摘要
研究背景
系统性红斑狼疮(systemic lupus erythematosus,SLE)是一种自身免疫介导的,以免疫性炎症为主要表现的弥漫性结缔组织病。临床主要特征是多个器官、系统受累和血清检测到多种自身抗体(以抗核抗体为代表)。SLE病程迁延,病死率高,病因至今未明,也无特异有效的治疗方法。
SLE发病过程中同时存在细胞免疫和体液免疫系统紊乱两种形式,而T淋巴细胞的异常并导致B淋巴细胞的过度活化,最终导致免疫系统的异常应答是其主要发病机制。近年来国内外对SLE的免疫紊乱研究趋于深入,多项研究认为由于SLE患者自身淋巴细胞平衡被打破,过度活化的T淋巴细胞导致多克隆B细胞分化和激活,从而产生大量自身抗体引起多系统损害受累。目前认为的T淋巴细胞的异常可能是SLE发病的重要因素。有研究表明SLE患者Th的凋亡显著增加,且凋亡水平与疾病活动性相关,因此,T辅助淋巴细胞(Th)的数量及功能失衡是T淋巴细胞免疫功能异常的重要方面。
Th淋巴细胞不是单一的一种细胞群,而是包含了一系列具有不同职能的细胞亚群,幼稚CD4+T淋巴细胞可以分化为Th1、Th2、Th17和调节性T细胞(Treg),四种细胞分泌的细胞因子以及在体内免疫作用各不相同。因此,这些不同职能的细胞亚群在分化、发育及功能执行等方面存在着错综复杂的关系,需要对Th细胞亚型在系统性红斑狼疮病因机制方面发挥的作用进行深入细致的研究,才能有助于进一步明确Th细胞在SLE的作用。
早期,人们仅仅关注于Th1/Th2的失衡状态与SLE的发病关系。认为Th1/Th2的平衡状态在系统性红斑狼疮中起着决定性的作用,其间,“Th2优势”、“Th1优势”等多种免疫紊乱状态均在狼疮动物模型及患者体内被证实。但随着对SLE研究的深入,发现很多SLE临床征象和实验室特点不能用已知的Th1/Th2失衡模式来解释。近年来对T辅助细胞亚群的认识更加全面,在原有Th1/Th2细胞亚群基础上,Sakaguchi和Harrington先后在1995年和2005年又发现了调节性T细胞(Treg)和Th17两个新成员。逐渐发现Th17/Treg的平衡状态在SLE的疾病过程中发挥着重要作用。两者同属于Th细胞,但分化相互抑制,生物学表现完全相反。Th17启动自身免疫疾病发生, Treg细胞可减轻自身免疫病反应,两者相互拮抗相互抑制维持机体免疫状态的相对稳定。
Th1、Th2、Th17、Treg细胞均参与了SLE的致病过程,并且Th1/Th2、Th17/Treg的失衡状态可能导致SLE患者细胞功能紊乱,进而引起B细胞异常活化,促发和加剧SLE病情变化。研究显示,Th17/Treg细胞亚群失衡状态在系统性红斑狼疮患者中可能更为严重,从而在某种程度上,两组细胞平衡状态可以作为SLE疾病活动和病情评价的可靠指标。故将Th1/Th2、Th17/Treg作为一个观察整体,可能比单纯研究某类细胞的变化来推断SLE疾病状态更加科学系统。
总之,本研究从Th细胞的凋亡情况入手,深入分析Th1/Th2,Th17/Treg在SLE中的失衡状态,并相对全面系统分析Th1/Th2,Th17/Treg相关细胞因子谱变化特点,并对新成员Th17/Treg的特异转录因子的平衡状态进一步探究,以期通过相对较大样本的SLE病例资料,联合测定Th细胞亚群细胞、蛋白、基因水平变化及相应的SLE的疾病活动指标,探讨Th细胞亚群与SLE疾病活动性临床症状的关系及其对病情评估的价值。
目的
(1)通过比较系统性红斑狼疮患者和正常对照之间,SLE轻型、中度活动及重型患者之间的Th细胞凋亡情况,分析SLE患者Th细胞的凋亡与SLE疾病活动指数的关联。
(2)通过比较系统性红斑狼疮患者和正常对照之间,SLE轻型、中度活动及重型患者之间的Th1/Th2、Th17/Treg细胞平衡情况,分析Th1/Th2、Th17/Treg细胞与SLE的关系。
(3)系统分析SLE患者和正常对照之间,SLE轻型、中度活动及重型患者之间的Th1、Th2、Th17、Treg细胞因子水平,进一步验证Th1/Th2、Th17/Treg细胞与SLE的关系。
(4)初步研究SLE患者和正常对照之间,SLE轻型、中度活动及重型患者之间的Th17/Treg分化路径上游特异的转录因子RORγt和Foxp3水平,进一步验证Th1/Th2、Th17/Treg细胞与SLE的关系。深入了解Th17和调节性T细胞在SLE发病及病情进展过程中的意义。
方法
病例选取2009年6月至2010年9月在山西医科大学第二医院和中国人民解放军第264医院风湿科就诊SLE患者89例,诊断均符合1997年修订的美国风湿学会(American College of Rheumatology,ACR)分类标准。收集SLE患者一般情况和临床资料,根据SLE疾病活动指数(systemic lupus erythematosus disease activity index,SLEDAI)积分评估疾病的活动性,并结合临床症状将患者分为轻型SLE、中度活动型、重型三组。同时选取正常对照组27名,均不符合SLE诊断标准;无其他重大疾病史;未长期应用激素或免疫抑制剂;受检者本人或直系的亲属未患有自身免疫性疾病。实验在获得研究对象知情同意后,抽取外周静脉血。
采用流式细胞术检测SLE患者和正常对照之间,SLE轻型、中度活动及重型患者之间Th细胞凋亡率和Th1/Th2、Th17/Treg细胞水平。
采用流式细胞微球捕获技术(CBA)和ELSIA测定SLE患者和正常对照之间,SLE轻型、中度活动及重型患者之间IL-2、IL-4、IL-6、IL-10、TNF-α、IFN-γ,IL-17A和TGF-β水平
采用RT-PCR检测SLE患者和正常对照之间,SLE轻型、中度活动及重型患者之间血清Th17/Treg特异转录因子RORγt和Foxp3 mRNA表达水平
采用SPSS 13.0软件进行统计分析。
结果
(1)流式细胞术来测定Th细胞的凋亡情况显示SLE患者和正常人CD4+T淋巴细胞凋亡率分别为(1.11 0.36)%和(0.36 0.15)%,两者比较差异有统计学意义(P<0.0001)。轻型、中型、重型SLE患者组和正常对照组Th细胞凋亡率分别为(0.81 0.19)%、(1.030.21)%、(1.51 0.31)%和(0.36 0.15)%,组间比较差异有统计学意义(P<0.0001)
(2)流式细胞术测定四种Th细胞情况:SLE患者组Th1显著低于正常组,差异有统计学意义(P =0.049)。SLE患者组Th2与正常组比较差异无统计学意义。重型SLE患者Th1/Th2与中度活动组及轻型组比较差异有统计学意义。相关性分析显示:Th2/CD4+与SLEDAI呈正相关(r=0.4012, P =0.0001),Th1/Th2与SLEDAI呈负相关(r=-0.2683,P =0.0130)。SLE患者组Th17与正常组比较差异无统计学意义,但Th17占CD4+T细胞两组比较差异有统计学意义(P =0.012),患者组比例较正常人高。SLE患者组Treg低于正常组,差异有统计学意义( P=0.023)。重型SLE患者Th17、Th17/CD4+、Treg与正常对照组及轻型组比较差异有统计学意义。相关性分析显示:SLEDAI与Th17、Th17/CD4+和Th17/Treg呈正相关(r =0.3608, P =0.0006, r=0.4522, P <0.001,r=0.4240, P <0.001),与Treg呈负相关(r=-0.352, P =0.0010)。
(3)相关细胞因子测定结果:流式细胞微球捕获技术(CBA)测定SLE患者组与正常对照组IL-17、IL-10、IL-6、IL-4比较显著增高(P均<0.05)其余细胞因子比较差异无统计学意义。相关性分析显示:IL-17、IL-6、IL-4与SLEDAI呈正相关,IL-2、IFN-γ与SLEDAI呈负相关,尚不能认为TNF、IL-10与SLEDAI有相关性。
IL-17在不同活动度组间比较差异均有统计学意义,且在SLE重型患者中增高最为明显(F=52.660,P <0.001)。IFN-γ、IL-10、IL-6、IL-4、IL-2在不同活动度组间比较差异均有统计学意义(P <0.05)。TNF-α水平在不同活动度组间比较差异无统计学意义。
ELISA测定TGF-β结果显示SLE患者和正常对照之间,SLE轻型、中度活动及重型患者之间比较差异无统计学意义(F=2.150,P =0.157)。相关性分析显示:尚不能认为TGF-β水平与SLEDAI有相关性(r=-0.071,P =0.5150)。
(4)RT-PCR实验结果显示SLE患者Th17、Treg细胞的特异性转录因子ROR-γt、Foxp3的mRNA经RT-PCR扩增后均有所表达。SLE组ROR-γt、Foxp3的mRNA表达变化倍率显著高于正常对照组,比较差异均有统计学意义(t=-2.656,P =0.012;t=-2.873,P =0.007)。不同活动度SLE患者组间ROR-γt mRNA表达组间比较差异有统计学意义(F=3.335,P =0.047),Foxp3 mRNA组间比较差异无统计学意义(F=2.119,P =0.129)。相关性分析显示:ROR-γt与SLEDAI、Th17呈正相关(r=0.4969, P =0.0038, r=0.3960, P =0.0992),尚不能认为与其他相关Th细胞及细胞因子有相关性,同时尚不能认为Foxp3与SLEDAI及相关Th细胞及细胞因子有相关性。
结论
SLE患者存在Th细胞凋亡异常情况,且Th细胞凋亡率与SLEDAI评分有显著相关性。Th2/CD4+、Th17、Th17/CD4+、Th17/Treg与SLEDAI呈正相关,Th1/Th2、Treg与SLEDAI呈负相关。细胞因子检测IL-17、IL-6、IL-4与SLEDAI呈正相关,IL-2、IFN-γ与SLEDAI呈负相关,Th17细胞特异转录因子ROR-γt mRNA检测结果与SLEDAI、Th17呈正相关。研究表明SLE患者存在Th1/Th2、Th17/Treg失衡状态,且与疾病活动有关,联合检测Th细胞亚群,可能对于SLE病情评价更有意义。
Backgroud
Systemic lupus erythematosus (SLE) is a prototypic systemic disease characterized by autoimmune involved autoimmune inflammation and, SLE multisystem-involved multi-organ-damaged autoantibody production (antinuclear antibody, ANA). It is a chronic processing, has a high death ratio. The etiology and pathogenesis mechanisms of SLE have not been clearly elucidated. At present, there is no specific effective treatment for SLE.
There are two forms disorders of the cellular immune and humoral immune system in the course of SLE. T lymphocyte abnormalities and B lymphocyte excessive activation cause the abnormal immune system response. The above situations are main pathogenesis of SLE.In recent years,the immune disorders research of SLE tends to be thorough, multiple studies SLE itself that since lymphocyte balance is broken, excessive actiated T lymphocytes led to many cloned B cell differentiation and activation, resulting in a massive autoantibody cause multi-system damage involvement.The current research found that T lymphocyte exception may be the important factors SLE morbidity.One research has shown that the apoptosis of Th cells in SLE increased significantly, and apoptosis level and disease activity related.Therefore, the imbalance of T helper cells number and function is an important aspect of the immune function abnormality.
T helper cells is not a single group of cells, but contains a series with various functions lymphocyte subsets.Naive CD4+ T lymphocytes may differentiate into four kinds of cells:T helper cell type 1(Th1),T helper cell type 2(Th2),T helper cell type 17(Th17)and regulatory T cells(Treg). Their cytokines and immune function each are not identical. Therefore, there are complicated relationships of the different functions of lymphocyte subsets in the differentiation and development and functional execution and other aspects. Need to study Th cell subtypes playing a role pathogenetic mechanisms in SLE, that can conduce to further clear understand the role of Th cells in SLE.
Early, many research only focus on therelationship of Th1/ Th2 imbalance state and SLE.Imbalance in Th1/Th2 plays an import role in pathogenesis of SLE.“Th2 prominence”and“Th1 prominence”were proved in animal model and lupus patients. But with the deepening of the research of SLE ,it was found that a lot of SLE clinical manifestations and laboratory characteristics cannot use Th1 / Th2 imbalances model to explain. In recent years ,the understanding of T helper cells subsets was more comprehensive. In 1995 and 2005, Harrington and Sakaguchi successively found the two new members: regulatory T cells (Treg) and Th17. Gradually, the important role of Th17 / Treg balance was discovered in SLE. Both with belong to Th cells, but Th17 and Treg are completely opposite in differentiation and biology performance. Th17 stimulate an autoimmune response and Treg cells can reduce autoimmune disease reaction. They maintain the immune state of relative stability.
Th1、Th2、Th17、Treg contribute to the pathogenesis in RA, the imbanlance of Th1/Th2、Th17/Treg lead cell function disorder activation of B cells,increase the degree of SLE. Th17/Treg cells may be involved in the occurrence of disease and development,in some degree it can be the index of exultation the disease .thus Th1/Th2、Th17/Treg as a whole may be a more scientific than one single index.
In short, this research from Th cell apoptosis, in-depth analysis the imbalance of Th1 / Th2, Th17 / Treg in SLE and related cytokines spectrum change characteristics. And we further study on Th17 / Treg specific transcription factors. Through the relatively large sample of SLE cases material, combined determination Th lymphocyte subsets cell, protein, gene level change and the corresponding SLE disease activity index, we discusses the relationship of Th lymphocyte subsets and SLE disease activity ,clinical symptoms of disease assessment and SLE's diagnosis value.
Objective
(1)To study the levels of lymphocyte apoptosis and disease activity index in systemic lupus erythematosus patients.
(2)To study the balance of Th1/Th2、Th17/Treg and disease activity index in systemic lupus erythematosus patients.
(3)To study the level of cytokines secreted by Th1/Th2、Th17/Treg and disease activity index in systemic lupus erythematosus patients.
(4)To study the level of RORγt and Foxp3 and disease activity index in systemic lupus erythematosus patients.
Methods
Eighty-nine SLE patients,diagnosis complied with ACR classification standard ,were recruitedin the second hospital shanxi medical university and the 264 hospital of PLA from June 2009 to September 2010. We collected SLE patients generally and clinical materials.According to systemic disease activity index (SLEDAI) integral evaluation, patients were divided into light SLE, moderate activity type, heavy three groups. Meanwhile, 27 patients normal were selected as control group. Th apoptosis rate and Th1 / Th2, Th17 / Treg cell level were detected with Flow cytometery. Concentrations of IL-2、IL-4、IL-6、IL-10、TNF-α、IFN-γ,IL-17A and TGF-βin plasma were measured by Cytometric Bead Array (CBA) and Enzyme-linked immunosorbent assay (ELISA). Expression Levels of RORγt and Foxp3 mRNA were measured by RT-PCR. The data was analyzed by SPSS 13.0.
Results
(1)SLE patients had higher apoptosis rate of CD4+T cells than health controls[(1.11±0.36)% vs(.0.36±0.15)%,P<0.001]. Apoptosis rate of Th cells in serious group was higher than that in the light group ,medium group and control group[(1.51±0.31)%、(0.81±0.19)%、(1.03±0.21)%vs.(0.36±0.15), P <0.001].
(2)The percentage of Th1 cells in peripheral lymphocyte in SLE was lower than that in the control, and there was no statistical significance in the percentage of Th2 cells between SLE and control. The ratio of Th1/Th2 in serious group was higher than that in the light group and medium group.Th2/CD4+ correlated positively with SLEDAI scores ( r=0.4012, P =0.0001).Th1/Th2 correlated negatively with SLEDAI scores(r=-0.2683,P =0.0130). There was no statistical significance in the percentage of Th17 cells between SLE and control. But the ratio of Th17/ CD4+ in SLE was higher than that in the control(P =0.012).The percentage of Treg cells in peripheral lymphocyte in SLE was lower than that in the control (P =0.023). The percentage of Th17、Th17/CD4+ in serious group were higher than that in the light group and medium group. The percentage of Treg cells in the serious group was lower than that in the light group and medium group.There were positive correlation between Th17、Th17/CD4+,Th17/Treg and SLEDAI scores(r=0.3608, P =0.0006, r =0.4522, P <0.001, r =0.4240, P <0.001).There was negative correlation between Treg and SLEDAI scores(r =-0.352, P =0.0010)
(3)IL-17、IL-10、IL-6、IL-4 detected by CBA were elevated compared to those in healthy control (P<0.05),and the levels of other cytokines showed no statistic differences between patients with SLE and healthy control. The levels of plasma IL-17、IL-6 andIL-4 were positively correlated with SLE disease activity index(SLEDAI)scores. The levels of plasma IL-2、IFN-γshowed negative correlation with SLEDAI. No significant correlation were observed between plasma TNF、IL-10 and SLEDAI.
The levels of plasma IL-17、IL-10、IL-6、IL-4 in the serious group were higher than that in the light group and medium group. The levels of plasma IFN-γ、IL-2 in the serious group were lower than that in the light group and medium group(F=52.660,P <0.001).The levels of TNF-αshowed no statistic differences between patients in serious group and other group.
The levels of TGF-βdetected by ELISA showed no statistic differences between patients in SLE and healthy control(F=2.150,P =0.157). No significant correlation was observed between plasma TGF-βand SLEDAI(r =-0.071,P =0.5150).
(4)The SLE patients showed higher levels of ROR-γt、Foxp3 mRNA than the healthy controls(t=-2.656,P =0.012;t =-2.873,P =0.007).Expression of ROR-γt mRNA in the serious group was higher than that in the light group and medium group(F=3.335,P =0.047). The levels of Foxp3 mRNA showed no statistic differences between patients and healthy controls(F=2.119,P =0.129). The levels of ROR-γt mRNA was positively correlated with SLEDAI and Th17(r =0.4969, P =0.0038, r =0.3960, P =0.0992) .No significant correlation was observed between ROR-γt mRNA and other Th cells and cytokines. No significant correlation was observed between Foxp3 mRNA and Th and cytokines realted. Conclusion
There is Th apoptosis abnormalities in SLE patients and Th apoptosis rate Th2/CD4+、Th17、Th17/CD4+、Th17/Treg were positively correlated with SLEDAI. Th1/Th2 and Treg were negatively correlated with SLEDAI. Concentration of IL-17、IL-6、IL-4 were positively correlated with SLEDAI , and IL-2、IFN-γwere negatively correlated with SLEDAI. ROR-gamma t mRNA was significantly correlated with the SLEDAI score and Th17. Our research shows that imbalance state of Th1 / Th2 and Th17/Treg was found in SLE., and united detection of Th lymphocyte subsets was possibly more meaningful for SLE condition evaluation.
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