系统性红斑狼疮患者Fas和CD40L表达的初步研究Word下载.docx

1、 _） 作者：徐红星, 邱德华,王慧娟 ,杨晓帆,季晓辉【摘要】 目的 探讨系统性红斑狼疮（SLE）患者凋亡调节蛋白受体Fas及共刺激分子CD40L的表达情况及其相互之间的调节。方法 采用酶联免疫吸附试验（ELISA）检测SLE患者和正常人血清sFas和sCD40L的水平并分析两者之间的相关性;同时分离10例SLE患者和10例正常对照的外周血淋巴细胞：将细胞培养48 h后，采用流式细胞仪检测T细胞亚群表面Fas及CD40L的表达情况;在培养过程中分别加入抗FasL抗体阻断Fas信号及抗CD40L抗体阻断CD40L信号后，观察T细胞亚群表面CD40L及Fas的表达。结果 SLE患者血清sFas和

2、sCD40L平均水平分别为4.18 g/L和5.87 g/L，显著高于正常对照组2.27 g/L和2.31 g/L,且SLE患者血清sFas活动期高于稳定期（P0.01或P0.05）;sFas、sCD40L水平与SLEDAI（疾病活动指数）之间存在一定的正相关性（r=0.688,r=0.253，P均0.05）,但sFas与sCD40L水平之间未显示明显相关（r=0.201，P0.05）。经细胞培养后，SLE患者CD4（+）和CD8（+） T细胞表面Fas表达增加，与正常对照组比较，差异有显著性（P0.01或P0.05）;SLE患者CD4（+）和CD8（+） T细胞表面CD40L表达增加，与正常

3、对照组比较，差异有显著性（P均0.05）。SLE患者CD4（+）及CD8（+） T细胞上Fas与CD40L表达均呈正相关关系（r=0.311和r=0.517，P均0.05）。加入抗FasL抗体阻断后，CD4（+）和CD8（+） T细胞表面CD40L表达下降，与阻断前比较，差异有显著性（P0.05）;加入抗CD40L抗体阻断后，CD4（+）和CD8（+） T细胞表面Fas表达与阻断前比较差异无显著性（P0.05）。结论 SLE患者血清sFas和sCD40L水平升高且呈正相关关系;T细胞亚群表面Fas及CD40L高表达且呈正相关关系;T细胞上Fas的表达调控CD40L的表达。说明凋亡调节蛋白Fas

4、及共刺激分子CD40L可能共同参与SLE的发病过程。 【关键词】 系统性红斑狼疮;Fas;CD40LAbstract: Objective To investigate the expressions of Fas and CD40L in patients with systemic lupus erythematosus （SLE）. Methods Serum sFas, sCD40L levels in patients with SLE and healthy controls were determined using a commercially available ELISA

5、system and the correlation between the levels of sFas and sCD40L was analyzed. Meanwhile, peripheral blood mononuclear cells （PBMC） of patients with SLE and health controls were isolated by gradient centrifugation of heparinized blood. The expressions of Fas and CD40L on CD4（+） and CD8（+） T lymphocy

6、tes in patients with SLE and controls were determined by flow cytometry （FCM） after cultivation for 48 h. The antibodies of FasL and CD40L were add in the process of cultivation, and then the expressions of CD40L and Fas of peripheral blood T lymphocytes were determined. Results The average levels o

7、f sFas and sCD40L in SLE patients were 4.18 g/L and 5.87 g/L, respectively, which were markedly higher than those of 2.27 g/L and 2.31 g/L in the healthy controls; the serum levels of sFas and sCD40L were significantly higher in active SLE patients than those inactive patients （P0.01, P0.05）; the se

8、rum levels of sFas and sCD40L were shown to have a positive correlation with SLEDAI scores （r=0.688, r=0.253,P0.05）, while no positive correlation was revealed between the levels of sFas and sCD40L （r=0.201,P0.05）. The expressions of Fas on CD4（+） and CD8（+） T lymphocytes in patients with SLE were s

9、ignificantly higher than those of the healthy controls （P0.01 and P0.05）; the expressions of CD40L on CD4（+） and CD8（+） T lymphocytes in patients with SLE were significantly higher than that of the healthy controls （P0.05）. There was no correlations between the expressions of Fas on CD4（+） and CD8（+

10、） T lymphocytes and those of CD40L （r=0.311, r=0.517）. The expressions of CD40L on CD4（+） and CD8（+） T lymphocytes in patients with SLE significantly decreased after blockage by the antibody of FasL （P0.05）; there was no difference in the expression of Fas in patients with SLE after blockage by the

11、antibody of CD40L （P0.05）. Conclusion The levels of sFas and sCD40L in SLE patients were higher than those of healthy controls and there was a positive correlation between them. The expressions of Fas and CD40L on CD4（+） and CD8（+） T lymphocytes in patients with SLE increased; and the expression of

12、Fas had a positive correlation with that of CD40L; meanwhile, the expression of Fas regulated that of CD40L, which suggests that both Fas and CD40L are involed in the process of SLE.Key words：systemic lupus erythematosus; Fas; CD40L系统性红斑狼疮（SLE）是一种慢性自身免疫性疾病，引起多器官损害，具有产生多种自身抗体的特点1。Fas和CD40L均属于TNF/NGF受

13、体超家族成员。Fas是一种细胞凋亡的调节蛋白受体，通过与Fas配体结合，介导细胞凋亡2。CD40L是表达于B细胞或其他抗原递呈细胞（APC）表面的CD40分子的配体，目前认为，共刺激分子CD40-CD40L是为激活淋巴细胞的第二信号系统的组成部分，其在T细胞介导的B细胞活化中具有重要作用3-4。本研究同时检测SLE患者血清sFas和sCD40L水平及外周血CD4（+）和CD8（+） T细胞表面CD40L和Fas的表达，并用抗FasL抗体、抗CD40L抗体分别阻断Fas信号和CD40信号测定CD4（+）和CD8（+） T细胞表面CD40L和Fas的表达，观察了淋巴细胞表面Fas与CD40L表达的

14、相关性及相互之间是否存在表达的调控情况，从而探讨Fas和CD40L参与SLE发病过程的可能尚未被认识的机制。1 资料和方法1.1 研究对象和分组 SLE组：南京医科大学附属苏州市立医院门诊或住院的SLE患者46例，其中女性42例，男性4例，年龄961岁，平均年龄32岁。其中36例进行血清sFas、sCD40L测定,10例进行T细胞亚群表面Fas与CD40L表达及相关性研究。所有SLE病例均符合美国风湿病协会（ARA）的诊断标准。根据临床表现及实验室检查指标，参照SLE疾病活动性指数（systemic lupus erythematosus disease activity index, SLE

15、DAI）5评分标准，判断SLE的病情活动性，将SLE组患者进一步分为活动期（SLEDAI 10）及缓解期（SLEDAI10）2个亚组。正常对照组:30例，其中男3例，女27例，平均年龄32.5岁，均为门诊健康体检者。1.2 主要仪器 MicroReader4型酶联免疫检测仪,HFsafe-900型生物安全柜;TC2323型二氧化碳培养箱,Coulter公司流式细胞仪。1.3 主要试剂 晶美生物工程有限公司sFas检测试剂盒,Bender MedSystems公司sCD40L检测试剂盒,上海试剂二厂淋巴细胞分离液,GIBCO公司RPMI 1640培养液,eBioscience公司抗人FasL、抗

16、人CD40L阻断性抗体,Caltag公司PE-CY5-抗CD3、FITC-抗CD4、FITC-抗CD8荧光标记抗体,eBioscience公司PE-抗CD40L、PE-抗Fas荧光标记抗体。1.3 方法1.3.1 sFas、sCD40L的检测 严格按说明书进行操作。结果判断：用MicroReader4酶标仪比色，得到光密度（D）值，以标准品浓度作横坐标，D值作纵坐标，以平滑线连接各标准品的坐标点，通过标本的D值可在标准曲线上查出其浓度。1.3.2 细胞分离培养 取正常健康体检者和SLE患者肝素抗凝外周静脉血，用淋巴细胞分离液分离单个核细胞，用RPMI 1640培养液调细胞密度至1109/L，加入24孔培养板中37、5% CO2培养48 h。SLE患者细胞培养液中纯化抗人FasL抗体和