【作者】 安湘杰；

【导师】 涂亚庭；

【作者基本信息】 华中科技大学 ， 皮肤病与性病学， 2013， 博士

【摘要】 第一部分RasGRP3在系统性红斑狼疮患者外周血单个核细胞及B细胞中的表达目的系统性红斑狼疮(systemic lupus erythematosus, SLE)是以自身抗体的产生为特征的累及多个器官和组织的自身免疫病,内源性的高反应性B淋巴细胞是狼疮发病的重要环节。SLE患者中存在免疫分子的表达异常可导致BCR通路持续性激活,而RasGRP3(Ras guanyl nucleotide releasing protein3, Ras鸟苷酸释放蛋白3)主要在B细胞中表达,为BCR信号通路下游重要分子。本实验研究RasGRP3在SLE患者外周血单个核细胞(Peripheral blood mononuclear cell, PBMC)中的表达,并进一步提取PBMC中的B细胞,研究RasGRP3在SLE患者外周血B细胞中的表达,并探讨其在SLE发病免疫机制的意义。方法收集临床上确诊SLE患者的外周血标本20例,以及正常志愿者20例,Ficoll法提取SLE患者及正常人外周血中的PBMC,采用免疫磁珠分选法纯化B细胞,流式检测B细胞的纯度。使用RT-PCR方法检测SLE患者及正常人PBMC和B细胞中RasGRP3的mRNA表达及用Western Blot方法检测蛋白的表达。其中10例标本用于PBMC中RasGRP3mRNA表达分析,5例标本用于RasGRP3蛋白表达分析,5例标本用于纯化B细胞,检测B细胞中RasGRP3mRNA的表达结果免疫磁珠分选法分选B细胞,其纯度能达到90%以上。SLE患者PBMC中RasGRP3的mRNA和蛋白质的表达水平明显高于正常人。在纯化后的B细胞中,与正常人相比,SLE患者RasGRP3的mRNA表达水平也明显升高。结论SLE患者外周血PBMC及B细胞中RasGRP3的mRNA和蛋白水平均较正常人高,提示B细胞中RasGRP3的异常表达可能参与狼疮发病。第二部分SLE患者PBMC中RasGRP3的mRNA水平与临床资料的关联目的研究RasGRP3的mRNA表达与SLE患者临床特征的联系,探讨其表达增高是否与SLE临床体征,实验室检查结果,病情活动程度有关。方法收集临床上确诊SLE患者26例用于分析PBMC中RasGRP3mRNA水平与临床特征的关联,Ficoll法提取PBMC, RT-PCR方法半定量检测其mRNA表达。通过回归统计分析SLE患者疾病活动程度与其PBMC中RasGRP3的mRNA表达的相关性。根据患者临床特征以及实验室检查结果分组,使用T检验研究RasGRP3的mRNA表达在每两组间有无差异。结果SLE患者PBMC中RasGRP3的mRNA表达水平与SLE疾病活动指数(SLEDAI)成正相关(p=0.0301),并且疾病活动期患者较静止期患者PBMC中RasGRP3的mRNA水平显著增加(p<0.05)。血尿、蛋白尿的SLE患者与非血尿、蛋白尿的患者相比,RasGRP3的mRNA表达显著增加(p<0.05)。结论RasGRP3可能为反应狼疮疾病活动的指标之一,并与肾脏损害有关,但本次研究样本量偏小,仍需大样本研究进一步验证。第三部分地塞米松对系统性红斑狼疮患者中RasGRP3表达的影响目的分别体内外研究糖皮质激素对SLE患者PBMC中RasGRP3的作用,探讨糖皮质激素通过抑制RasGRP3的表达治疗SLE的机制。方法选取临床上使用糖皮质激素(约lmg/kg强的松)的SLE患者10例,抽取其糖皮质激素治疗前及治疗后两周的外周血,提取PBMC。其中5例检测治疗前后PBMC中RasGRP3的mRNA表达水平,5例检测蛋白表达水平。在体外实验中,使用两种不同浓度地塞米松(1.10-5、1×10-6mol/L),与正常人PBMC细胞共同培养,在孵育时间为0h,12h,24h提取细胞,使用RT-PCR的方法检测RasGRP3的mRNA水平,选取合适的浓度。另外收集未经治疗的SLE患者6例,使用该浓度的地塞米松共孵育,观察不同时间点(0h、12h、24h)的地塞米松对PBMC中RasGRP3的mRNA水平的影响。并在地塞米松孵育0h,24h,48h提取细胞,使用western blot方法检测RasGRP3蛋白水平及下游信号通路p-ERK, p-AKT的水平。结果未使用糖皮质激素治疗时,SLE患者PBMC中RasGRP3的表达高于正常人,使用中等剂量糖皮质激素治疗2周后,RasGRP3的表达明显下降,接近正常人的表达水平。地塞米松能抑制正常人PBMC中RasGRP3的mRNA表达,1×10-6mol/L浓度的地塞米松对RasGRP3表达的有抑制作用且未明显引起细胞凋亡。使用该浓度的地塞米松与SLE患者PBMC共培养,观察不同时间点地塞米松对RasGRP3的抑制作用。我们发现,24h时地塞米松对PBMC内RasGRP3的mRNA及蛋白水平抑制作用最强。同时在24h地塞米松能明显抑制PBMC中pERK及p-AKT的表达。结论SLE患者在临床治疗时,使用糖皮质激素治疗2周后,PBMC中RasGRP3的水平下降。地塞米松能抑制正常人及SLE患者PBMC中RasGRP3及下游信号通路pERK及p-AKT的表达。这可能为激素的抗炎机制之一。更多还原

【Abstract】 Part Ⅰ The expressions of RasGRP3in peripheral blood lymphocytes and B lymphocytes of SLE patientsObjective Systemic lupus erythematosus (SLE) is a chronic disease with diverse clinical presentations and immunological abnormalities, involving multiple tissues and organs. The immunological mechanism for lupus has not been fully classified. In order to explore the role of Ras guanine nucleotide releasing protein (RasGRP3) pathogenesis of autoimmunity in SLE, we detected the expressions of RasGRP3in peripheral blood mononuclear cells (PBMCs) of patients with SLE and further study the RasGRP3expressions in purified B cells.Methods The blood samples from patients diagnosed as SLE and healthy volunteers were collected. Human PBMCs were obtained by Ficoll method. B cells were purified from PBMCs by immunomagnetic beads separation. The purity of B cells was confirmed by FCM and the expressions of RasGRP3were detected by RT-PCR and Western Blot.Results The purity of B cell could reach more than90%by immunomagnetic beads separation. Compared with healthy volunteers, the mRNA and protein expressions of RasGRP3were elevated both in PBMCs and B cells.Conclusions The expressions of RasGRP3in both PBMC and B cells of SLE patients were much higher compared with healthy volunteers. Aberrant expressions of RasGRP3might contribute to the pathogenesis of autoimmunity in lupus. Part II The relationship between the mRNA expressions of RasGRP3in PBMCs and the clinical data of SLE patientsObjective:SLE patients manifest diverse clinical presentations and laboratory results. In order to explore whether the expression of RasGRP3is related to the clinical data in SLE patients, such as clinical signs, laboratory tests, disease activity, and so on.Methods:The peripheral blood samples from patients diagnosed as SLE were collected, as well as the detailed clinical data (basic information, complaims, symptoms, clinical signs, and laboratory test results). The PBMCs were extracted by Ficoll method, and further detected the RasGRP3mRNA expressions by semi-quantitative RT-PCR. We grouped the data by clinical features and laboratory test results, and compared the RasGRP3mRNA expressions between different two groups by Ttest.Results The mRNA expression of RasGRP3was positively correlated with SLE disease activity index (SLEDAI)(p=0.0301). The mRNA levels of RasGRP3in the patients with active disease were much higher than that in remission stage (p<0.05). In addition, the mRNA levels of RasGRP3in the patients with hematuria or proteinuria were significantly higher than that without kidney damage (p<0.05).Conclusion The mRNA expression of RasGRP3may be used as an indicator for lupus disease activity, and RasGRP3may be related to kidney damages. Due to limited sample size, further samples are needed to confirm this conclusion. Part Ⅲ Dexamethasone inhibits the expression of RasGRP3in PBMC of SLE patients both in Vitro and VivoObjective In order to observe the RasGRP3expression after dexamethasone treatment in vivo and in vitro, as well as explore the role of dexamethasone in the treatment of lupus.Methods The SLE patients who treated with moderate-dose corticosteroids (1mg/kg prednisone) were recruited in. The blood samples were acquired before and two weeks after the corticosteroids treatment to obtain PBMCs. The mRNA expression of RasGRP3was measured by RT-PCR; the protein level was examined by Western Blot. In vitro, dexamethasone of two different concentrations (1×10-5、1×10-6mol/L) was applied to co-culture with PBMC from healthy volunteers at different incubation time (Oh,12h,24h), and then we observed the RasGRP3mRNA expressions by RT-PCR to choose a concentration for better inhibition effect. We co-cultured the PBMC from untreated SLE patients with dexamethasone in this appropriated concentration and observed the mRNA expression of RasGRP3at different time points (Oh,12h,24h). In addition, the protein expressions of RasGPR3, p-ERK and p-AKT were also detected by Western Blot.Results The RasGRP3expression of SLE patients without corticosteroids treatment was much higher than that of normal people. After2weeks application of moderate-dose corticosteroids, the expression of RasGRP3was obviously inhibited. In vitro, dexamethasone could inhibit the mRNA expression of RasGRP3in normal people, and the1×10-6mol/L concentration of dexamethasone had obvious inhibited effect and did not induce cell apoptosis. We co-cultured the PBMC from SLE patients with1×10-6mol/L dexamethasone and observed the expression of RasGRP3at different time points, and found that dexamethasone showed the strongest inhibition on the expression of RasGRP3at the time points of24h;and at this time point, dexamethasone also inhibited pERK and p-AKT which were the downstream signaling pathway of RasGRP3. Conclusion:In vivo,2-week corticosteroids treatment could inhibit the RasGRP3expression in SLE patients; in vitro, dexamethasone could reduce the RasGRP3expression as well as p-ERK and p-AKT of its downstream signaling pathway of PBMCs from both healthy people and SLE patients. This may be one of the mechanisms of corticosteroids treatment of SLE.更多还原