發(fā)布時間：2018-03-15 07:34

  本文選題：瑞香素　切入點：系統(tǒng)性紅斑狼瘡　出處：《第三軍醫(yī)大學》2016年博士論文　論文類型：學位論文

【摘要】：研究背景：SLE (systemic lupus erythematosus,系統(tǒng)性紅斑狼瘡)是一種自身免疫性炎癥性疾病,可累及腎、血管、皮膚、關(guān)節(jié)和腦等全身多個器官。在SLE進展過程中,對核酸及其相互作用蛋白的免疫耐受缺失導致自身抗體產(chǎn)生最終引起全身炎癥和組織損傷。盡管在控制感染和腎衰的醫(yī)療進展提高了紅斑狼瘡患者的生存時間,但目前仍缺乏有效的治療手段阻止SLE自身免疫炎癥的發(fā)生。傳統(tǒng)的免疫抑制劑缺乏靶向免疫抑制作用,療效不能保證,且副作用較多限制其應(yīng)用。一些生物制劑在類風濕性關(guān)節(jié)炎、銀屑病包括關(guān)節(jié)病型銀屑病等應(yīng)用獲得成功,但應(yīng)用于SLE并沒有獲得肯定的療效。因此發(fā)展新的治療藥物控制SLE自身炎癥的發(fā)生有著重要的意義。A20又稱為TNFAIP3 (tumor necrosis factor alpha induced protein 3,腫瘤壞死因子α誘導蛋白3),是腫瘤壞死因子α誘導的鋅指蛋白。全基因組關(guān)聯(lián)研究證實TNFAIP3 (A20)是SLE的易感基因,A20異常表達與SLE的發(fā)病有著密切關(guān)系。之前的研究發(fā)現(xiàn)TNF-α刺激后紅斑狼瘡患者單核細胞異常低水平表達的A20導致NF-κB持續(xù)活化的炎癥反應(yīng)。因此我們推測系統(tǒng)性紅斑狼瘡患者炎癥活化階段A20表達相對不足導致NF-κB持續(xù)活化引起全身炎癥反應(yīng)可能是SLE發(fā)病機制之一,而通過藥物干預(yù)手段提高A20的表達水平就可能成為治療紅斑狼瘡的新策略。瑞香素(Daphnetin)是瑞香科屬植物的自然提取物屬于香豆素類化合物,它能顯著提高小鼠體內(nèi)A20的表達,且對自身免疫性關(guān)節(jié)炎發(fā)揮抗炎治療作用。目前以重組腺病毒為載體在動物體內(nèi)過表達目的基因的研究方法已經(jīng)被廣泛應(yīng)用于基礎(chǔ)研究和基因治療中。本研究利用瑞香素干預(yù)和重組腺病毒兩種方法在系統(tǒng)性紅斑狼瘡小鼠模型中高表達A20蛋白,觀察在系統(tǒng)性狼瘡小鼠模型體內(nèi)提高A20的表達是否能緩解SLE炎癥反應(yīng)以及深入探討A20對SLE炎癥的治療機制,從而加深對SLE發(fā)病機理的認識,為SLE的治療提供新的治療策略。研究一瑞香素誘導A20高表達抑制NZB/W F1小鼠炎癥反應(yīng)目的：研究瑞香素誘導的高表達A20是否對NZB/W F1狼瘡模型小鼠的炎癥有潛在的治療效應(yīng)。方法：16-18周NZB/W F1和BABL/c隨機分為三組：NZB/W F1小鼠未治療組(SLE; n= 20)、NZB/W F1小鼠瑞香素治療組(Daphnetin; n=20)、BABL/c陰性對照組(Sham;n=10)。利用試劑盒檢測血尿素氮水平評估小鼠腎臟功能；酶聯(lián)免疫吸附試驗檢測血清anti-nRNP IgG、anti-dsDNA IgG、TNF-α和IL-6；蛋白質(zhì)印跡法檢測外周血單核細胞pNFKB-p65和A20蛋白表達水平；曼-惠特尼u檢驗或單因素方差分析統(tǒng)計組間差異,結(jié)果表示為x±s。結(jié)果：瑞香素治療后A20表達比未治療組顯著升高,SLE組小鼠A20表達比陰性對照組小鼠高；SLE模型小鼠血清TNF-α和IL-6比正常小鼠顯著升高,瑞香素治療后血清TNF-α和IL-6水平明顯降低；SLE組小鼠血清anti-nRNP IgG和anti-dsDNA IgG高于陰性對照組小組,瑞香素治療組小鼠血清anti-nRNP IgG和anti-dsDNA IgG低于SLE組小鼠；瑞香素治療后小鼠尿素氮水平降低表明腎臟損傷減輕；SLE組pNFKB-p65蛋白表達比對照組提高,而瑞香素治療后pNFKB-p65蛋白表達顯著下降。結(jié)論：瑞香素誘導的高表達A20對NZB/W F1狼瘡模型小鼠的炎癥有治療效果,高表達的A20使NF-KB活性下調(diào)可能其治療機制。研究二腺病毒介導A20過表達緩解pristane誘導的狼瘡腎炎目的：研究腺病毒介導的過表達A20是否對pristane誘導的狼瘡模型小鼠腎炎癥有治療效應(yīng)。方法：6-8周齡BALB/c雌性小鼠腹腔內(nèi)注射0.5mL pristine或者PBS,注射PBS的小鼠則成為正常對照組,3個月后注射pristane的小鼠隨機分為Ad-A20、Ad-control和pristane組并在腹腔內(nèi)分別注射1.0×109pfu Ad-A20、Ad-control和PBS；利用ELISA試劑盒檢測血清IL-1β、IL-6IgG、anti-nRNP、anti-dsDNA和尿白蛋白；腎臟石蠟切片進行HE染色并在光鏡下觀察,以雙盲的方式對每只小鼠8個視野腎小球和腎小管評分進行腎臟病理評估；腎臟冰凍切片進行IgG和C3免疫熒光染色并在熒光顯微鏡下進行觀察,熒光強度利用Image J軟件評估并評分；蛋白質(zhì)印跡法檢測A20、NLRP3、 ASC、p-IκB、IκB、F4/80、CCL2、Caspase-1p20、p-NF-κBp65和NF-κBp65蛋白表達水平；曼-惠特尼“檢驗或單因素方差分析統(tǒng)計組間差異,結(jié)果表示為x±s。結(jié)果：Pristane組和Ad-control組小鼠腹腔巨噬細胞A20蛋白較PBS組小鼠比較而言表達輕度上調(diào),而Ad-A20治療組小鼠腹腔巨噬細胞A20蛋白較其他三組比較而言顯著上調(diào)；Pristane組和Ad-control組小鼠血清抗dsDNA和抗nRNP抗體水平顯著高于PBS組小鼠,而Ad-A20治療組小鼠血清抗dsDNA和抗nRNP抗體水平則較Ad-control組顯著降低但仍高于PBS組小鼠；病理半定量分析表明Ad-A20組小鼠腎小球和腎小管活動度計分僅稍高于BPS組小鼠,而Ad-A20組小鼠腎小球和腎小管活動度計分顯著低于Pristane組和Ad-control組小鼠。小鼠24小時尿液白蛋白檢測表明Pristane組小鼠尿液白蛋白顯著高于PBS組小鼠,而Ad-A20組小鼠尿液白蛋白低于Ad-control組小鼠；Pristane組和Ad-control組小鼠腎臟CCL2和F4/80表達水平顯著高于PBS組小鼠,而Ad-A20治療組小鼠CCL2和F4/80表達水平則較Ad-control組顯著降低；Pristane組和Ad-control組小鼠β-IκB和p-NF-KBp65表達水平顯著高于PBS組小鼠,而Ad-A20治療組小鼠β-IκB和p-NF-KBp65表達水平則較Ad-control組顯著降低；Pristane組和Ad-control組小鼠NLRP3、ASC和Caspase-1p20表達水平顯著高于PBS組小鼠,而Ad-A20治療組小鼠NLRP3、ASC和Caspase-1p20表達水平則較Ad-control組顯著降低。結(jié)論：腺病毒介導的過表達A20緩解了pristane誘導的SLE模型小鼠炎癥和腎臟損傷,其治療機制可能與抑制巨噬細胞NLRP3炎性小體和NF-κB活性相關(guān)。由于能對NLRP3炎性小體和NF-κB活性進行雙重抑制,過表達A20有望成為治療SLE的新選擇。
[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.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R-332;R593.241

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