Th细胞在类风湿关节炎中的作用及其意义

【作者】 陈俊伟；

【导师】 李小峰；

【作者基本信息】 山西医科大学 ， 内科学， 2010， 博士

【摘要】 类风湿关节炎(rheumatoid arthritis, RA)是一种以关节炎为主要临床表现的自身免疫性疾病,其特征是以大量T淋巴细胞浸润的慢性滑膜炎。迄今为止,发病机制还不甚明确,但发病机制中未知抗原被递呈给T辅助细胞(T Helper Cell, Th细胞)是一重要环节,已有大量文献证明Th细胞和细胞因子在RA发病中起重要作用。CD4+T细胞与不同的初始抗原接触,接受不同的信号模式,可分化为4种不同的Th细胞亚型,分别为Th1、Th2、Th17细胞以及调节性T细胞(Treg细胞)。尽管Th细胞的活化是一个关键的保护性免疫反应过程,但不同的Th细胞亚型在类风湿关节炎中发挥着不同的作用,Th细胞除了通过分泌细胞因子和趋化因子发挥作用以外,细胞与细胞之间的直接接触也是其作用方式之一。总之,Th细胞分化过程的紊乱就会引起免疫病理反应,从而导致自身免疫性疾病。19世纪80年代认为Th细胞在类风湿关节炎中起着重要作用,其中,Th1细胞驱动疾病的发生与疾病炎症有关,而Th2细胞在一定程度上对抗炎症。由于Th1和Th2细胞呈极化分化,在正常情况下,Th1和Th2细胞相互促进,相互制约,使机体Th1／Th2细胞维持一定的平衡。在某种特定的病理环境中,可能导致Th1／Th2的失衡,首先可能是Th1和Th2细胞数量分化不平衡,使得Th1和Th2细胞所分泌的炎性细胞因子和抗炎细胞因子失衡,从而启动疾病的发生。自从上世纪末和本世纪初,先后认识Treg细胞和Th17细胞以来,发现Th17细胞加重炎症和延续破坏进程,而Treg细胞在对抗炎症和维持自身免疫耐受以及调节免疫反应中起着关键的作用,相关的研究认为Th17细胞和Treg细胞在RA的发病过程中,尤其是在疾病的后期起着更为关键的作用,因此,我们推测认为：Th17／Treg细胞的失衡可能与Th1／Th2细胞一样参与了疾病的发生和发展。但鉴于Thl、Th2、Th17细胞以及Treg细胞均有CD4+T细胞分化而来,并且相互作用,相互抑制,形成一个非常复杂的网络,所以,这一新的发病模式必须经过进一步的证实。目前国内外均未发现这方面的研究,本研究旨在细胞、蛋白以及基因水平上,对Th1细胞、Th2细胞、Th17细胞以及Treg细胞对类风湿关节炎作用进行初步探讨。目的本研究通过分离RA患者外周血淋巴细胞、血清并提取RNA,检测其淋巴细胞的凋亡和Th1、Th2、Th17细胞以及Treg细胞数量、相关细胞因子血清浓度和各亚型的特异性转录因子表达状况,初步探讨Th亚型细胞在RA的作用及其意义。方法首先将入选的RA患者根据DAS28,分为DAS28≤3.2组,3.2<DAS28≤5.1组和DAS28>5.1组；将外周血淋巴细胞,加入3H-TdR培养24h,过滤、固定、烘干后,用液体闪烁计数仪测定每分钟衰变值；同时将淋巴细胞加入双层琼脂糖凝胶中,裂解、解旋、电泳、染色,应用CASP彗星图像分析软件进行自动分析,初步分析RA患者淋巴细胞代谢与疾病的关系；对淋巴细胞CD4、PI、AnnexV进行标记,运用FCM检测CD4+淋巴细胞的凋亡率,分析CD4+T淋巴细胞凋亡与疾病的关系：对外周血淋巴细胞进行培养、刺激以及胞内外标记,运用FCM检测Th1细胞、Th2细胞、Th17细胞以及Treg细胞数量,进一步分析Th细胞与疾病的关系；分离外周血清,应用CBA和ELISA法检测Th细胞相关细胞因子水平,进一步说明Th细胞功能与疾病的关系；同时运用RT-PCR测定Th细胞特异性转录因子,在基因水平上,验证Th细胞与疾病的关系。各组实验数据以均数±标准差(x±s)或中位数±四分位数间距(M±QR)表示。两组数据间的差异用t检验或Wilcoxon检验和Kruskal-Wallis H检验,P<0.05或P<0.0083为差异有显著性。结果1.RA患者外周淋巴细胞DNA代谢状况比正常对照者活跃,两者比较无统计学意义(P>0.05)；RA患者外周淋巴细胞DNA损伤低于正常对照者,两者比较无统计学意义(P>0.05)；RA患者外周CD4+T淋巴细胞凋亡率低于正常对照者,两者比较有统计学意义(P<0.05)。2.Th1细胞在RA患者外周淋巴细胞中的百分率高于正常人,两者比较有统计学意义(P<0.05)；Th2细胞在RA患者外周淋巴细胞中的百分率高于正常人,两者比较无统计学意义(P>0.05)；Th17细胞在RA患者外周淋巴细胞中的百分率高于正常人,两者比较有统计学意义(P<0.05)；Treg细胞在RA患者外周淋巴细胞中的百分率低于正常人,两者比较有统计学意义(P<0.05)。3. DAS28≤3.2的RA患者Th1和Th2细胞在外周淋巴细胞所占的百分率大于正常人,两者比较无统计学意义(P>0.05),Th17细胞在外周淋巴细胞所占的百分率小于正常人,两者比较有统计学意义(P<0.05),Treg细胞在外周淋巴细胞所占的百分率小于正常人,两者比较无统计学意义(P>0.05);3.2<DAS28≤5.1的RA患者Th1和Th2细胞在外周淋巴细胞所占的百分率大于正常人,两者比较无统计学意义(P>0.05),Th17细胞在外周淋巴细胞所占的百分率大于正常人,两者比较有统计学意义(P<0.05),Treg细胞在外周淋巴细胞所占的百分率小于正常人,两者比较无统计学意义(P>0.05)；DAS28>5.1的RA患者Th1和Th17细胞在外周淋巴细胞所占的百分率为大于正常人,两者比较有统计学意义(P<0.05),Treg细胞在外周淋巴细胞所占的百分率小于正常人,两者比较有统计学意义(P<0.05),Th2细胞在外周淋巴细胞所占的百分率大于正常人,两者比较无统计学意义(P>0.05)。4.RA患者IL-2、IL-4、IL-6、IL-10、IL-17A, IFN-y和TNF-a的血清浓度均增高,与正常人比较有统计学意义(P<0.0083);TGF-β血清浓度也增高,与正常人比较无统计学意义(P>0.083)。5. DAS28≤3.2的RA患者IL-2. IL-4和TNF-α血清浓度大于正常人,两者比较有统计学意义(P<0.0083),IL-6、IL-10和IL-17A血清浓度大于正常人,两者比较无统计学意义(P>0.0083),IFN-γ和TGF-β血清浓度小于正常人,两者比较无统计学意义(P>0.0083)；3.2<DAS28≤5.1的RA患者IL-2、IL-6、IL-10和IL-17A血清浓度大于正常人,两者比较有统计学意义(P<0.0083), IL-4、IFN-γ、TNF-a和TGF-β血清浓度大于正常人,两者比较无统计学意义(>0.0083);DAS28>5.1的RA患者IL-2、IL-4、IL-6、IL-10、IL-17A、IFN-y和TNF-a血清浓度均大于正常人,两者比较有统计学意义(P<0.0083)；TGF-β血清浓度大于正常人,与正常人比较无统计学意义(P>0.0083)。6.RA患者Th1、Th2、Th17、Treg细胞四种特异性转录因子T-bet、GATA-3、ROR-γt、Foxp3 mRNA在RA的表达量分别为17.01、26.12、28.31、29.11。结论1.RA患者外周血CD4+T淋巴细胞异常增殖,可能是由于凋亡受阻造成的。2. Th17/Treg细胞与Thl/Th2-一样在RA中发挥着重要的作用,可能在不同的时期参与疾病的发生和发展。3.Th细胞分泌的细胞因子促使促炎因子与抗炎因子的失衡,从而参与RA的发病。4. Th1、Th2和Th17细胞是通过分泌细胞因子起作用的,而Treg细胞是通过细胞间的直接接触来对抗炎症的。5. Th1、Th2、Th17和Treg细胞的特异性转录因子T-bet. ROR-γt、GATA-3、Foxp3 mRNA在RA患者中均有表达。更多还原

【Abstract】 Rheumatoid arthritis is a kind of chronic autoimmune disease with the joints inflammation characterized by a large number of T lymphocytes infiltration in the synovial. To now, the pathogenic mechanism in RA is also unclear. However that the unknown antigen is presented to T helper (Th) cells is at the hub in the pathogenesis.There are a lot of data that suggest Th types and cytokines contribute to the pathogenesis in RA. Naive conventional CD4 T cells have open to their 4 distinct fates that are determined by the pattern of signals during their initial interaction with antigen.These 4 population are Thl,Th2, Th17 and regulatory T (Treg) cells. They exert such functions mainly through secreting cytokines and chemokines that play different functions. Th cells play a central role in cellular defence against pathogens as well as in cellular self-tolerance. The activation of Th cells is a crucial process determining the course of a protective immune response. Dysregulated activation processes can lead to pathologic immune reactions and may induce autoimmune diseases.Th cells are at the hub of this paradigm, which was developed in the 1980s. Many studies led to the belief that RA was a Thl-driven disease. The Thl phenotype is associated with inflammation in RA, whereas the Th2 phenotype copes with inflammation to some extent. The excess of pro-inflammatory cytokines and relative deficiency in anti-inflammatory cytokines define the Thl/Th2 imbalance, which was believed to drive RA. The Th17 cells increase the process of inflammation and joints destruction in RA. Treg cells play a critical role in competing with inflammation and maintaining self-tolerance as well as regulating immune responses. The study belive that Th17 cells and Treg cells play a important role, especially in the latter process. Although Treg cells play a critical role in the pathogenesis in RA, the imbalance of Thl/Th2 and Thl7/Treg may be the pathogenic mechanism of RA. It has been speculated that Thl7/Treg cells may be involved in the occurrence of disease and development as Thl/Th2 cells. Given Thl, Th2, Thl7 cells and Treg cells are differentiated from CD4+T cell, it is a very complex network which is mutuall interaction and inhibition in the four phenotype cells. The new paradigm must be further confirmed. But the speculation haven’t been studied by our or foreign workers. To further explore the action of the Th1,Th2, Th17 and regulatory T cells to rheumatoid arthritis, we have the study in cell, protein and gene level.ObjectiveTo investigate the effect of lymphocyte apoptosis, Thl cells,Th2 cells, Th17 cells, Treg cells and related cytokines in RA.The study detected the number and function of the four phenotype cells and the expression of transcription factor subtypes by detecting the peripheral blood lymphocytes,blood serum and lymphocytic mRNA in patients with RA.MethodsFirstly,all the RA patients selected into this study were divided into different disease activity groups according to Disease Activity Score.The 3H-TdR was added to the peripheral blood lymphocytes for 24 hours and then filtered,fixed, dried and measured the decay of values per minute using liquid scintillation counting. At the same time the lymphocytes were added into the dual-layer agarose gel, cracked, unwinded, electrophored and stained. Analysis CASP comet image automatic analysis software illustrated the relationship between lymphocytes metabolism and the disease preliminarily. The lymphocytes were marked by CD4, PI, AnnexinⅤand then detected apoptosis of CD4+lymphocyte by flow cytometry to analysis the relationship between the CD4+lymphocyte apoptosis and diseases in RA furtherly.The peripheral blood mononuclear cells were cultured, stimulated, as well as in vivo markered and then detected the number of the Thl phenotype cells, Th2 phenotype cells, Th17 phenotype cells and Treg phenotypes cell number to analysis the relationship between the various subtypes of Th cell and disease in RA furtherly. The relevant levels of cytokines in peripheral blood were found through the Cytometric Bead Array and Enzyme-linked Immunosorbent Assay to detect the relationship between function of the various subtypes of Th cell and the desease in RA. Finally, The RNA in Peripheral blood mononuclear cells were extracted and measured expression of transcription factors of the four phenotypes cells by Real Time PCR. All data were expressed as Mean±standard deviation (x±s) or median±quartile range(M±QR). The difference between different groups was assessed with t-test or Wilcoxon-test and Kruskal-Wallis H-test. If the pvalue was less than 0.05 or 0.0083,It was considered to be a statistically significant difference.Results1. The DNA metabolic activity of peripheral lymphocyte was higher in RA compared with the control, but there was no statistical significance (P> 0.05); The DNA damage of peripheral lymphocytes in RA was higher compared with the control,but there was no statistical significance (P> 0.05); The apoptosis of CD4+T lymphocyte was lower than the control,but there was no statistical significance (P<0.05).2. The percentage of Thl cells in peripheral lymphocyte in RA was higher than that in the control, but there was statistical significance (P<0.05); The percentage of Th2 cells in peripheral lymphocyte in RA was higher than that in the control, but there was no statistical significance (P>0.05); The percentage of Thl7 cells in peripheral lymphocyte in RA was higher than that in the control, but there was statistical significance (P<0.05); The percentage of Treg cells in peripheral lymphocyte in RA was higher than that in the control, but there was statistical significance (P<0.05);3. The percentage of Thl and Th2 cells in peripheral lymphocyte in RA with DAS28≤3.2 was higher than that in the control, but there was no statistical significance (P>0.05), The percentage of Th17 cells in peripheral lymphocyte in RA was higher than that in norma and there was statistical significance (P<0.05), The percentage of Treg cells in peripheral lymphocyte in RA was lower than that in the control and there was no statistical significance (P>0.05); The percentage of Thl and Th2 cells in peripheral lymphocyte in RA with 3.2 <DAS28≤5.1 was higher than that in the control, but there was no statistical significance (P >0.05), The percentage of Th17 cells in peripheral lymphocyte in RA was higher than that in the control and there was statistical significance (P<0.05), The percentage of Treg cells in peripheral lymphocyte in RA was lower than that in the control, but there was no statistical significance (P>0.05); The percentage of Thl and Thl7 cells in peripheral lymphocyte in RA with DAS28>5.1 was higher than that in the control and there was statistical significance (P <0.05); The percentage of Treg cells in peripheral lymphocyte in RA was lower than that in the control and there was statistical significance (P>0.05),The percentage of Th2 cells in peripheral lymphocyte in RA was higher than that in the control, but there was no statistical significance (p>0.05).4. The serum concentration of IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-y and TNF-a in RA were increased compared with the control,and there was statistically significant (p<0.05); The level of TGF-βwas also increased compared with the control,but there was no statistical significance (P> 0.05).5. The serum concentration of IL-2, IL-4 and TNF-a in RA with DAS28≤3.2 was increased compared with the control, and there was statistical significance (P<0.0083); The serum concentration of IL-6, IL-10 and IL-17A was greater than the control,but there was no statistical significance (p> 0.0083);The serum concentration of IFN-γand TGF-βwas lower than control,but there was no statistical significance (p> 0.0083); The serum concentration of IL-2, IL-6, IL-10 and IL-17A in RA with 3.2<DAS28≤5.1 was increased compared with the control, and there was statistical significance (p<0.0083);The serum concentration of IL-4, TNF-a,IFN-y and TGF-βwas greater than the control,but there was no statistical significance (p> 0.0083);The serum concentration of IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-y and TNF-a in RA patients with DAS28> 5.1 was increasd compared with the control,and there was statistical significance (p＜0.0083); Althoght the serum concentration of TGF-P was higher compared with the control,but there was no statistical significance (P> 0.0083). 6. The expression level of T-bet, ROR-yt, GATA-3, Foxp3 mRNA that is dividely specific transcription factor of Thl, Th2, Th17 and Treg cells in RA is 17.01,26.12,28.31,29.11.Conclusion1. The proliferation CD4+T lymphocytes in RA patients are abnormal.It may be due to obstruction caused by apoptosis.2. The Th17/Treg cells play an important role in RA as Thl/Th2 cells. They may be involved in different stages of occurrence and development in the disease.3. TheTh cells secrete cytokines to promote pro-inflammatory cytokines and anti-inflammatory factor in the imbalance, which participate in the pathogenesis of RA.4. The Thl, Th2 and Th17 cells work by secreting cytokines, while Treg cells cope with the inflammation through direct contact between cells.5. The T-bet, ROR-yt, GATA-3 and Foxp3 mRNA which are special transcription factor of the Thl, Th2,Th17 and Treg cells are expressed respectively in RA patients.更多还原