本文選題：系統(tǒng)性紅斑狼瘡　切入點：漿細胞　出處：《第三軍醫(yī)大學》2015年碩士論文　論文類型：學位論文

【摘要】：背景系統(tǒng)性紅斑狼瘡(systemic lupus erythematosus,SLE)是一種以高水平自身抗體、B細胞高反應性、累及多個臟器為特點的復雜自身免疫性疾病。目前臨床治療SLE多采用糖皮質(zhì)激素、非甾體類抗炎藥、免疫抑制劑等非特異性免疫抑制療法,抑制自身免疫反應的同時不可避免的帶來了肝毒性、胃腸炎等副作用。自身抗體是SLE的重要致病因素,漿細胞是抗體的直接來源,提示漿細胞靶向治療SLE可能直接抑制或減少自身抗體的產(chǎn)生。B淋巴細胞誘導成熟蛋白1(B lymphocyte induced maturation protein1,Blimp1)是誘導漿細胞發(fā)育成熟及維持漿細胞存活和抗體分泌能力的關(guān)鍵轉(zhuǎn)錄因子,我們課題組前期實驗證實Blimp1在SLE中高表達,并與SLE疾病活動度、自身抗體滴度及漿細胞數(shù)量正相關(guān),提示Blimp1可能成為診斷SLE并判斷其病情嚴重程度的指標,抑制Blimp1表達可能是漿細胞靶向治療SLE的新方向。目的本研究旨在通過RNA干擾(small interfering RNA,si RNA)技術(shù)及小分子化學藥物2-甲氧基雌二醇(2-methoxyestradiol,2-ME2)抑制轉(zhuǎn)錄因子Blimp1在SLE中的高表達,觀察Blimp1被抑制后MRL-lpr狼瘡小鼠的24小時尿蛋白、外周血抗雙鏈DNA抗體、腎臟病理改變、淋巴器官病理腫大、漿細胞及雙陰性T細胞等SLE疾病特征的改變情況,同時分析抗體產(chǎn)生相關(guān)轉(zhuǎn)錄因子BCMA、XBP-1、J-chain的表達變化情況,以探索抑制Blimp1在SLE中的作用及可能分子機制。方法1.購買MRL-lpr狼瘡小鼠,構(gòu)建慢病毒介導的Blimp1 siRNA(實驗組)及空白載體(對照組),包裝并滴定慢病毒,尾靜脈注射MRL-lpr狼瘡小鼠3周后,提取小鼠PBMC中總RNA,RT-PCR檢測Blimp1、BCMA、XBP-1、J-chain、C-myc mRNA表達水平,western blot及IHC分析小鼠肝臟、腎臟、脾臟、淋巴結(jié)中Blimp1蛋白的表達及組織分布情況。同時考馬斯亮藍染色法分析實驗組及對照組小鼠24小時尿蛋白水平,ELISA技術(shù)定期檢測外周血抗雙鏈DNA抗體水平,并用HE染色病理切片評價腎臟病理改變情況。2.購買mrl-lpr狼瘡小鼠及b6正常小鼠,隨機等分為實驗組(2-me2處理)和對照組(dmso處理),按組灌胃給藥4周,每周記錄小鼠體重及皮膚潰爛面積、生化儀器檢測隨機尿尿蛋白及尿肌酐水平,而后處死小鼠并記錄小鼠淋巴器官重量、背部皮損面積評分和淋巴結(jié)腫大評分情況,qrt-pcr及westernblot檢測小鼠blimp1mrna及蛋白表達情況,流式細胞術(shù)分析小鼠脾臟淋巴細胞亞群變化情況。結(jié)果1.blimp1mrna及蛋白在mrl-lpr狼瘡小鼠外周血、脾臟、腎臟及淋巴結(jié)中高表達,blimp1sirna注射3周后,狼瘡小鼠外周血blimp1表達水平和抗雙鏈dna抗體水平分別下降78%和28%(p0.05),同時伴隨抗體分泌相關(guān)轉(zhuǎn)錄因子xbp-1、j-chain和bcmamrna水平下降。相比對照組,雖然blimp1蛋白水平在blimp1sirna處理狼瘡小鼠肝臟中未見明顯變化,但在腎臟、脾臟和淋巴結(jié)中分別下降95%、72%和47%(p0.05)。此外,在blimp1sirna的作用下,狼瘡小鼠的腎臟病理改變減輕,24小時尿蛋白水平也顯著下降(p0.05)。2.blimp1在mrl-lpr狼瘡小鼠外周血、腎臟、脾臟中高表達,2-me2可顯著抑制狼瘡小鼠外周血中blimp1mrna表達(Δct值6.99±0.69vs7.36±0.50,p0.05)、腎臟中blimp1蛋白表達(od值0.46±0.07vs0.15±0.16,p0.05)、脾臟中blimp1蛋白表達(od值0.88±0.11vs0.26±0.18,p0.05),并降低其尿蛋白/尿肌酐比值(0.77±0.29vs0.27±0.17,p0.05)、抑制其脾臟的過度增生腫大(脾臟/體重比值11.91±2.59vs7.04±3.30,p0.05),減少狼瘡小鼠致病性t細胞數(shù)量(cd3+cd4-cd8-細胞數(shù)量11.93±2.70x106vs2.13±1.74x106,p0.05),同時降低小鼠背部皮損面積大小及評分和淋巴結(jié)腫大評分、緩解其腎臟病變。結(jié)論我們前期實驗發(fā)現(xiàn)blimp1在sle患者中高表達,并與sle疾病活動度、自身抗體滴度及漿細胞數(shù)量正相關(guān)。本次研究證實blimp1在mrl-lpr狼瘡小鼠中高表達,blimp1sirna可顯著降低狼瘡小鼠blimp1mrna及蛋白表達水平、24小時尿蛋白及外周血抗雙鏈dna水平,緩解其腎臟病變。2-me2可抑制mrl-lpr狼瘡小鼠腎臟、脾臟、外周血中高表達的blimp1,降低狼瘡小鼠隨機尿尿蛋白/尿肌酐比值、抑制脾臟及淋巴結(jié)過度增生、減少其脾臟中致病性t細胞數(shù)量并緩解其腎臟病理改變,提示抑制blimp1可以緩解狼瘡小鼠的sle癥狀,其作用的產(chǎn)生機制可能是通過抑制blimp1進而抑制漿細胞分化成熟相關(guān)轉(zhuǎn)錄因子xbp-1、bcma、j-chain的表達,進而造成漿細胞成熟障礙、漿細胞數(shù)量減少、自身抗體分泌水平下降。綜合以上證據(jù)表明,Blimp1有可能成為判斷SLE病情程度的實驗室新指標,抑制Blimp1有可能為治療SLE提供新策略。
[Abstract]:The background of systemic lupus erythematosus (systemic lupus, erythematosus, SLE) is a kind of high levels of autoantibodies, B cell hyperreactivity, multiple organ involvement characterized by complicated autoimmune disease. The clinical treatment of SLE by glucocorticoids, non steroidal anti-inflammatory drugs, immunosuppressive agents such as non specific immunity suppression therapy, suppression of autoimmune reaction and inevitably bring about liver toxicity, gastroenteritis and other side effects. Autoantibodies are important pathogenic factors of SLE plasma cells is a direct source of antibodies, suggesting that plasma cell targeting SLE can directly inhibit or reduce the production of autoantibodies of.B lymphocytes induced by mature protein 1 (B lymphocyte induced maturation protein1, Blimp1) is a key transcription factor induced plasma cell maturation and maintenance of plasma cell survival and antibody secretion ability, our previous experiments confirmed that Blimp1 The high expression in SLE, and the SLE disease activity, the number of antibodies and plasma cells are positively related, which suggests that Blimp1 may be SLE diagnosis and judge the severity, inhibition of Blimp1 expression may be a plasma cell targeted therapy for SLE. The purpose of this study aims at using RNA interference (small interfering RNA, Si, RNA) and small molecule drug 2- methoxyestradiol (2-methoxyestradiol, 2-ME2) high expression inhibition of transcription factor Blimp1 in SLE in the observation of Blimp1 was inhibited after MRL-lpr mice 24 hour urine protein, anti double stranded DNA antibody in peripheral blood and renal pathological changes, pathological enlargement of lymphoid organs the change of plasma cells, and double negative T cells SLE disease characteristics, and analysis of antibody related transcription factor BCMA, XBP-1, expression of J-chain, to explore the inhibition of Blimp1 in SLE may play a role in the molecular The mechanism of MRL-lpr lupus mice. Methods 1. buy, the construction of lentivirus mediated Blimp1 siRNA (experimental group) and blank vector (control group), lentivirus packaging and titration, 3 weeks after the intravenous injection of MRL-lpr mice, PBMC mice total RNA extraction, RT-PCR detection of Blimp1 BCMA, XBP-1, J-chain, C-myc, mRNA the expression level of western, blot and IHC analysis of mouse liver, kidney, spleen, Blimp1 protein expression and tissue distribution of lymph nodes. At the same time, Coomassie brilliant blue staining of the experimental group and control group of mice 24 hours urine protein level, regular ELISA detection of peripheral blood anti double stranded DNA antibody level and HE staining the renal pathological changes of.2. were evaluated to buy MRL-lpr and B6 lupus mice in normal mice, were randomly divided into experimental group (2-me2 treatment) and control group (DMSO treatment group), by gavage for 4 weeks, body weight was recorded weekly and the skin of mice ulcer surface Product, biochemical instrument to detect random pee protein and creatinine level, and then the mice were sacrificed and record the weight of lymphoid organs of mice, the back of the lesion area score and lymph node enlargement score, the expression of qRT-PCR and Westernblot were detected blimp1mrna and protein analysis, the changes of mice spleen lymphocyte subsets by flow cytometry. Results of 1.blimp1mrna and protein in MRL-lpr lupus mice peripheral blood, spleen, kidney and high expression in lymph nodes, 3 weeks after blimp1sirna injection, mice peripheral blood BLIMP1 expression and anti double stranded DNA antibody levels were decreased by 78% and 28% (P0.05), accompanied by the antibody secreting transcription factor XBP-1, J-chain and bcmamrna decreased compared to the control group. Although, the protein level of BLIMP1 showed no significant changes in the liver in blimp1sirna treated mice, but in the kidney, spleen and lymph nodes decreased by 95%, 72% and 47 % (P0.05). In addition, in the presence of blimp1sirna, the renal pathological changes alleviated lupus mice, 24 hours urinary protein levels decreased significantly (P0.05).2.blimp1 in MRL-lpr mice peripheral blood, kidney, high expression in the spleen, 2-me2 can significantly inhibit the expression of blimp1mrna in peripheral blood of mice (CT value 6.99 + 0.69vs7.36 + 0.50, P0.05), the expression of BLIMP1 protein in the kidney (OD = 0.46 + 0.07vs0.15 + 0.16, P0.05), the expression of BLIMP1 protein in the spleen (OD = 0.88 + 0.11vs0.26 + 0.18, P0.05), and reduce the urinary protein / creatinine ratio (0.77 + 0.29vs0.27 + 0.17, P0.05), hyperplasia inhibition of the spleen enlargement (spleen / body weight ratio 11.91 + 2.59vs7.04 + 3.30, P0.05), reduce the number of mice of pathogenic T cells (cd3+cd4-cd8- cell number 11.93 + 2.70x106vs2.13 + 1.74x106, P0.05), while reducing the small rat back lesion size and score and lymph nodes Score, alleviate the renal lesions. Conclusion our previous studies demonstrated that high expression of BLIMP1 in SLE patients, and SLE disease activity, the number of antibodies and plasma cells are related. This study confirmed that the high expression of BLIMP1 in MRL-lpr mice, blimp1sirna significantly decreased the blimp1mrna and protein expression level of lupus mice, anti double the chain of DNA level 24 hours urine protein and peripheral blood, alleviate the renal lesion in.2-me2 can inhibit the kidney of MRL-lpr mice, the spleen, the high expression of BLIMP1 in peripheral blood, reduce lupus mice were urinary protein / creatinine ratio, inhibition of spleen and lymph node hyperplasia, reduce the number of pathogenic T cells in the spleen and to alleviate the renal pathological changes, suggesting that inhibition of BLIMP1 can alleviate the symptoms of SLE mice, produced its mechanism may be through inhibition of BLIMP1 and inhibition of plasma cell differentiation related to mature With factor XBP-1, BCMA, J-chain expression, resulting in plasma cell dysmaturity, plasma cell number decreased, autoantibody secretion levels decreased. The above evidence suggests that Blimp1 may become a new index of SLE severity judgment laboratory, inhibition of Blimp1 may provide a new strategy for the treatment of SLE.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R593.241

【共引文獻】

相關(guān)碩士學位論文 前1條

1 葉青;基于免疫機理的化工園區(qū)安全生產(chǎn)應急研究[D];華南理工大學;2012年

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本文編號：1610462