CD40siRNA对自身免疫性心肌炎大鼠Th17细胞及IL-17与IL-23的作用
发布时间：2019-06-13 18:45
【摘要】：目的病毒性心肌炎是儿童的常见疾病之一,可导致心律失常、扩张型心肌病、心力衰竭、心源性休克甚至猝死等严重后果。其发病机制尚不明确,但越来越多的研究资料证明自身免疫性损伤是造成病毒性心肌炎后期心肌损害的重要原因。因此建立自身免疫性心肌炎的动物模型,模拟病毒性心肌炎后期免疫性损伤的发病过程,是研究病毒性心肌炎的自身免疫损伤过程、探讨病毒性心肌炎发病机制的较好的方法。Thl7细胞是近年来新发现的CD4+T细胞的一个亚群,其分化的特异性转录因子是维甲酸相关孤儿受体。Th17细胞以主要分泌细胞因子IL-17为特点,参与了许多自身免疫性疾病的发病过程,比如系统性红斑狼疮、多发性硬化、病毒性肝炎的后期等。有研究发现,在病毒性心肌炎和自身免疫性心肌炎中Thl7细胞亚群及外周血IL-17、IL-23浓度有明显变化,表明Th17细胞及其细胞因子IL-17、IL-23在病毒性心肌炎及免疫性心肌炎炎性反应及免疫损伤中发挥了重要作用。小干扰RNA (small interfering RNA, siRNA)也被称为短干扰RNA (short interfering RNA)或沉默RNA (silencing RNA),一般含有20-25个核苷酸,主要参与RNA干扰现象,专一性的调节基因的表达。使用小干扰RNA (siRNA)特异性的敲除分子靶点是一种新兴的基因沉默的方法。因此使用小干扰RNA治疗自身免疫性疾病也许是一种极有前景的治疗方法。目前小干扰RNA的干扰效果已经在越来越多的细胞实验中被验证,但是,关于小干扰RNA在动物实验中的干扰效果的研究还比较少见,使用小于扰RNA治疗大鼠自身免疫性心肌炎尚未有人研究报道。本研究第一次应用CD40小干扰RNA(CD40siRNA)慢病毒表达载体对猪心肌球蛋白免疫诱导的实验性自身免疫性心肌炎(experimental autoimmune myocarditis, EAM)大鼠进行免疫治疗,并观察CD40siRNA慢病毒表达载体对EAM大鼠心肌组织病理积分、心肌组织RORC mRNA表达水平、外周血中IL-17及IL-23浓度的影响,分析其潜在的作用机制,以便帮助医务工作者找到更有效的治疗病毒性心肌炎的方法。 方法选取40只6-8周龄健康雄性Lewis大鼠,大鼠均为无特殊病原体的SPF级实验动物,体质量在185-210g之间,平均体质量为197.25±5.82g。40只大鼠被随机分为EAM模型组、正常对照组、CD40siRNA治疗组及siRNA对照组四组,每组10只大鼠。EAM模型组、CD40siRNA治疗组及siRNA对照组大鼠以猪心肌球蛋白与完全弗氏佐剂的混合液0.2m1于大鼠双后足足垫区皮下注射,正常对照组大鼠仅在双后足足垫区皮下注射PBS缓冲液0.2ml/只。CD40siRNA治疗组大鼠与siRNA对照组大鼠于免疫后第8天分别尾静脉注射CD40siRNA慢病毒表达载体、siRNA慢病毒表达载体。免疫后第21天处死全部大鼠,光镜下观察大鼠心肌组织病理变化并计算心肌组织病理积分；实时定量PCR法(RQ-PCR)检测心肌组织Th17细胞分化的特异性转录因子RORC mRNA表达水平；酶联免疫吸附法(ELISA)检测大鼠外周血中细胞因子IL-17与IL-23浓度。 结果 1. CD40siRNA治疗组心肌组织病理积分比EAM模型组心肌组织病理积分明显下降(2.34±0.60vs.3.40±0.35,p0.05),siRNA对照组心肌组织病理积分与EAM模型组心肌组织病理积分相比无差异(3.56±0.21vs.3.40±0.35,p0.05); 2. CD40siRNA治疗组心肌组织RORC mRNA表达比EAM模型组心肌组织RORC mRNA表达明显下降(2.13±0.28vs.2.93±0.36, p0.05), siRNA对照组心肌组织RORC mRNA与EAM模型组心肌组织RORC mRNA相比无差异(3.05±0.16vs.2.93±0.36,p0.05)； 3. CD40siRNA治疗组外周血IL-17浓度较EAM模型组外周血IL-17浓度明显下降(114.38±8.29vs.148.70±5.04, p0.05), siRNA对照组外周血IL-17浓度与EAM模型组外周血IL-17浓度相比无差异(144.15±5.82vs.148.70±5.04.p0.05)； 4.CD40siRNA治疗组外周血IL-23浓度较EAM模型组外周血IL-23浓度明显下降(107.00±7.69vs.136.98±23.16, p0.05), siRNA对照组外周血IL-23浓度与EAM模型组外周血IL-23浓度相比差异无统计学意义(142.11±15.87vs.136.98±23.16,p0.05)。 结论 1.CD40siRNA可减轻实验性自身免疫性心肌炎大鼠的心肌炎症反应。 2.CD40siRNA可以下调实验性自身免疫性心肌炎大鼠RORC mRNA的表达,减少外周血IL-17及IL-23的分泌水平,具体机制可能与其干扰共刺激分子CD40的生成,阻断CD40-CD40L共刺激信号通路有关。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R725.4
【共引文献】
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1 李淑清;刘巍;李为民;;Th17细胞在病毒性心肌炎发病机制中的作用[J];国际心血管病杂志;2011年01期
2 许玉霞;;黄芪颗粒联合维生素C辅助治疗小儿病毒性心肌炎临床研究[J];儿科药学杂志;2013年02期
3 雷茜;钟家蓉;;Th17细胞在病毒性心肌炎中的作用[J];重庆医学;2013年23期
4 金春杰;陈丽娟;丁旭;宋义祥;;病毒性心肌炎的临床诊治进展[J];中国循证心血管医学杂志;2011年04期
5 余红;韩淑英;包巨太;储金秀;张搏男;;病毒性心肌炎临床治疗进展[J];中国全科医学;2010年24期
6 杨帆;伍伟锋;;白细胞介素17与心血管疾病[J];实用医学杂志;2009年24期
7 林英辉;;Th17细胞与临床疾病的研究进展[J];实用医学杂志;2011年02期
8 刘海英;刘丹莉;高顺利;;黄芪注射液对急性病毒性心肌炎患儿血清IL-23、IL-17水平及Th17细胞的影响[J];实用医学杂志;2013年16期
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