【作者】 冯媛；

【导师】 朱平；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 博士

【摘要】 目的:脊柱关节病(spondyloarthropathy,SpA)是一组以中轴和/或外周关节受累、具有家族聚集倾向、血清类风湿因子(RF)阴性为特点的相互关联的多系统炎症性疾病。该病在我国的发病率和致残率都较高,但因为各种SpA的病因和发病机制均未完全明了,缺乏有效的病因治疗。因此研究SpA的病因具有重要意义。目前有关其参与SpA发病主要有以下几个假说:1)分子模拟假说:HLA-B27经典的HLA-I类分子结构通过将关节炎多肽提呈至CD8~+T细胞的TCR上而诱导关节炎;2)HLA-B27异常形式的免疫识别:在多种因素影响下,细胞表面存在多种B27的异常形式,包括自由重链(FHC),重链同源二聚体等,被特异的CD4~+T细胞、NK细胞等识别从而诱发炎症损伤;3)蛋白错误折叠,内质网应激与炎症反应:HLA-B27在内质网的折叠速率减慢,造成内质网应激,引发未折叠蛋白反应(UPR)导致炎症反应。目前理论上有关SpA的发病机理更倾向于后两种假说,而这两种假说均与B27的蛋白表达量相关。虽然SpA的发病机制目前尚不清楚,但可以确定的是除了外界环境因素的影响,HLA-B27与SpA的发病关系非常密切,且有HLA-B27转基因大鼠试验表明HLA-B27的拷贝量与转基因鼠的发病有直接关系。另外,也有报道表明HLA-B27阳性的AS患者比HLA-B27阳性正常人的HLA-B27表达量明显增高。以上证据均表明HLA-B27的表达高低与SpA的易感性有关,而启动子的转录调控是影响HLA-B27表达的重要途径。因此本实验拟通过对HLA-B27的启动子(promoter)区序列及活性进行分析,试图发现一些对HLA-B27启动子有活化作用的因子,并在体外研究HLA-B27表达阳性的人单核细胞系在这些激活因子的作用下其UPR相关分子表达的情况。以初步判定HLA-B27启动子激活因子与UPR反应参与SpA的发病机理,为进一步揭示SpA的发病机制提供线索和依据。方法:1.通过搜索IMGT/HLA数据库,建立了包括HLA-B27*05序列在内的所有有效HLA A类和B类分子启动子区序列数据库。通过对HLA-B27*05与其他分子的5’端非编码区特别是转录因子结合位点的序列进行序列分析比对,检测B27与其他HLA I类分子的差异,试图寻求一定规律。2.为了研究SpA患者滑液内是否有活化B27启动子的因子,本实验采用25例SpA与8例RA患者的滑液对转染了B27启动子的Hela细胞进行孵育,48小时后通过检测转染载体的荧光素酶活性研究B27启动子对滑液的反应。进而用anti-TNF-α抗体进行阻断试验,以确定TNF-α是否具有激活B27启动子的作用。3.采用CBA和Real time PCR的方法集中检测炎性因子在SpA患者外周血单个核细胞中的表达情况,以挑选相关细胞因子进行HLA-B27启动子的活性实验。为了进一步研究B27启动子在人单核细胞的活性,以及TNF-α和其他致炎因子的激活作用,我们将HLA-B27的启动子序列重组入带有黄色荧光蛋白报告基因的载体pmax-YF-PRL内。选用人单核细胞系U937作为宿主细胞,引入重组载体,建立稳定转染细胞株。用25种细胞因子(IL-1α,IL-1β,IL-2,IL-4,IL-5,IL-6,IL-7,IL-8,IL-12-p70,IL-13,IL-15,IL-17,IL-18,TNF-α,M-CSF,GM-CSF,IP-10,MCP-1,RANTES,MIP-1α,IFN-α,IFN-β,IFN-γ,TGF-β1和OSM)及12种TLR配体(MDP,PGN,Zyn,PIC,LPS,Flag,FSL,Lox,ssRNA,EcDNA,Pams和Decap KP)作用于该转染子,分别在孵育后24,48及72小时运用流式细胞仪检测细胞的黄色荧光表达强度,筛选对B27启动子有激活作用的因子。4.为了证明在SpA患者外周血单个核细胞中可能存在UPR反应,首先采用定量PCR的方法对UPR主要相关蛋白BIP和CHOP的mRNA表达进行检测,进而我们将HLA-B2704基因和HLA-A2对照基因重组入带有绿色荧光蛋白(GFP)报告基因的真核表达载体pEGFP-N1内,并将以上重组质粒和pEGFP-N1原质粒转入人单核细胞系U937,建立表达HLA-B2704全长的人单核细胞稳定转染细胞株B27-U937,表达HLA-A2的对照细胞株A2-U937以及只表达GFP的细胞株GFP-U937。随后,用筛选出的HLA-B27启动子激活因子作用于以上转染细胞株,并采用real-time PCR检测B27-U937,A2-U937和EGFP-U937细胞株的UPR相关蛋白(Grp78,CHOP,XBP-1)mRNA的表达情况。结果:1.所有I类分子中只有6个等位基因与HLA-B27的promoter区完全相同,且其中三种是HLA-B27的亚型。同时还发现ISRE和kB2在B类分子中高度保守。2.发现25例SpA患者滑液中有5例可明显增高Hela细胞中HLA-B27 promoter的活性,而RA患者滑液无此作用。且这些增强作用均可被TNF-α拮抗剂部分阻断。3.发现AS患者外周血单个核细胞与健康对照相比显著高表达多种炎症性细胞因子,包括TNF-α,IL-10,IL-6和IL-1β,可能在AS中发挥着重要作用。研究还显示,TNF-α,INF-α,INF-β,INF-γ可激活U937细胞中HLA-B27启动子的活性(P<0.05),且以上因子的激活作用在72小时内与孵育时间成正相关。孵育72小时后,以INF-γ的激活作用最强。不同剂量细胞因子对启动子活性影响无显著差异(P>0.05)。4. AS患者外周血单个核细胞中GRP78和CHOP基因的mRNA表达量比健康对照组显著增高,提示存在UPR反应。GRP78,CHOP和XBP-1基因mRNA的表达量在人单核细胞U937转染子B27-U937和A2-U937中无显著差异(P>0.05)。当用PMA、IFN-γ、LPS和TNF-α等细胞因子对以上细胞进行孵育后,real-time PCR结果显示IFN-γ刺激8小时后,CHOP和GRP78的mRNA水平相对于未刺激细胞均明显增加,尤其以GRP78显著(P<0.05)。但B27-U937,A2-U937和EGFP-U937细胞间无明显差异。LPS和TNF-α刺激后均得到相似的结果。而半定量PCR结果也显示在细胞因子的作用下HLA-B27并不能调节XBP-1剪切体的含量。结论:1. TNF-α,IL-10,IL-6和IL-1β在AS患者外周血单个核细胞中高表达,提示这些因子在SpA发病中起重要作用。同时我们还发现SpA患者滑液中确实存在可刺激HLA-B27表达的成分,且与TNF-α相关。这些结果都明确指出了细胞因子极有可能参与了SpA的发病机制。2.通过序列比对及多种细胞因子和TLR配体对HLA-B27启动子活性作用的筛选,我们发现在HLA-B类分子启动子区高度保守的ISRE、kB2转录因子结合位点对HLA-B27 promoter的活化起到了决定性的作用,仅与该位点相关的细胞因子INF、TNF对HLA-B27启动子有明显活化作用。提示以上因子有可能通过刺激HLA-B27启动子的活性,从而增加HLA-B27的表达而诱发细胞的UPR参与SpA的发病。3.为了探明这些HLA-B27启动子激活因子是否是通过增加HLA-B27的表达来诱导细胞的UPR而参与致病,我们首先检测了AS患者外周血单个核细胞中GRP78和CHOP基因的mRNA表达量,结果显示比健康对照组显著增高,提示存在UPR反应。进而我们检测了HLA-B27基因全长转染U937细胞中CHOP、GRP78和XBP-1在这些启动子激活因子作用下的表达,结果显示较未刺激细胞有显著增加,但与转染了A2和EGFP的细胞无显著差异(P>0.05)。这些结果提示在多种炎症因子刺激情况下,HLA-B27转染细胞可能并没有异常增高的UPR反应存在,这与本实验室前期结果仍有差异。因此我们将采用多种细胞系,特别是人原代单核/巨噬细胞来研究HLA-B27对细胞因子的反应,从而结合已有结果对SpA发病机理进行进一步的研究。更多还原

【Abstract】 Objectives:The spondyloarthropathies (SpAs) are the second-commonest cause of inflammatory arthritis in humans, after rheumatoid arthritis. One genetic factor shared by all the varieties of SpA has already been identified, that is, HLA-B27. Although the presence of the HLA-B27 allele marks a strong predisposition for SpA, its role in disease pathogenesis is still unclear. Until now, there are three main hypotheses explaining the role of HLA-B27 in SpA: 1) Molecular mimicy: HLA-B27 or HLA-B27 bound peptides were thought to resemble bacterial peptides, and thus become the target of cross-reactive antibodies and/or CD8~+ cells. 2) Immunological recognition of aberrant forms of HLA-B27: the aberrant forms of HLA-B27 such as free heavy chains or HLA-B27 homodimers are specifically recognized by the receptors on NK, CD4~+ T cells or other immune cells. 3) Protein misfolding, ER stress, and inflammatory disease: HLA-B27 misfolding results in heavy chain retension and cause unfolded preotein response (UPR) activation that perhaps initiate or perpetuates a chronic inflammatory process. These hypotheses are based on the activities of the HLA-B27 protein, and hence would require the HLA-B27 gene to be transcribed.It is believed in addition to certain other genes and/or environmental factors, the expression degree of the HLA-B27 gene is also responsible for susceptibility to this disease. HLA-B27 positivity strongly influences spondyloarthropathy (SpA) susceptibility and phenotype, and experiments of HLA-B27 transgenic rats showed the gene level is probably linked to its arthritis-causing potential. Other people reported that the expression of the HLA-B27 molecule on the surface of PBMCs is higher in patients with AS than in HLA-B27-positive healthy volunteers. However, very little research has been conducted to investigate HLA-B27 gene regulation.Based on several observations suggesting that factors modulating HLA-B27 transcription may play a role in the pathogenesis of SpA, we designed this study to identify factors that might be responsible for the transcription of HLA-B27 in human monocytic cells and to assess whether any differences in the UPR can be detected following incubation with these promoter-activating factors.Methods:1. The IMGT/HLA Database was used to compare HLA-B2705 with other HLA B alleles and HLA A alleles at the 5’untranslated region to find out the similarities and differences especially at transcription binding sites.2. After that we used 25 specimens of synovial fluid of SpA patients to incubate the Hela B27 promoter transfectants for 48 hours, and TNF-αinhibitor was added into those positive specimens.3. Then the promoter region of HLA-B27 was transfected into U937 cell line to reveal the respond of B27 promoter in human monocyte cell line. The transfectant was challenged separately with 25 kinds of cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12-p70, IL-13, IL-15, IL-17, IL-18, TNF-α, M-CSF, GM-CSF, IP-10, MCP-1, RANTES, MIP-1α, IFN-α, IFN-β, IFN-γ, TGF-β1 and OSM) and 12 kinds of Toll Like Receptor ligands (MDP, PGN, Zyn, PIC, LPS, Flag, FSL, Lox, ssRNA, EcDNA, Pams and Decap KP). Promoter activity is measured by Flow Cytometry.4. Quantitive RT-PCR was used to study the mRNA expression of UPR related genes (GRP78, CHOP) in PBMC of SpA patients. Meanwhile, we constructed HLA-B27-EGFP, HLA-A2-EGFP and EGFP-alone stable transfected U937 cells and examined the FHC expression on them. Moreover, the UPR in B27 and A2 transfected U937 cells were studied by RT-PCR. HLA-B27 promoter-activating factors were used to incubate these cell lines.Resultes:1. There are just 6 B alleles including 3 B27 subtypes having the same sequence with HLA-B2705. X1/X2, S and CAAT Box binding site of B alleles have more variability than others. ISRE and kB1/2 are highly conservative in HLA B alleles.2. 5 out of 25 specimens of SF from SpA patients could significantly increase the activity of the HLA-B27 promoter. None of the RA specimens caused the same effect. The promoter activation caused by SF could be significantly inhibited by anti-TNF-αantibody3. The CBA and RT-PCR results showed that these cytokines, TNF-α,IL-10,IL-6 and IL-1, were significantly higher in the PBMC of AS patients than that of normal control.Out of TNF-α, only INF-α, -β, -γcan activate the B27 promoter in U937 cell line. And all the increase effects and the incubate time have positive correlation in 72 hours. The dose response relationship for HLA-B27 promoter activity in response to these cytokines was tested, and there were no significant differences in this cell line. When the peaks were compared, the most effective cytokine was INF-γ.4. Compared with the PBMC of normal control, the GRP78 and CHOP expression of SpA was significantly higher (P<0.05). The mRNA expression of GRP78, XBP-1 and CHOP were same in EGFP-U937, B27-U937 and A2-U937 cells. Then we then used IFN-γ, LPS, and TNF-αas stimulators to activate these U937 cells. The PCR results showed that after IFN stimulation for 8 h, CHOP and GRP78 expression were all increased compared with unstimulated cells, especially the GRP78 expression. But there were no significant differences between B27, A2-U937, and EGFP-U937 cells. LPS and TNF-αstimulation demonstrated the same results and we found that HLA-B27 does not modulate XBP-1 splicing following cytokine stimulation also.Conclusion:1. Our results confirmed that the cytokines including TNF-α,IL-10,IL-6 and IL-1βsecreted by PBMC play important roles in SpA. We found that Synovial fluids of spondyloarthropathy patients can significantly increased activity of HLA-B27 promoter and the increase was inhibited by TNF-αinhibitor. All these results indicate that cytokines play a very important role in pathogenesy of SpA.2. After compared HLA-B2705 with other HLA B alleles and HLA A alleles and screened cytokines and Toll Like Receptor ligands, we found out that only INF and TNF-αwhich associated with ISRE and kB1/2 binding sites can activate B27 promoter significantly. These results inferred that the HLA-B27 promoter activating factors could possibly stimulate the expression of HLA-B27 to induce the UPR in B27 expression cells.3. Quantitive RT-PCR was used to study the mRNA expression of UPR related genes (GRP78, CHOP) in PBMC of SpA patients. Compared with the PBMC of normal control, the GRP78 and CHOP expression of SpA was significantly higher. The result indicated there is UPR in SpA patients. Then we used these HLA-B27 promoter activating factors as stimulators to activate these transfected U937 cells. The PCR results showed that after stimulation, GRP78, CHOP and XBP-1 expression were all increased compared with unstimulated cells. But there were no significant differences between B27, A2-U937, and EGFP-U937 cells. These results were different with our early research. So, further studies on the response of the HLA-B27 promoter to cytokines in different kinds of cells, especially primary human monocytes/macrophages, combined with our findings will provide further understanding of the roles of HLA-B27 in the pathogenesis of SpA.更多还原