脊柱关节病患者外周血和关节液单个核细胞基因谱的研究

目的：了解脊柱关节病（SpA）患者外周血和关节液单个核细胞（SFMC）基因的变化及其特点，探讨与SpA关节炎相关的基因及可能的意义。方法：采用含1176个基因的cDNA微阵列，检测6名健康志愿者外周血单个核细胞（PBMC），5例SpA和5例类风湿关节炎（RA）病人PBMC和关节液SFMC的基因表达，挑选SpA SFMC表达异常的9个致炎、抗炎、信号传导基因或受体和粘附分子，扩大病例数以半定量PCR再验证微阵列检测结果。结果：cDNA微阵列和PCR结果显示，1176cDNA微阵列基因图谱和阳性基因数量在RA和SpA病人SFMC组无明显区别，比较SpA或RA病人的SFMC和PBMC，发现SpA和RA的SFMC中的阳性基因均少于各自的PBMC。在RA的PBMC有53个基因明显高于RA SFM（P＜0．05），但在SpA PBMC仅有5个基因明显高于SpA SFMC(P＜0．05）。SpA病人SFMC的IL－1β、TNF-α、TGF-β、TGF-β2、c-jun、JAK-显著高于健康人PBMC（P＜0．05）。结论：SpA患者的SFMC基因表达谱异常，但与RA的SFM未见明显特征型区别，IL－1β、TNF-α、TGF-β、TGF-β2、c-jun、JAK-3与SpA关节炎的发生和发展相关。     

慢性心力衰竭患者单核细胞趋化蛋白-3的表达改变

目的 :测定 β型趋化因子单核细胞趋化蛋白 3(monocytechemotacticprotein 3,MCP 3)在慢性心力衰竭 (心衰 )患者中的表达改变 ,探讨其在慢性心衰发病中的作用。方法 :收集 2 5例慢性心衰患者 (慢性心衰组 )和 10例正常人 (正常对照组 )外周血单个核细胞。用逆转录聚合酶链式反应检测MCP 3的表达 ,Bio Rad图像分析系统进行相对定量后 ,分析MCP 3的表达改变及与外周血白细胞计数的关系。结果 :慢性心衰组MCP 3表达阳性率及定量表达水平与正常对照组比较均有显著性差异 (P <0 0 5～ 0 0 1) ,原发病为扩张型心肌病、冠心病和原发性高血压的心衰患者MCP 3表达水平均显著高于正常对照组 (P均 <0 0 1) ,且扩张型心肌病心衰患者的MCP 3表达水平显著高于冠心病和原发性高血压心衰患者 ,均有显著性差异 (P均 <0 0 5 ) ;相关分析显示MCP 3表达水平与外周血白细胞和淋巴细胞计数不相关 ,但与中性粒细胞计数呈正相关 (γ =0 42 0 ,P <0 0 5 )。结论 :慢性心衰患者外周血趋化因子MCP 3表达上调 ,提示免疫因素可能参与了慢性心衰的发生发展     

单核细胞趋化蛋白-1对血压与酰托普利治疗的影响

目的:观察原发性高血压患者应用血管紧张素转换酶抑制剂(ACEI)酰托普利(Ceptopril)治疗前后血浆单核细胞趋化蛋白-1(MCP-1)浓度、MCP-1信使核糖核酸(mRNA)表达及血压的变化,初步探讨MCP-1与高血压的可能关系。方法:选择轻、中度原发性高血压患者60例,随机分入酰托普利组(n=30)和双氢克尿噻组(n=30),分别予酰托普利和双氢克尿噻治疗8周。测定了治疗前后的血压、MCP-1浓度以及外周血单个核细胞MCP-1的mRNA表达水平。另选30例健康人为正常对照组。结果:与正常对照组相比,高血压患者血浆MCP-1浓度及单个核细胞MCP-1的mRNA表达明显升高;酰托普利组治疗后,血压明显降低同时血浆MCP-1浓度及其mRNA表达也明显下降;双氢克尿噻组虽降压效果与酰托普利组类似,但无相应的MCP-1变化。结论:MCP-1是影响高血压患者血压的因素之一,酰托普利除有效降压外还可通过降低炎症因子MCP-1的表达使患者获益。     

血管内皮生长因子在脊柱关节病外周关节病变发病机制中的作用研究

目的 探讨血管内皮生长因子(VEGF)在脊柱关节病(SpA)发病机制中的作用以及与疾病活动性的相关性。方法 收集外周血单个核细胞(PBMC)和关节液中的单个核细胞(SFMC)，经脂多糖(LPS)刺激后，采用反转录聚合酶链反应(RT-PCR)检测VEGF mRNA的表达，并与疾病活动性进行相关性比较，同时采用酶联免疫吸附试验(ELISA)方法测定关节液和血清中的VEGF(SF-VEGF／s-VEGF)，并与VEGF mRNA以及疾病活动性进行相关性比较。结果 未经任何刺激的PBMC无VEGF mRNA表达，经LPS刺激的SFMC其VEGF mRNA的表达比未经刺激的SFMC显著增高，未经刺激的SFMC VEGF mRNA表达与临床常用的疾病活动性指标C反应蛋白(CRP)显著相关，经LPS刺激后的SFMC VEGF mRNA表达还与Bath强直性脊柱炎(AS)疾病活动性指数(BASDAI)、外周关节肿胀数．SF-VEGF、s-VEGF显著相关。SF-VEGF水平较s-VEGF明显增高，SF-VEGF水平与BASDAI、外周关节肿胀数以及外周血白细胞计数的相关有显著性；s-VEGF水平显示出与BASDAI、Bath强直性脊柱炎功能指数(BASFI)、外周关节肿胀数、疲倦程度以及外周血小板计数有相关性。结论 SpA患者SFMC表达VEGF mRNA，LPS可上调其表达；SF-VEGF水平高于S-VEGF，与血沉(ESR)、CRP相比s-VEGF更能准确反应机体疾病活动性。     

类风湿关节炎患者外周血CD4 T细胞关节局部趋化机制

目的探讨类风湿关节炎(RA)患者外周血CD4 T细胞比例变化情况及其对关节局部趋化的机制。方法流式细胞术分析RA患者外周血单个核细胞(PBMC)中CD4 T细胞的比例,并分析CD4 T细胞比例与RA疾病活动性评分系统(DAS28)评分间的关系。激光共聚焦方法检测RA患者滑膜局部浸润CD4 T细胞情况。流式细胞术分析RA患者CD4 T细胞表面趋化因子受体表达情况。结果与对照组相比,RA患者外周血PBMC中CD4 T细胞的比例显著降低(P0.01),且RA患者外周血CD4 T细胞比例与DAS28评分呈明显负相关(r=-0.753 0,P=0.001 2)。关节局部CD4 T细胞浸润的激光共聚焦结果显示,与对照组相比,RA患者滑膜中具有更多的CD4 T细胞浸润。趋化因子受体分析结果显示,RA患者外周血CD4 T细胞表面CCR5、CXCR1和CXCR3的表达显著升高(P0.01)。结论 RA患者外周血CD4 T细胞表面表达升高的CCR5、CXCR1和CXCR3可能参与外周血CD4 T细胞向滑膜局部的趋化。     

急性心肌梗死患者炎症因子C反应蛋白对单核细胞趋化蛋白-1表达的影响

目的探讨急性心肌梗死患者炎症因子C反应蛋白(CRP)与黏附分子单核细胞趋化蛋白-1(MCP-1)表达的关系。方法检测20例急性心肌梗死患者(均选白2007年2～7月在北京医院心内科心脏监护室住院的患者)及27例健康对照者血清生化指标、超敏C反应蛋白(Hs-CRP)及MCP-1水平,分析Hs-CRP与MCP-1的相关性。体外分离培养外周血单核细胞,以不同浓度CRP刺激单核细胞,用定量PCR测定MCP-1表达变化。结果与对照组比较,急性心肌梗死组血清Hs-CRP及MCP-1水平明显升高。急性心肌梗死组与健康对照组Hs-CRP水平分别为:(82.84±43.79)nmol/L和(2.09±5.31)nmol/L(P<0.01);急性心肌梗死组与健康对照组MCP-1水平分别为:(19.61±10.59)nmol/L和(10.78±5.10)nmol/L(P<0.05),且两者呈正相关(r=0.451,P<0.05)。CRP浓度依赖性地增加外周血单核细胞中MCP-1的表达。在CRP水平为47.0～188.0 nmol/L时差异具有统计学意义(P<0.05或P<0.01)。在47.0、94.0、188.0 nmol/L浓度刺激组中MCP-1相对表达量较未刺激组高44%、124%、142%。结论急性心肌梗死患者中,黏附分子MCP-1表达水平升高可能是由于炎症因子CRP增加导致。     

类风湿关节炎患者Th1/Th2细胞平衡的研究

目的　在单个细胞水平上研究类风湿关节炎 (RA)患者滑液及外周血中Th1/Th2细胞平衡状态及其与疾病活动程度的关系。方法　用酶联免疫斑点法 (ELISPOT)检测RA患者滑液单个核细胞 (SFMC)和外周血单个核细胞 (PBMC)的细胞内细胞因子IFN γ和IL 4的表达情况。结果　RA患者SFMC中Th1及Th1/Th2比值与自身及正常人PBMC相比显著升高 (P <0 0 1) ,Th1/Th2比值与患者Stoke指数呈正相关 (r =0 893,P <0 0 1)。结论　在RA患者关节中细胞因子分泌模式朝Th1偏移 ,Th1/Th2比值与疾病活动性相关 ,Th1/Th2平衡在RA发病机制及疾病进展中起重要作用。     

活动性系统性红斑狼疮自噬及相关基因研究

目的 观察活动性系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMC)的自噬现象并检测相关基因的表达水平. 方法 以初诊及新发作的活动性SLE患者20例为病例组,类风湿关节炎(RA)患者10例和健康志愿者10名分别作为对照组.密度梯度离心法分离PBMC,并利用贴壁法除去单核细胞.运用透射电镜(TEM)观察细胞的超微结构,半定量反转录-聚合酶链反应(RT-PCR)及实时定量RT-PCR检测各组PBMC中Beclin1、微管相关蛋白1轻链3(MAPLC3)mRNA的表达水平. 结果 TEM发现活动性SLE患者PBMC中存在自噬现象;Beclinl和MAPLC3 mRNA在活动性SLE患者表达量均增高,且分别较RA组和健康对照组表达差异有统计学意义. 结论 活动性SLE患者外周血PBMC存在自噬现象,且细胞自噬相关基因表达较健康对照组和RA组增强,这可能与SLE的发生、发展相关.     

人类免疫缺陷病毒-1感染者血清趋化因子的检测及克隆构建

目的 探讨调节活化正常T细胞表达与分泌趋化因子(RANTES)及单核细胞趋化蛋白-1(MCP-1)在HIV-1感染中的作用.方法 以ELISA检测HIV-1感染者(治疗组和未治疗组)和健康对照组人群外周血清RANTES及MCP-1的浓度;构建hRANTES-pcDNA3.1,hMCP-pcDNA3.1及hMCP/hRANTES-pcDNA3.1重组质粒,在CHO细胞中体外高效表达,获得重组蛋白,并研究三者对人外周血单个核细胞的趋化功能.结果 RANTES在健康对照组为(164.3±21.3)pg/mL,未治疗组为(1 224.1±62.0)pg/mL,治疗组为(475.3±36.2)pg/mL;MCP-1分别为(90.6±28.5)、(335.0±30.3)和(807.2±62.6)pg/mL.HIV-1感染后RANTES和MCP-1均升高,有效抗病毒治疗后RANTES有所下降,而MCP-1则明显上升.重组蛋白经Western印迹鉴定,能与各自单克隆抗体结合,且三者对人外周血单个核细胞都有趋化作用,MCP/RANTES融合蛋白的趋化作用更强.在重组蛋白浓度为50、200、400和800 pg/mL时,MCP/RANTES融合蛋白趋化外周血单个核细胞数分别为52×104/mL、102×104/mL、132×104/mL和184×104/mL;RANTES趋化外周血单个核细胞数分别为27×104/mL、51×104/mL、65×104/mL和96×104/mL;MCP-1趋化外周血单个核细胞数分别为18×104/mL、44×104/mL、54×104/mL和74×104/mL.结论 RANTES和MCP-1可能都参与了HIV-1的感染和机体对HIV-1的免疫反应.     

血糖控制对高血糖危象患者外周血MCP-1水平的影响

目的观察血糖控制对高血糖危象患者外周血单核细胞趋化蛋白(MCP)-1水平的影响。方法对54例诊断为糖尿病酮症酸中毒(DKA)以及非酮症高血糖高渗(NKH)患者的MCP-1进行检测,并选择40例健康体检者作为对照组。结果治疗前,高血糖危象患者MCP-1水平明显高于对照组(P<0.05)。在血糖得到有效控制后,患者MCP-1与治疗前比较明显下降(P<0.05)。相关分析提示MCP-1水平与空腹血糖(FPG)、2 h餐后血糖(2 h PG)、胰岛素抵抗指数(HOMA-IR)有明显的正相关。结论对高血糖危象患者积极进行血糖控制,能够有效降低MCP-1水平。     

Overexpression of osteopontin in rheumatoid synovial mononuclear cells is associated with joint inflammation, not with genetic polymorphism

OBJECTIVE: Osteopontin (OPN) is thought to play an important role in rheumatoid synovitis. We investigated the expression of OPN in rheumatoid synovial fluid mononuclear cells (SFMC) and its potential association with genetic polymorphism of the OPN gene and joint inflammation in rheumatoid arthritis (RA). METHODS: 1. The expression of OPN mRNA in peripheral blood mononuclear cells (PBMC) and SFMC of patients with RA was analyzed quantitatively by real-time polymerase chain reaction (PCR). Results were analyzed in paired PBMC and SFMC and control PBMC. 2. Six single nuclear acid polymorphisms of the OPN gene were genotyped in a cohort of 192 Chinese patients with RA and controls (n = 288) by restriction fragment length polymorphism PCR or direct DNA sequencing. 3. SF derived from RA patients was examined for the stimulating effect on mRNA expression of the OPN gene in PBMC. RESULTS: The expression of OPN gene was significantly increased in SFMC and, to a lesser degree, in PBMC of patients with RA compared to control PBMC (p < 0.01). However, the prevalence of OPN genotype and allele frequencies at the selected positions did not differ significantly between RA patients and the control group (p > 0.05). Further characterization indicated that SF known to contain a variety of proinflammatory factors significantly stimulated mRNA expression of OPN in PBMC obtained from RA patients or healthy controls. CONCLUSION: Overexpression of OPN mRNA in SFMC is associated with proinflammatory factors produced in inflamed joints, but not with OPN genetic polymorphisms. OPN gene polymorphisms do not correlate with susceptibility to RA.     

Quantitation of IL-2Rp75 (CD122) expression on mononuclear cells in rheumatic disease.

OBJECTIVE: To compare expression of the p75 chain of the interleukin-2 receptor (IL-2Rp75, CD122) on peripheral and synovial mononuclear cells in rheumatoid and non-rheumatoid inflammatory arthritis. METHODS: Peripheral blood (PBMC) and synovial (SFMC) mononuclear cells were isolated from subjects with rheumatoid arthritis (n = 16) and non-rheumatoid inflammatory arthritis (n = 12). PBMC were isolated from six healthy controls. Expression of CD122 was examined using indirect immunofluorescence and quantitative flow cytometry. RESULTS: There was no difference in IL-2Rp75 expression on PBMC from rheumatoid arthritis patients, non-rheumatoid arthritis patients, and controls. In subjects with rheumatoid arthritis there was no difference in IL-2Rp75 expression on PBMC and SFMC. However, in the non-rheumatoid arthritis group there was an increase in IL-2Rp75 expression on SFMC compared with PBMC (P = 0.0032). On SFMC there was a greater expression of IL-2Rp75 in non-rheumatoid arthritis than in rheumatoid arthritis (P = 0.0007). Expression was greater on CD8 positive cells and in subjects with shorter duration of disease. CONCLUSIONS: The p75 chain of the IL-2 receptor, an important T cell activation antigen, is not upregulated in synovial fluid. This appears to be a disease specific defect and provides further support for the concept of "frustrated" or incomplete T cell activation in this disease.     

Gene expression in juvenile arthritis and spondyloarthropathy: pro-angiogenic ELR+ chemokine genes relate to course of arthritis

OBJECTIVE: To evaluate the ability of microarray-based methods to identify genes with disease-specific expression patterns in peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) of juvenile arthritis patients and healthy controls. METHODS: Microarray data (Affymetrix U95Av2) from 26 PBMC and 20 SFMC samples collected from patients with active disease (classified by course according to ACR criteria) were analysed for expression patterns that correlated with disease characteristics. For comparison, PBMC gene expression profiles were obtained from 15 healthy controls. Real-time PCR was used for confirmation of gene expression differences. RESULTS: Statistical analysis of gene expression patterns in PBMC identified 378 probe sets corresponding to 342 unique genes with differing expression levels between polyarticular course patients and controls (t test, P<0.0001). The genes represented by these probe sets were enriched for functions related to regulation of immune cell functions, receptor signalling as well as protein metabolism and degradation. Included in these probe sets were a group of CXCL chemokines with functions related to angiogenesis. Further analysis showed that, whereas angiogenic CXCL (ELR+) gene expression was elevated in polyarticular PBMC, expression of angiostatic CXCL (ELR-) chemokines was lower in polyarticular SFMC compared with corresponding pauciarticular samples (t test, P<0.05). CONCLUSIONS: This pilot study demonstrates that juvenile arthritis patients exhibit complex patterns of gene expression in PBMC and SFMC. The presence of disease-correlated biologically relevant gene expression patterns suggests that the power of this approach will allow better understanding of disease mechanisms, identify distinct clinical phenotypes in disease subtypes, and suggest new therapeutic approaches.     

High percentage of CD8+, Leu-7+ cells in rheumatoid arthritis synovial fluid.

OBJECTIVE. While analyzing the phenotype of the synovial fluid mononuclear cells (SFMC) clustered about dendritic cells in rheumatoid arthritis (RA) joint effusions, it was noted that most of the clustering cells were CD8+ and coexpressed Leu-7. Therefore, the present study was conducted to investigate the frequency of CD8+, Leu-7+ cells in RA SF. METHODS. SFMC from 13 patients with RA and from 12 patients with non-RA inflammatory arthritides were examined for CD8 and Leu-7 expression using 2-color immunofluorescence flow cytometry. RESULTS. RA SFMC had statistically significantly greater percentages of total CD8+ cells, total Leu-7+ cells, and CD8+, Leu-7+ cells, compared with SFMC from the non-RA patients. These RA CD8+, Leu-7+ SFMC had a distinctive electron microscopic appearance compared with CD8+, Leu-7- SFMC. When peripheral blood mononuclear cells (PBMC) from 31 RA patients (including 7 from the SFMC group) were compared with PBMC from 15 normal controls, the percentage of CD8+, Leu-7+ cells was not significantly greater in the RA patients. However, the combination of a modest increase in CD8+, Leu-7+ cells and a decrease in total CD8 cells in RA PBMC altered the composition of the RA CD8 population compared with normal PBMC, such that over 40% of RA peripheral blood CD8 cells coexpressed Leu-7. CONCLUSION. The increased frequency of CD8+, Leu-7+ cells in RA SFMC may arise from the fact that a high percentage of the CD8+ PBMC in RA patients are also Leu-7+. This altered composition of CD8 cells in RA SF may have a role in the pathogenesis of the disease.     

Production of T-cell cytokines at the single-cell level in patients with inflammatory arthritides: enhanced activity in synovial fluid compared to blood

We used a newly developed, sensitive ELISPOT technique in order to estimate the number of cells producing interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) in patients with rheumatoid arthritis (RA) and other inflammatory arthritides, and to correlate the results with clinical and laboratory parameters of disease activity. SFMC and PBMC were cultured either without stimuli or with a standardized dose of phytohaemagglutinin (PHA) for 6 h. Twenty-nine patients, 16 with RA and 13 with other inflammatory joint diseases, were investigated and compared to PBMC from 25 healthy controls. The mean number of IFN-gamma-producing cells was 37.1/10(5) plated SFMC (range 0-121.5). The corresponding value for PBMC was 5.1 (0-39). The difference was highly significant (P = 0.0033 for RA patients, P = 0.0050 for non-RA patients and P < 0.0001 for all patients). Forty-five per cent of SFMC samples (range for all samples 0-38.5 SFC/10(5) MNC) and 25% of PBMC samples (0-20.5) exhibited spontaneous IL-4 production, yielding a significant difference for all patients treated collectively (P = 0.021). Although the cells that spontaneously secrete these cytokines are relatively few, quantification of these cells thus shows increased functional T-cell activation and decreased ratio of cells spontaneously producing IL-4 vs IFN-gamma in the joint fluid as compared to blood of arthritis patients.      

High percentage of CD8+, Leu-7+ cells in rheumatoid arthritis synovial fluid

Objective. While analyzing the phenotype of the synovial fluid mononuclear cells (SFMC) clustered about dendritic cells in rheumatoid arthritis (RA) joint effusions, it was noted that most of the clustering cells were CD8+ and coexpressed Leu-7. Therefore, the present study was conducted to investigate the frequency of CD8+, Leu-7+ cells in RA SF. Methods. SFMC from 13 patients with RA and from 12 patients with non-RA inflammatory arthritides were examined for CD8 and Leu-7 expression using 2-color immunofluorescence flow cytometry. Results. RA SFMC had statistically significantly greater percentages of total CD8+ cells, total Leu-7+ cells, and CD8+, Leu-7+ cells, compared with SFMC from the non-RA patients. These RA CD8+, Leu-7+ SFMC had a distinctive electron microscopic appearance compared with CD8+, Leu-7– SFMC. When peripheral blood mononuclear cells (PBMC) from 31 RA patients (including 7 from the SFMC group) were compared with PBMC from 15 normal controls, the percentage of CD8+, Leu-7+ cells was not significantly greater in the RA patients. However, the combination of a modest increase in CD8+, Leu-7+ cells and a decrease in total CD8 cells in RA PBMC altered the composition of the RA CD8 population compared with normal PBMC, such that over 40% of RA peripheral blood CD8 cells coexpressed Leu-7. Conclusion. The increased frequency of CD8+, Leu-7+ cells in RA SFMC may arise from the fact that a high percentage of the CD8+ PBMC in RA patients are also Leu-7+. This altered composition of CD8 cells in RA SF may have a role in the pathogenesis of the disease.     

Enhanced T cell proliferative response to type II collagen and synthetic peptide CII (255-274) in patients with rheumatoid arthritis.

OBJECTIVE: To determine the presence of specific immune recognition of type II collagen (CII) and its immunodominant epitope CII (255-274) in patients with rheumatoid arthritis (RA). METHODS: T cell proliferative responses to bovine CII and a synthetic peptide encompassing CII (255-274) in peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) from RA patients, and in PBMC from osteoarthritis (OA) patients and healthy controls were assayed by mixed lymphocyte culture. RESULTS: The stimulation index (SI) and the number of positive (SI > or = 2) T cell responses to CII were higher in RA patients (n = 106) than in OA patients (n = 26) and healthy controls (n = 34). T cell responses to CII (255-274) were also enhanced in RA patients and correlated well with those to CII. In SFMC, positive responses to CII or CII (255-274) were detected in 61.9% of 42 RA patients. T cell responses to CII in SFMC were stronger and more prevalent than peripheral responses. The SI and positive responses to CII were higher in early RA than in late RA. Levels of IgG antibodies to CII in synovial fluid inversely correlated with T cell responses to CII. CONCLUSION: T cell responses to CII or CII (255-274) were enhanced in RA, especially in early disease. Synthetic peptide CII (255-274), as well as native CII, could be recognized as immunogenic antigens by T cells, particularly in the synovial fluid. These observations suggest that CII-reactive T cells play an important role in the pathogenesis of RA. Peripheral tolerance induction using CII (255-274) might be useful in the treatment of RA.