A20治疗系统性红斑狼疮小鼠模型机制研究

发布时间：2018-03-15 07:34

本文选题：瑞香素　切入点：系统性红斑狼疮　出处：《第三军医大学》2016年博士论文　论文类型：学位论文

【摘要】：研究背景：SLE (systemic lupus erythematosus,系统性红斑狼疮)是一种自身免疫性炎症性疾病,可累及肾、血管、皮肤、关节和脑等全身多个器官。在SLE进展过程中,对核酸及其相互作用蛋白的免疫耐受缺失导致自身抗体产生最终引起全身炎症和组织损伤。尽管在控制感染和肾衰的医疗进展提高了红斑狼疮患者的生存时间,但目前仍缺乏有效的治疗手段阻止SLE自身免疫炎症的发生。传统的免疫抑制剂缺乏靶向免疫抑制作用,疗效不能保证,且副作用较多限制其应用。一些生物制剂在类风湿性关节炎、银屑病包括关节病型银屑病等应用获得成功,但应用于SLE并没有获得肯定的疗效。因此发展新的治疗药物控制SLE自身炎症的发生有着重要的意义。A20又称为TNFAIP3 (tumor necrosis factor alpha induced protein 3,肿瘤坏死因子α诱导蛋白3),是肿瘤坏死因子α诱导的锌指蛋白。全基因组关联研究证实TNFAIP3 (A20)是SLE的易感基因,A20异常表达与SLE的发病有着密切关系。之前的研究发现TNF-α刺激后红斑狼疮患者单核细胞异常低水平表达的A20导致NF-κB持续活化的炎症反应。因此我们推测系统性红斑狼疮患者炎症活化阶段A20表达相对不足导致NF-κB持续活化引起全身炎症反应可能是SLE发病机制之一,而通过药物干预手段提高A20的表达水平就可能成为治疗红斑狼疮的新策略。瑞香素(Daphnetin)是瑞香科属植物的自然提取物属于香豆素类化合物,它能显著提高小鼠体内A20的表达,且对自身免疫性关节炎发挥抗炎治疗作用。目前以重组腺病毒为载体在动物体内过表达目的基因的研究方法已经被广泛应用于基础研究和基因治疗中。本研究利用瑞香素干预和重组腺病毒两种方法在系统性红斑狼疮小鼠模型中高表达A20蛋白,观察在系统性狼疮小鼠模型体内提高A20的表达是否能缓解SLE炎症反应以及深入探讨A20对SLE炎症的治疗机制,从而加深对SLE发病机理的认识,为SLE的治疗提供新的治疗策略。研究一瑞香素诱导A20高表达抑制NZB/W F1小鼠炎症反应目的：研究瑞香素诱导的高表达A20是否对NZB/W F1狼疮模型小鼠的炎症有潜在的治疗效应。方法：16-18周NZB/W F1和BABL/c随机分为三组：NZB/W F1小鼠未治疗组(SLE; n= 20)、NZB/W F1小鼠瑞香素治疗组(Daphnetin; n=20)、BABL/c阴性对照组(Sham;n=10)。利用试剂盒检测血尿素氮水平评估小鼠肾脏功能；酶联免疫吸附试验检测血清anti-nRNP IgG、anti-dsDNA IgG、TNF-α和IL-6；蛋白质印迹法检测外周血单核细胞pNFKB-p65和A20蛋白表达水平；曼-惠特尼u检验或单因素方差分析统计组间差异,结果表示为x±s。结果：瑞香素治疗后A20表达比未治疗组显著升高,SLE组小鼠A20表达比阴性对照组小鼠高；SLE模型小鼠血清TNF-α和IL-6比正常小鼠显著升高,瑞香素治疗后血清TNF-α和IL-6水平明显降低；SLE组小鼠血清anti-nRNP IgG和anti-dsDNA IgG高于阴性对照组小组,瑞香素治疗组小鼠血清anti-nRNP IgG和anti-dsDNA IgG低于SLE组小鼠；瑞香素治疗后小鼠尿素氮水平降低表明肾脏损伤减轻；SLE组pNFKB-p65蛋白表达比对照组提高,而瑞香素治疗后pNFKB-p65蛋白表达显著下降。结论：瑞香素诱导的高表达A20对NZB/W F1狼疮模型小鼠的炎症有治疗效果,高表达的A20使NF-KB活性下调可能其治疗机制。研究二腺病毒介导A20过表达缓解pristane诱导的狼疮肾炎目的：研究腺病毒介导的过表达A20是否对pristane诱导的狼疮模型小鼠肾炎症有治疗效应。方法：6-8周龄BALB/c雌性小鼠腹腔内注射0.5mL pristine或者PBS,注射PBS的小鼠则成为正常对照组,3个月后注射pristane的小鼠随机分为Ad-A20、Ad-control和pristane组并在腹腔内分别注射1.0×109pfu Ad-A20、Ad-control和PBS；利用ELISA试剂盒检测血清IL-1β、IL-6IgG、anti-nRNP、anti-dsDNA和尿白蛋白；肾脏石蜡切片进行HE染色并在光镜下观察,以双盲的方式对每只小鼠8个视野肾小球和肾小管评分进行肾脏病理评估；肾脏冰冻切片进行IgG和C3免疫荧光染色并在荧光显微镜下进行观察,荧光强度利用Image J软件评估并评分；蛋白质印迹法检测A20、NLRP3、 ASC、p-IκB、IκB、F4/80、CCL2、Caspase-1p20、p-NF-κBp65和NF-κBp65蛋白表达水平；曼-惠特尼“检验或单因素方差分析统计组间差异,结果表示为x±s。结果：Pristane组和Ad-control组小鼠腹腔巨噬细胞A20蛋白较PBS组小鼠比较而言表达轻度上调,而Ad-A20治疗组小鼠腹腔巨噬细胞A20蛋白较其他三组比较而言显著上调；Pristane组和Ad-control组小鼠血清抗dsDNA和抗nRNP抗体水平显著高于PBS组小鼠,而Ad-A20治疗组小鼠血清抗dsDNA和抗nRNP抗体水平则较Ad-control组显著降低但仍高于PBS组小鼠；病理半定量分析表明Ad-A20组小鼠肾小球和肾小管活动度计分仅稍高于BPS组小鼠,而Ad-A20组小鼠肾小球和肾小管活动度计分显著低于Pristane组和Ad-control组小鼠。小鼠24小时尿液白蛋白检测表明Pristane组小鼠尿液白蛋白显著高于PBS组小鼠,而Ad-A20组小鼠尿液白蛋白低于Ad-control组小鼠；Pristane组和Ad-control组小鼠肾脏CCL2和F4/80表达水平显著高于PBS组小鼠,而Ad-A20治疗组小鼠CCL2和F4/80表达水平则较Ad-control组显著降低；Pristane组和Ad-control组小鼠β-IκB和p-NF-KBp65表达水平显著高于PBS组小鼠,而Ad-A20治疗组小鼠β-IκB和p-NF-KBp65表达水平则较Ad-control组显著降低；Pristane组和Ad-control组小鼠NLRP3、ASC和Caspase-1p20表达水平显著高于PBS组小鼠,而Ad-A20治疗组小鼠NLRP3、ASC和Caspase-1p20表达水平则较Ad-control组显著降低。结论：腺病毒介导的过表达A20缓解了pristane诱导的SLE模型小鼠炎症和肾脏损伤,其治疗机制可能与抑制巨噬细胞NLRP3炎性小体和NF-κB活性相关。由于能对NLRP3炎性小体和NF-κB活性进行双重抑制,过表达A20有望成为治疗SLE的新选择。
[Abstract]:Background: SLE (systemic lupus erythematosus, systemic lupus erythematosus) is an autoimmune inflammatory disease involving the kidney, blood vessels, skin, joints and brain multiple body organs. In the progression of SLE, immune to the nucleic acid and its interacting protein resistance led to a lack of autoantibody production and eventually cause systemic inflammation and tissue injury. Despite the increase in medical progress to control infection and kidney failure in lupus patients survival time, but there is still lack of effective therapy to prevent SLE autoimmune inflammation. Traditional immunosuppressants lack of targeted immunosuppressive effects, efficacy can not be guaranteed, and have many side effects limit its application. Biological agents in rheumatoid arthritis, psoriasis, psoriatic arthritis, including the application of success, but applied to SLE did not have a positive effect. Therefore, the development of new SLE control of inflammation treatment occurs has important significance of.A20 is also called TNFAIP3 (tumor necrosis factor alpha induced protein 3, tumor necrosis factor alpha induced protein 3), tumor necrosis factor alpha induced zinc finger protein. A genome-wide association study confirmed that TNFAIP3 (A20) is the susceptibility gene of SLE and A20 the abnormal expression and the pathogenesis of SLE has a close relationship. Previous studies found that TNF- stimulated monocytes after systemic lupus erythematosus patients with abnormal low expression of A20 may lead to inflammation, NF- kappa B sustained activation. So we speculate that the systemic lupus erythematosus patients with inflammatory activation stage A20 expression relative deficiency of NF- kappa B activation induced by continuous systemic inflammatory reaction may be one of the pathogenesis of SLE, and by means of drug intervention to improve the expression level of A20 may become a new strategy for the treatment of lupus erythematosus. Daphnetin (Daphnetin) is. Natural extracts of plants belonging to the genus of coumarin compounds, it can significantly increase the expression of A20 in mice, and play a role in treatment of inflammatory autoimmune arthritis. The recombinant adenovirus as the research method of target gene expression in animal vector has been widely used in basic research and gene therapy in this study. The use of daphnetin intervention and two methods of recombinant adenovirus with high expression of A20 protein in systemic lupus erythematosus mouse model, observe the improvement of the expression of A20 in systemic lupus mice can alleviate SLE inflammatory reaction and probing into the treatment mechanism of A20 on SLE of inflammation, so as to deepen the understanding of SLE pathogenesis, provide a new therapeutic strategy for the treatment of SLE. The high expression of A20 inhibited NZB/W F1 mice induced inflammatory response to a study of daphnetin: daphnetin induced higher expression of A20 The NZB/W F1 lupus model mice inflammation has a potential therapeutic effect. Methods: 16-18 weeks of NZB/W F1 and BABL/c were randomly divided into three groups: NZB/W F1 mice without treatment group (SLE; n= 20), NZB/W F1 mice daphnetin group (Daphnetin; n=20), BABL/c negative control group (Sham; n=10) assessment of renal function in mice. Using the kit to detect the levels of blood urea nitrogen; enzyme-linked immunosorbent assay for detection of serum anti-nRNP IgG, anti-dsDNA IgG, TNF- alpha and IL-6; mononuclear cells pNFKB-p65 and the expression level of A20 protein in peripheral blood by Western blot; the Mann Whitney U test or ANOVA statistical difference between groups. The results are expressed as x + s. RESULTS: daphnetin after treatment A20 was significantly higher than the untreated group, the expression of SLE mice A20 mice in control group was higher than the negative; SLE model of mouse serum TNF- alpha and IL-6 were significantly higher than normal mice, daphnetin after treatment Serum TNF- and IL-6 levels were significantly lower in group SLE; anti-nRNP IgG and anti-dsDNA IgG in serum was higher than the negative control group, daphnetin group serum anti-nRNP IgG and anti-dsDNA IgG lower than SLE mice; reducing blood urea nitrogen level in mice after treatment showed that daphnetin reduce kidney injury; the expression of SLE protein in pNFKB-p65 group than the control group increased however, daphnetin after treatment, the expression of pNFKB-p65 was significantly decreased. Conclusion: daphnetin induced high expression of A20 in inflammatory F1 lupus model mice of NZB/W treatment, A20 expression and NF-KB activity decreased to its therapeutic mechanism. Two adenovirus mediated A20 overexpression of pristane induced remission in lupus nephritis Objective: over expression whether A20 has therapeutic effect on pristane induced lupus nephritis in mice mediated by adenovirus on. Methods: 6-8 week old female BALB/c mice Injection of 0.5mL pristine or PBS, injection of PBS mice as normal control group, 3 months after the injection of pristane mice were randomly divided into Ad-A20, Ad-control and pristane in group and intraperitoneal injection of 1 * 109pfu Ad-A20, Ad-control and PBS; IL-1 beta, using ELISA kit to detect serum IL-6IgG, anti-nRNP, anti-dsDNA the kidney and urinary albumin; paraffin sections were stained with HE and observed under optical microscope in a blinded fashion for each mouse 8 horizons of glomerular and tubular scores of renal pathological assessment; kidney frozen sections of IgG and C3 immunofluorescence staining were observed under the fluorescence microscope, the fluorescence intensity by using Image J software evaluation and score; Western blotting detection of A20, NLRP3, ASC, p-I kappa B, I kappa B, F4/80, CCL2, Caspase-1p20, p-NF-, Bp65 and NF- kappa kappa Bp65 protein expression level; the Mann Whitney test and single factor variance Analysis of statistical difference between groups, the results were expressed as x + s. RESULTS: Pristane group and Ad-control group A20 protein in peritoneal macrophages of mice compared with PBS mice compared to expression slightly raised, while Ad-A20 treatment group of mouse peritoneal macrophages A20 protein than the other three groups significantly increased compared; Pristane group and Ad-control group of mice serum anti dsDNA and anti nRNP the antibody level was significantly higher than that in PBS group, and Ad-A20 group of mice serum anti dsDNA and anti nRNP antibody level was significantly lower than that of Ad-control group but still higher than that in PBS group; semi quantitative pathological analysis showed that Ad-A20 mice glomerular and tubular activity scores only slightly higher than that of BPS group and Ad-A20 group of mice, mice glomeruli and renal tubules the activity score was significantly lower than that of Pristane group and Ad-control group. Mice were detected 24 h urine albumin showed that Pristane mice urinary albumin significantly Higher than the PBS group, and Ad-A20 group were lower than those in group Ad-control mice urinary albumin; the expression level of Pristane group and Ad-control group of mice kidney CCL2 and F4/80 were significantly higher than that in PBS group, and Ad-A20 group were CCL2 and F4/80 expression level was significantly lower than that of Ad-control group; the expression level of Pristane group and Ad-control group mouse Beta Kappa B and -I p-NF-KBp65 was significantly higher than that in PBS group, Ad-A20 treatment group and -I mouse Beta Kappa B and the expression level of p-NF-KBp65 was significantly lower than that of Ad-control group; Pristane group and Ad-control group NLRP3, the expression level of ASC and Caspase-1p20 were significantly higher than that in PBS group, while Ad-A20 mice treated with NLRP3, the expression level of ASC and Caspase-1p20 were significantly lower than those in Ad-control group conclusion: overexpression of A20 reduced pristane induced SLE mice model of inflammation and renal injury mediated by adenovirus, and its therapeutic mechanism may inhibit Macrophage NLRP3 inflammatory corpuscle and NF- kappa B activity are related. Due to double inhibition of NLRP3 inflammatory corpuscle and NF- kappa B activity, over expression of A20 is expected to become a new choice for SLE treatment.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R-332;R593.241

【相似文献】