune diseases may therefore be a highly promising treatment. At present, the interference effect of small interfering RNA has been verified in an increasing number of cell experiments, but the study on the interference effect of small interfering RNA in animal experiments is still rare, and the use of less than the interfering RNA in the treatment of the autoimmune myocarditis in the rat has not been reported. The first time in this study, the CD40siRNA lentiviral expression vector was used to immunize the experimental autoimmune myocarditis (EAM) induced by the porcine cardiac myosin and to observe the pathological integration of the CD40siRNA lentiviral expression vector to the myocardial tissue of the EAM rats. The effect of the level of RORC mRNA expression, the level of IL-17 and IL-23 in the peripheral blood of the myocardial tissue and its potential mechanism of action were analyzed in order to help the medical workers find a more effective method to treat viral myocarditis. Methods 40 healthy male Lewis rats from 6 to 8 weeks of age were selected. The body mass was 185-210 g, the mean mass was 197.25 and 5.82 g.40 rats were randomly divided into EAM model group, normal control group, CD40siRNA treatment group and siRNA control group. Group,10 for each group The rat. EAM model group, the CD40siRNA treatment group and the siRNA control group were injected subcutaneously with the mixed solution of porcine cardiac myosin and complete Freund's adjuvant, 0.2 ml, and the normal control group was injected with PBS buffer at 0.2 ml/ min only in the two-posterior foot pad area. CD40siRNA lentiviral expression vector and siRNA lentiviral expression vector were injected respectively at the end of the 8th day after the immunization with the CD40siRNA treatment group and the siRNA control group. Body. All the rats were sacrificed on the 21st day after immunization. The pathological changes of the myocardial tissue of the rat were observed under light microscope and the pathological score of the myocardial tissue was calculated. The specific transcription factor, RORC mRNA expression water, was detected by real-time quantitative PCR (RQ-PCR) in the detection of the differentiation of the Th17 cells in the myocardial tissue. Determination of IL-17 and IL-23 in peripheral blood of rats by enzyme-linked immunosorbent assay (ELISA) Degree. Results 1. The pathological score of the myocardial tissue in the treatment group of the CD40siRNA group was significantly lower than that of the myocardial tissue in the EAM model group (2.34, 0.60 vs. 3.40, 0.35, p0.05). The pathological score of the myocardial tissue in the siRNA control group was not different from that of the myocardial tissue in the EAM model group (3.56, 0.21 vs. 3.40, 0.35, p 2. The expression of RORC mRNA in the myocardium of the treatment group of the 2. CD40siRNA group was significantly lower than that of the RORC mRNA in the EAM model group (2.13, 0.28 vs. 2.93, 0.36, p0.05). The RORC mRNA in the myocardial tissue of the siRNA control group was not different from the RORC mRNA in the EAM model group (3.05-0.16 vs. 2.93-0.36). The IL-17 concentration in the peripheral blood of the treatment group of the CD40siRNA group was significantly lower than that in the EAM model group (114.38, 8.29 vs. 148.70, 5.04, p0.05), and the IL-17 concentration in the peripheral blood of the siRNA control group was not different from that of the peripheral blood IL-17 in the EAM model group (144.15, 5.82 vs. 148.70). The level of IL-23 in the peripheral blood of the treatment group was significantly lower than that of the EAM model group (107.00, 7.69 vs. 136.98, 23.16, p0.05), and the IL-23 concentration in the peripheral blood of the siRNA control group was not statistically significant (142.11-15.87 vs. 136.98-2) compared with the concentration of the IL-23 in the peripheral blood of the EAM model group. 3. Conclusion 1. CD40siRNA can reduce the experimental self. 2. CD40siRNA can reduce the expression of RORC mRNA in experimental autoimmune myocarditis and reduce the level of IL-17 and IL-23 in peripheral blood.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R725.4
[Abstract]:Objective Viral myocarditis is one of the common diseases of children, which can lead to serious consequences such as arrhythmia, dilated cardiomyopathy, heart failure, cardiogenic shock and sudden death. The pathogenesis of viral myocarditis is not clear, but more and more research data prove that the autoimmune damage is an important cause of the late-stage myocardial damage of viral myocarditis. Therefore, the animal model of the autoimmune myocarditis is established, the pathogenesis of the late-stage immune injury of the viral myocarditis is simulated, the self-immune injury process of the viral myocarditis is studied, and the better method of the pathogenesis of the viral myocarditis is discussed. Thl7 cells are a subset of newly discovered CD4 + T cells in recent years, and the specific transcription factor of its differentiation is a retinoic acid-related orphan receptor. Th17 cells are characterized by mainly secreted cytokines IL-17, and are involved in the pathogenesis of many autoimmune diseases, such as systemic lupus erythematosus, multiple sclerosis, late stage of viral hepatitis, and the like. It was found that Th17 cells and their cytokines IL-17 and IL-23 played an important role in the inflammatory reaction and immune injury of viral myocarditis and immune myocarditis. Small interfering RNA (siRNA) is also referred to as short interfering RNA or silent RNA, generally containing 20-25 nucleotides, mainly involved in the expression of RNA interference and specific regulatory genes. The use of a small interfering RNA (siRNA)-specific knockout molecule target is a new method of gene silencing. The use of small interfering RNA in the treatment of autoimmune diseases may therefore be a highly promising treatment. At present, the interference effect of small interfering RNA has been verified in an increasing number of cell experiments, but the study on the interference effect of small interfering RNA in animal experiments is still rare, and the use of less than the interfering RNA in the treatment of the autoimmune myocarditis in the rat has not been reported. The first time in this study, the CD40siRNA lentiviral expression vector was used to immunize the experimental autoimmune myocarditis (EAM) induced by the porcine cardiac myosin and to observe the pathological integration of the CD40siRNA lentiviral expression vector to the myocardial tissue of the EAM rats. The effect of the level of RORC mRNA expression, the level of IL-17 and IL-23 in the peripheral blood of the myocardial tissue and its potential mechanism of action were analyzed in order to help the medical workers find a more effective method to treat viral myocarditis. Methods 40 healthy male Lewis rats from 6 to 8 weeks of age were selected. The body mass was 185-210 g, the mean mass was 197.25 and 5.82 g.40 rats were randomly divided into EAM model group, normal control group, CD40siRNA treatment group and siRNA control group. Group,10 for each group The rat. EAM model group, the CD40siRNA treatment group and the siRNA control group were injected subcutaneously with the mixed solution of porcine cardiac myosin and complete Freund's adjuvant, 0.2 ml, and the normal control group was injected with PBS buffer at 0.2 ml/ min only in the two-posterior foot pad area. CD40siRNA lentiviral expression vector and siRNA lentiviral expression vector were injected respectively at the end of the 8th day after the immunization with the CD40siRNA treatment group and the siRNA control group. Body. All the rats were sacrificed on the 21st day after immunization. The pathological changes of the myocardial tissue of the rat were observed under light microscope and the pathological score of the myocardial tissue was calculated. The specific transcription factor, RORC mRNA expression water, was detected by real-time quantitative PCR (RQ-PCR) in the detection of the differentiation of the Th17 cells in the myocardial tissue. Determination of IL-17 and IL-23 in peripheral blood of rats by enzyme-linked immunosorbent assay (ELISA) Degree. Results 1. The pathological score of the myocardial tissue in the treatment group of the CD40siRNA group was significantly lower than that of the myocardial tissue in the EAM model group (2.34, 0.60 vs. 3.40, 0.35, p0.05). The pathological score of the myocardial tissue in the siRNA control group was not different from that of the myocardial tissue in the EAM model group (3.56, 0.21 vs. 3.40, 0.35, p 2. The expression of RORC mRNA in the myocardium of the treatment group of the 2. CD40siRNA group was significantly lower than that of the RORC mRNA in the EAM model group (2.13, 0.28 vs. 2.93, 0.36, p0.05). The RORC mRNA in the myocardial tissue of the siRNA control group was not different from the RORC mRNA in the EAM model group (3.05-0.16 vs. 2.93-0.36). The IL-17 concentration in the peripheral blood of the treatment group of the CD40siRNA group was significantly lower than that in the EAM model group (114.38, 8.29 vs. 148.70, 5.04, p0.05), and the IL-17 concentration in the peripheral blood of the siRNA control group was not different from that of the peripheral blood IL-17 in the EAM model group (144.15, 5.82 vs. 148.70). The level of IL-23 in the peripheral blood of the treatment group was significantly lower than that of the EAM model group (107.00, 7.69 vs. 136.98, 23.16, p0.05), and the IL-23 concentration in the peripheral blood of the siRNA control group was not statistically significant (142.11-15.87 vs. 136.98-2) compared with the concentration of the IL-23 in the peripheral blood of the EAM model group. 3. Conclusion 1. CD40siRNA can reduce the experimental self. 2. CD40siRNA can reduce the expression of RORC mRNA in experimental autoimmune myocarditis and reduce the level of IL-17 and IL-23 in peripheral blood.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R725.4
【共引文獻(xiàn)】
相關(guān)期刊論文 前8條
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1 俞p芑，
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