摘要
类风湿性关节炎(rheumatoid arthritis, RA)是一种以关节滑膜炎为特征的慢性全身性自身免疫性疾病。滑膜炎反复发作,导致关节内软骨和软骨下骨组织的破坏,关节功能障碍,最终造成关节畸形和强直。RA是最常见的风湿性疾病之一。不同RA患者的发病、病程及预后差异较大,严重者伴有多种并发症,且病变持续恶化。因此,RA的早期诊断和积极治疗尤为关键。
RA的详细发病机制仍未阐明。一般认为是某种未知抗原作用于有遗传倾向的个体,诱导自身免疫应答,引发滑膜增生性病变,导致关节的破坏。自身免疫应答包括抗原递呈、免疫细胞相互作用和免疫效应三个阶段,是一个由多种免疫细胞(抗原递呈细胞、T细胞、B细胞)、分子(膜分子和可溶性分子)以及生物因子(细胞因子、炎性因子)参与并受到严格调控及制约的复杂生理过程。
越来越多的实验证明,细胞因子参与RA的整个病理过程,并在其中发挥重要作用。各种生物因子间的关系密切、作用复杂,其特点是网络性和多相性。在RA不同的病理过程中参与的生物因子不同。IFN-γ、GM-CSF等在RA的触发阶段起关键作用;T细胞向滑膜迁移受IL-15、IL-16的调控;IL-4、IL-10则作用于B细胞的激活和分化生长。而IL-1、IL-8和TNF-α等炎症因子在病变中起主导作用。
为进一步探讨细胞因子在RA中的免疫效应机制,本文第一部分建立细胞因子实时荧光定量聚合酶链反应(fluorescence quantitative PCR, FQ-PCR)检测方法。将分离获得的人外周血PBMC置激发型CD3抗体包被的培养板中培养,收取不同培养时间点的培养上清和单个核细胞。分离获得单个核细胞的总RNA并逆转录成cDNA。特异性扩增IL-2、IFN-γ和IL-10的基因片段,与PMD18-T载体连接,制备重组质粒并定量。在优化的PCR条件下,建立目的基因和内参照基因(β-Actin)的检测标准曲线,并检测单个核细胞中目的基因的mRNA表达。同时检测培养上清中相应细胞因子的含量,研究其相关性。结果显示:1.在优化的PCR条件下,目的基因和内参照基因的检测标准曲线稳定可靠,在106、105、104和103 copies/ml浓度范围内具有良好线性关系;2.受CD3抗体的激发,外周血单个核细胞胞浆目的基因的mRNA和相应的培养上清中蛋白质表达水平明显高于健康对照;3.目的基因的mRNA水平和蛋白质表达有一定的相关性。
第二部分通过对活动期和缓解期RA患者外周血中CD4+T细胞表面的共刺激分子和凋亡分子、多种细胞因子及其相应的mRNA表达水平的检测,进而探讨这些免疫效应分子与类风湿性关节炎病程发展、转归以及预后的相关性。结果显示:1.急性活动期RA患者外周血CD4+T细胞表面CD154、CD69的表达明显高于健康对照组和RA稳定期组,而健康对照组和RA稳定期组之间无显著差异;2.急性活动期RA患者的血清和血浆sCD154水平均高于健康对照组和稳定期RA患者组;3.不同细胞因子在健康对照组血清中的基准水平不同,同样外周血PBMC中其相应的mRNA基准水平也有显著差异,细胞因子血清蛋白质基准水平与其mRNA基准水平完全不相关;4.急性活动期RA患者IL-6、IFN-γ以及IL-10的血清含量与其外周血PBMC中其相应的mRNA水平显著升高,两者之间有良好的相关性。
综上所述,本研建究立了细胞因子荧光定量PCR的检测方法。采用该检测技术,分析急性活动期RA患者外周血细胞因子及其mRNA的表达变化,证实细胞因子在基因转录水平和蛋白质表达水平之间的相关性。RA患者细胞因子及其mRNA的检测,能为RA的临床诊断、治疗和愈后提供新的实验指标,具有潜在的应用价值。
Rheumatoid arthritis (RA) is a systemic, inflammatory autoimmune disorder that presents as a symmetric polyarthritis associated with swelling and pain in multiple joints, often initially occurring in the joints of the hands and feet. Articular inflammation causes activation and proliferation of the synovial lining, expression of inflammatory cytokines, chemokine-mediated recruitment of additional inflammatory cells, as well as B cell activation with autoantibody production. A vicious cycle of altered cytokine and signal transduction pathways and inhibition of programmed cell death contribute to synoviocyte and osteoclast mediated cartilage and bone destruction. In the last few years, the study of serum cytokine profiles and cytokine mRNA expressions in PBMC in patients of RA have become a hot spot in the pathogenesis of RA.
The study was divided into two parts. In the first part, this study was designed to investigate the mRNA expression and protein level of cytokines in PBMCs stimulated with CD3 mAb, and to compare the pattern of cytokine protein and cytokine mRNA elevations in PBMCs with that of controls. The PBMCs of healthy adult were isolated by Ficoll and incubated with anti-CD3 mAb coated microwell plates. Cytokine levels of IL-2、IFN-γand IL-10 in culture supernatant were tested by ELISA. PBMCs stimulated by CD3 mAb were selected to express mRNA of cytokines. Cytokine gene fragments of were amplified using specific primers. PCR products were inserted into the PMD18-T vector. Recombinant plasmids were selected after sequencing. Target genes and house keeping gene used asβ-Actin were amplified on fluorescence quantitative PCR. Standard amplification curves were developed at the concentration of target genes andβ-Actin ranging from 103、104、105 and 106 copies/ml. The results showed that: 1. Standard amplification curves were established with high specificity, reliability and stability. 2. The expression of mRNA in PBMCs stimulated by CD3 mAb was significantly higher than that of controls. 3. The levels of in supernatant showed significant difference between the PBMCs stimulated with CD3 mAb and controls. Compared with controls, mean cytokine levels of IL-2、IFN-γand IL-10 were all higher in CD3 mAb co-cultured group. 4. Cytokine levels of IL-2、IFN-γand IL-10 have great relation with the mRNA expression of cytokine in PBMCs.
In the second part, we continuingly established standard amplification curves of IL-1β、IL-6、IL-8 and TNF-α. The study was proposed to investigate cytokine mRNA expression in PBMCs and protein level of cytokines in active RA patients, and to compare the pattern of cytokine protein and cytokine mRNA elevations in active RA patients with that of stable RA patients and healthy controls. We also explore the functions of molecules expressed on CD4+T cells inducing activation of cells and apoptosis in causing imbalanced immunity of patients with RA. The results showed that: 1. Expression of CD154 significantly increased on CD4+T cells in patients with active RA, comparing with stable RA and healthy controls. 2. Soluble CD154 in plasma with active RA was higher than that of stable RA patients and controls. 3. Compared with controls and stable RA patients, mean serum levels of IL-1β, IL-6, IL-8, IL-10 and IFN-γwere all higher in patients with active RA. In contrast, stable RA patients show higher level of IL-10 compared with controls. 4. Basal levels of cytokine mRNA expression in PBMCs of controls were different. mRNA expression of IL-6, IFN-γ, IL-10 and TNF-αshowed a more general increase in patients with active RA comparing with controls and stable RA patients.
In summary, mRNA expression of cytokines in PBMCs could be measured by fluorescence quantitative PCR. Both the levels of cytokine in serum and mRNA cytokine expression in PBMCs were significantly higher in patients with active RA than that of stable RA patients and controls. All these results showed the measurement of mRNA expression in PBMCs and serum level of cytokines in patients with active might play an important role in RA diagnosis and prognosis in clinic.
引文
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