抑制转录因子Blimp1在系统性红斑狼疮中的作用及机制研究

发布时间：2018-03-14 08:44

本文选题：系统性红斑狼疮　切入点：浆细胞　出处：《第三军医大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景系统性红斑狼疮(systemic lupus erythematosus,SLE)是一种以高水平自身抗体、B细胞高反应性、累及多个脏器为特点的复杂自身免疫性疾病。目前临床治疗SLE多采用糖皮质激素、非甾体类抗炎药、免疫抑制剂等非特异性免疫抑制疗法,抑制自身免疫反应的同时不可避免的带来了肝毒性、胃肠炎等副作用。自身抗体是SLE的重要致病因素,浆细胞是抗体的直接来源,提示浆细胞靶向治疗SLE可能直接抑制或减少自身抗体的产生。B淋巴细胞诱导成熟蛋白1(B lymphocyte induced maturation protein1,Blimp1)是诱导浆细胞发育成熟及维持浆细胞存活和抗体分泌能力的关键转录因子,我们课题组前期实验证实Blimp1在SLE中高表达,并与SLE疾病活动度、自身抗体滴度及浆细胞数量正相关,提示Blimp1可能成为诊断SLE并判断其病情严重程度的指标,抑制Blimp1表达可能是浆细胞靶向治疗SLE的新方向。目的本研究旨在通过RNA干扰(small interfering RNA,si RNA)技术及小分子化学药物2-甲氧基雌二醇(2-methoxyestradiol,2-ME2)抑制转录因子Blimp1在SLE中的高表达,观察Blimp1被抑制后MRL-lpr狼疮小鼠的24小时尿蛋白、外周血抗双链DNA抗体、肾脏病理改变、淋巴器官病理肿大、浆细胞及双阴性T细胞等SLE疾病特征的改变情况,同时分析抗体产生相关转录因子BCMA、XBP-1、J-chain的表达变化情况,以探索抑制Blimp1在SLE中的作用及可能分子机制。方法1.购买MRL-lpr狼疮小鼠,构建慢病毒介导的Blimp1 siRNA(实验组)及空白载体(对照组),包装并滴定慢病毒,尾静脉注射MRL-lpr狼疮小鼠3周后,提取小鼠PBMC中总RNA,RT-PCR检测Blimp1、BCMA、XBP-1、J-chain、C-myc mRNA表达水平,western blot及IHC分析小鼠肝脏、肾脏、脾脏、淋巴结中Blimp1蛋白的表达及组织分布情况。同时考马斯亮蓝染色法分析实验组及对照组小鼠24小时尿蛋白水平,ELISA技术定期检测外周血抗双链DNA抗体水平,并用HE染色病理切片评价肾脏病理改变情况。2.购买mrl-lpr狼疮小鼠及b6正常小鼠,随机等分为实验组(2-me2处理)和对照组(dmso处理),按组灌胃给药4周,每周记录小鼠体重及皮肤溃烂面积、生化仪器检测随机尿尿蛋白及尿肌酐水平,而后处死小鼠并记录小鼠淋巴器官重量、背部皮损面积评分和淋巴结肿大评分情况,qrt-pcr及westernblot检测小鼠blimp1mrna及蛋白表达情况,流式细胞术分析小鼠脾脏淋巴细胞亚群变化情况。结果1.blimp1mrna及蛋白在mrl-lpr狼疮小鼠外周血、脾脏、肾脏及淋巴结中高表达,blimp1sirna注射3周后,狼疮小鼠外周血blimp1表达水平和抗双链dna抗体水平分别下降78%和28%(p0.05),同时伴随抗体分泌相关转录因子xbp-1、j-chain和bcmamrna水平下降。相比对照组,虽然blimp1蛋白水平在blimp1sirna处理狼疮小鼠肝脏中未见明显变化,但在肾脏、脾脏和淋巴结中分别下降95%、72%和47%(p0.05)。此外,在blimp1sirna的作用下,狼疮小鼠的肾脏病理改变减轻,24小时尿蛋白水平也显著下降(p0.05)。2.blimp1在mrl-lpr狼疮小鼠外周血、肾脏、脾脏中高表达,2-me2可显著抑制狼疮小鼠外周血中blimp1mrna表达(Δct值6.99±0.69vs7.36±0.50,p0.05)、肾脏中blimp1蛋白表达(od值0.46±0.07vs0.15±0.16,p0.05)、脾脏中blimp1蛋白表达(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)、抑制其脾脏的过度增生肿大(脾脏/体重比值11.91±2.59vs7.04±3.30,p0.05),减少狼疮小鼠致病性t细胞数量(cd3+cd4-cd8-细胞数量11.93±2.70x106vs2.13±1.74x106,p0.05),同时降低小鼠背部皮损面积大小及评分和淋巴结肿大评分、缓解其肾脏病变。结论我们前期实验发现blimp1在sle患者中高表达,并与sle疾病活动度、自身抗体滴度及浆细胞数量正相关。本次研究证实blimp1在mrl-lpr狼疮小鼠中高表达,blimp1sirna可显著降低狼疮小鼠blimp1mrna及蛋白表达水平、24小时尿蛋白及外周血抗双链dna水平,缓解其肾脏病变。2-me2可抑制mrl-lpr狼疮小鼠肾脏、脾脏、外周血中高表达的blimp1,降低狼疮小鼠随机尿尿蛋白/尿肌酐比值、抑制脾脏及淋巴结过度增生、减少其脾脏中致病性t细胞数量并缓解其肾脏病理改变,提示抑制blimp1可以缓解狼疮小鼠的sle症状,其作用的产生机制可能是通过抑制blimp1进而抑制浆细胞分化成熟相关转录因子xbp-1、bcma、j-chain的表达,进而造成浆细胞成熟障碍、浆细胞数量减少、自身抗体分泌水平下降。综合以上证据表明,Blimp1有可能成为判断SLE病情程度的实验室新指标,抑制Blimp1有可能为治疗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.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.241

【共引文献】