携带hIL-4逆转录病毒重组体的构建及在类风湿关节炎中体外表达的研究

目的构建携带人白介素4(hIL-4)表达序列的重组逆转录病毒pLXSN-hIL-4并检测目的基因的表达。方法2004年6月至12月在中国医科大学基础医学院设计带有酶切位点的引物,含有目的基因的DNA进行聚合酶链反应扩增并纯化目的基因片段。pLXSN与目的基因片段hIL-4进行定向克隆连接,筛出阳性克隆并进行鉴定。扩增重组逆转录病毒载体pLXSN-hIL-4,并转染体外培养的人滑膜成纤维样细胞。Westernblotting法测定目的基因蛋白表达水平。结果成功构建了携带治疗基因的逆转录病毒重组体:rRV-hIL-4。Westernblotting法检测到了hIL-4的表达。结论成功构建了携带治疗基因的逆转录病毒重组体:rRV-hIL-4。以逆转录病毒为载体可以将hIL-4基因导入体外培养的人滑膜成纤维样细胞,转染后的细胞可以表达hIL-4蛋白。     

含LacZ报告基因的腺病毒载体在体外培养类风湿关节炎滑膜细胞中表达的实验观察

目的：观察含报告基因的腺病毒对体外培养的类风湿关节炎滑膜细胞的感染及其表达情况，方法：将含LacZ报告基因腺病毒感染类风湿关节滑膜细胞，通过X－gal显色的方法观察腺病毒感染滑细胞后基因的表达情况，绘制感染效率曲线。结果：腺病毒可以有效感染类风湿关节炎滑膜细胞，并表达携带的基因；报告基因，在一定范围内，腺病毒滴度越高，基因表达信号越强。结论：腺病毒载体可以携带外源性基因在类风湿关节炎滑膜细胞中表达。     

逆转录病毒感染与类风湿关节炎的相关性研究

目的：为了探讨逆转录病毒与类风湿湿关节炎发病的相关性。方法；采用细胞培养，免疫组织化学技术等牟３３例ＲＡ患者的外周血要本进行了人类嗜Ｔ淋巴细胞病毒Ｉ型相关抗原，逆转录酶活性测定及观察自发性合胞体形成。结果：７５．８％ＲＡ样本阳性表达ＨＴＬＶ－Ｉ相关抗原，对照组为阴性，ＨＴＬＶ－Ｉ相关抗原阳笥标本同时表达ＲＴ活性，且两才之间存在相关性；３６．４％ＲＡ样本出现自发性合胞体形成，对照组无类似现象，且自发     

IL-33在类风湿关节炎合并间质性肺炎患者中的表达及意义

目的探讨类风湿关节炎合并肺间质病变(RA-ILD)患者的临床血清中白介素-33(IL-33)表达,特征外周血单个核细胞(PBMC)。方法类风湿关节炎(RA)患者115例,分为RA-ILD组(29例)和RA组(86例),另选取健康自愿者20例为对照组,类风湿因子(RF)、抗环瓜氨酸抗体(anti-CCP)及抗角蛋白抗体(AKA)的浓度,检测PBMC中IL-33 mRNA表达,同时通过酶联免疫吸附法(ELISA)测定各组血清中IL-33的表达。结果与RA患者比,RA-ILD组除性别外平均年龄、病程比较差异均有统计学意义,RF、anti-CCP及AKA的浓度在RA-ILD与RA两组之间无统计学差异(P0.05),RA-ILD组PBMC中IL-33 mRNA及血清中IL-33表达含量明显比RA及正常对照组升高(P0.01)。结论 RA-ILD易发生于年龄大、病程较长的RA患者,一般实验检查不能预测RA患者ILD发生的可能性;PBMC和血清中IL-33可能与RA-ILD的发病有关;IL-33水平增高可能为RA-ILD的预测指标。     

天然免疫分子TLR2 TLR4在类风湿关节炎的表达模式研究

目的 探讨天然免疫分子TLR2、TLR4在类风湿关节炎(RA)发病中的作用。方法 采用多色免疫荧光流式细胞仪技术，观察RA患者和正常对照外周血CD14阳性单核细胞上TLR2、TLR4分子的表达率和平均荧光强度。结果 ①TLR2和TLR4在RA患者外周血CDl4阳性单核细胞阳性率和平均荧光强度显著高于对照组；②二者表达模式在早期RA组和晚期RA组差异无统计学意义：③TLR2平均荧光强度与C反应蛋白(CRP)水平有关联。结论天然免疫分子TLR2、TLR4在RA的表达模式，提示其在RA发病和病情进展中有重要作用。     

HLA-DR_4基因检测在类风湿关节炎诊治中的意义探讨

为了研究HLA-DR_4基因检测在类风湿关节炎诊治中的意义,对50例类风湿关节炎患者进行了HLA-DR_4的(PCR-SSP方法)检测,同时结合患者的临床表现和实验室指标进行分析。结果显示:HLA-DR_4阳性者31例(阳性率为62％);比较HLA-DR_4阳性和阴性两组患者其他指标,可见,HLA-DR_4阳性组的关节疼痛指数、ESR、类风湿因子(RF)滴度均明显高于HLA-DR_4阴性组(P值分别<0.05或0.01),手腕部X线在Ⅱ级～Ⅲ级异常变化者也以HLA-DR_4阳性组较多(P<0.005)。结果提示:HLA-DR_4基因检测是临床作为类风湿关节炎患者判断病情和估计预后有价值的指标之一,值得进一步探讨。     

人白细胞介素-1受体拮抗剂或白细胞介素-10逆转录病毒载体的构建与体外实验

目的 构建携带人白细胞介素-1受体拮抗剂(hIL-1Ra)或人白细胞介素-10(hIL-10)基因的重组逆转录病毒(rRv)，体外转染免滑膜成纤维样细胞，检测目的基因的表达水平与持续时间。方法 提取人外周血单个核细胞总RNA，反转录聚合酶链反应(RT-PCR)扩增目的基因；酶切、连接目的基因与逆转录病毒载体pLXSN，筛出阳性克隆，经GP2-293细胞包装，收集病毒并鉴定；rRV-hlL-1Ra、rRV-hlI-10分别体外转染兔滑膜成纤维样细胞，RT-PcR测定目的基因mRNA表达水平与时间的关系。免疫组织化学与免疫印迹测定蛋白水平的表达与持续时间。结果 成功构建了携带hIL-1Ra或hIL-10基因的重组逆转录病毒，rRv-hIL-1Ra与rRV-hIL-10均能有效转染体外培养的兔滑膜成纤维样细胞，RT-PcR测定目的基因mRNA高峰均出现于转染后第5天左右。免疫组织化学和免疫印迹均检测到hIL-lRa、hlL-10的表达。经G418筛选后的细胞中，hIL-1Ra的表达在转染后第30天内达高峰，至少持续60d：hIL-10的表达至少持续40d。结论 以重组逆转录病毒为载体可以成功地将hIL-lRa或hIL-10基因导人体外培养的兔滑膜成纤维样细胞并实现稳定表达。     

类风湿关节炎与TLR4单核苷酸多态性关联研究的Meta分析

目的探讨类风湿关节炎(RA)与TLR4基因Asp299Gly多态性的关联情况。方法检索已发表的有关RA和TLR4基因Asp299Gly多态性的文献．进行Meta分析。结果3项研究共纳入718例RA患者和1392名正常对照，综合分析显示TLR4基因Asp299Gly多态性不是RA的关联基因，OR=1．23(0．67，2．25)，P=0．5；TLR4基因Asp299Gly多态性分布在男女性RA患者分布差异无统计学意义，OR=0．48(0．22．1．03)，P=0．06：在共同表位基因阳性和阴性患者组差异无统计学意义，OR=0．67(0．40，1．13)，P=0．13；在类风湿因子(RF)阳性和阴性患者组差异无统计学意义，OR=I．02(0．51，2．02)，P=-I；RA患者中，Asp299Asp基因型患者发病早于Asp299Gly基因型者，OR=-4．35(-7．45，-1．25)，P=0．006。结论Meta分析显示，TLR4基因Asp299Gly多态性与RA易感性无关联．但Asp299Asp基因型者起病早于Asp299Gly基因型者。应在东方人群进一步开展随机对照和前瞻的队列研究揭示其在RA发病中的作用。     

趋化因子受体CCR4在活动期类风湿关节炎患者外周血CD4+T细胞表达的研究

目的 探讨活动期类风湿关节炎(aRA)患者趋化因子受体CCR4在外周血CD4+T细胞的表达及其意义,并对该表达与血清重要细胞因子水平的相关性进行研究.方法流式细胞计数方法对12例aRA患者和10名健康对照者外周血CD4+T细胞表面CCR4的表达情况进行检测;酶联免疫吸附试验(ELISA)方法检测患者血清肿瘤坏死因子(TNF)-α、白细胞介素(IL)-10及干扰素(IFN)-γ的水平并记录患者相关临床及实验室资料.分析及评价aRA患者外周血淋巴细胞中CD4+CCR4+T细胞百分数(CD4+CCR4+T%)与TNF-α、IL-10、IFN-γ的血清水平及临床指标的相关性.结果 aRA组外周血CD4+CCR4+T%明显高于健康对照组(P＜0.01).aRA组血清TNF-α(P＜0.01)、IL-10(P＜0.01)及IFN-γ(P＜0.01)水平明显高于健康对照组.在检测的细胞因子中,aRA患者血清IL-10水平与CD4+CCR4+T%呈明显正相关关系(r＝0.66,P＜0.05).在临床指标中,CD4+CCR4+T%与aRA患者Lunsbury关节指数、血沉、C反应蛋白及血小板计数呈明显正相关关系(P＜0.05).结论 CCR4在CD4+T细胞趋化至病变关节过程中可能发挥重要作用;外周血CD4+CCR4+T%与血清IL-10水平密切相关;该百分数可以作为临床评价RA活动性的一个敏感指标.     

贵州汉族类风湿关节炎患者HLA-DR4亚型多态性的研究

类风湿关节炎 (ｒｈｅｕｍａｔｏｉｄａｒｔｈｒｉｔｉｓ ,ＲＡ)的发病机制尚未彻底阐明。研究发现ＲＡ与ＨＬＡ ＤＲ4有不同程度的相关性 ,但ＤＲ4至少包括 2 2种亚型[1] ,不同亚型与ＲＡ的相关性不尽相同。为此我们采用序列特异性引物聚合酶链反应 (ＰＣＲ ＳＳＰ)技术 ,检测了贵州地区汉族ＲＡ患者ＤＲ4基因亚型的多态性 ,现报告如下。1　材料和方法1 1　研究对象本院风湿科 2 0 0 0— 2 0 0 1年住院ＲＡ患者 35例 ,诊断符合美国风湿协会 (ＡＣＲ) 1987年诊断标准[2 ] ,其中男性 7例 ,女性 2 8例 ,年龄 9～ 75岁 ,平均 4 6岁。 5 1份对照…     

Serum IgG4 anti-Fab antibodies in rheumatoid arthritis are constitutively expressed

Summary IgG4 comprises a significant proportion of the total anti-Fab antibody (aFABA) response in many but not all patients with rheumatoid arthritis (RA). Analyses of the dynamics of IgG aFABA subclass expression in 11 RA patients for periods of up to 11 months demonstrated that IgG4 aFABA was restricted to 6 of the 11 RA patients' sera initially studied and comprised approximately 25% (or more) of the total IgG aFABA response. Quantities of IgG4 aFABA in subsequent, serially obtained serum samples from these patients remained stable throughout the study period, whereas the remaining RA patients whose initial sera possessed small quantities of serum IgG4 aFABA failed to generate any augmented IgG4 aFABA response during the study. Elevated expression of IgG4 aFABA did not appear to be a consequence of a generalized polyclonal gammopathy or a generalized increase in autoantibody expression, though patients with higher total IgG4 serum levels expressed significantly greater quantities of IgG4 aFABA. These results indicate that the differential expression of IgG4 aFABA among RA patients reflects constitutive production within a subset of RA patients in whom IgG4 appears to comprise a significant proportion of the total IgG aFABA response.     

Anti-CCP antibodies in rheumatoid arthritis and psoriatic arthritis

Our aim is to assess the prevalence and associated clinical features of anti-CCP (cyclic citrullinated peptide) antibodies for RF (rheumatoid factor)-positive and RF-negative rheumatoid arthritis (RA) and psoriatic arthritis (PsA). In a prospective, cross-sectional, multi-centre study, we determined the titres of anti-CCP antibodies in 208 RA patients (129 RF-positive, 79 RF-negative), 56 PsA patients and 39 healthy controls (HC). Clinical parameters including disease activity (disease activity score 28-DAS28), physical disability (health assessment questionnaire-HAQ), functional capacity (functional class) and radiological erosions were investigated in patients with RA. In PsA patients, clinical and radiological features were determined. Anti-CCP2 antibodies were measured using a second-generation anti-CCP enzyme-linked immunosorbent assay (Euro-Diagnostica, Netherlands). One-hundred four of 129 RF-positive RA (81%), 16 of 79 RF-negative RA (20%), seven of 56 PsA patients (12.5%) and none of the HC had anti-CCP antibodies. RA patients with anti-CCP antibodies had significantly higher disease activity, greater loss of function and more frequent erosive disease than anti-CCP antibody-negative group. In subgroup analysis, anti-CCP antibodies in RF-negative patients were also associated with erosive disease. All PsA patients with anti-CCP antibodies had symmetric arthritis with higher number of swollen joints. The prevalence of anti-CCP antibodies in RF-positive RA patients was significantly higher than in RF-negative RA and PsA patients. Anti-CCP antibodies were also associated with erosive disease in RF-negative RA patients. Both anti-CCP and RF tests were negative in 30% of the patients. Anti-CCP positivity was a frequent finding in PsA and associated with symmetrical polyarthritis.     

Prevalence of antihuman parvovirus B19 IgG antibodies in patients with refractory rheumatoid arthritis and polyarticular juvenile rheumatoid arthritis

We investigated the prevalence of antihuman parvovirus B19 immunoglobulin G (IgG) antibody in 108 Japanese patients with rheumatoid arthritis (RA) and 11 patients with polyarticular juvenile rheumatoid arthritis (JRA). Seropositivity of anti-B19 was significantly higher in patients with refractory RA (57.6%, 38/66) compared with patients with remittent RA (19.0%, 8/42; P>0.001) or age-matched controls (24.3%, 19/78; P>0.001). Patients with refractory polyarticular JRA had a significantly higher frequency of anti-B19 seropositivity (71.4%, 5/7) than age-matched controls (8.3%, 5/60; P>0.001), while none of the remittent group was positive for the antibody (0/4).     

Enhanced in vitro IgM rheumatoid factor synthesis in seropositive rheumatoid arthritis families

Summary We studied the patterns of in vitro IgM RF production by peripheral blood mononuclear leukocytes (MNL) in first- and second-degree relatives and non-blood relatives of 14 RA patients. In all these included 25 individuals with RA (14 probands, 11 other affected members), 88 non-affected first- and second-degree relatives and 15 non-affected non-blood relatives. In vitro production of IgM RF and IgM by MNL from RA patients was similar to what we and others have previously described. Spontaneous production of IgM RF was demonstrated by MNL from some RA patients and the ratio of IgM RF to total IgM was higher for patients than controls. The non-blood relatives failed to demonstrate spontaneous in vitro IgM RF production. In contrast, MNL from approximately 16% of non-affected blood relatives spontaneously produced IgM RF. Spontaneous IgM RF production in this group did not correlate with HLA-DR3 or HLA-DR4 and was found in Caucasoids, American blacks, simplex (one affected individual), and multiplex (more than one affected individual) families. Linkage analysis using either a recessive or dominant model of inheritance failed to detect linkage among affected and non-affected family members.     

Pericarditis in rheumatoid arthritis

Summary The annual incidence of clinically manifest pericarditis was found to be 0.34% in 157 females and 0.44% in 77 males with rheumatoid arthritis, observed for a mean time of 5.7 years. The development of pericarditis was independent of disease duration, but was related to extensive joint involvement, subcutaneous nodules, and a high Waaler Rose titre. Concomitant pleural effusion was present in four of five patients. Pericarditis in rheumatoid arthritis may indicate a serious prognosis quo ad vitam. Four of the five patients died within eight years, but their age at death was relatively high (62–73 years).     

Association of CTLA4 gene A-G polymorphism with rheumatoid arthritis in Chinese

The aim of this study was to investigate the allelic association of a single nucleotide polymorphism in exon 1 of the cytotoxic T-lymphocyte antigen-4 (CTLA4) gene with rheumatoid arthritis (RA) in Chinese people. One hundred and eighty-six unrelated adults with RA and 203 randomly selected normal adults were studied. All were ethnic Chinese living in Taiwan. The CTLA4 A-G polymorphism was genotyped with a polymerase chain reaction (PCR) and digestion with the restriction enzyme BstEII. The genotype and allele frequencies of CTLA4 in patients with rheumatoid arthritis differed significantly from those of adult controls (#E5/E5#=0.022 and #E5/E5#=0.037, respectively). Genotype CTLA4 49 G/G and allele G were associated with an increased risk of RA (RR=1.72, 95% CI=1.15–2.57, #E5/E5#=0.008; RR=1.39, 95% CI=1.02–1.89, #E5/E5#=0.037, respectively), whereas genotype A/G and allele A were associated with protection against RA (RR=0.58, 95% CI=0.39–0.87, #E5/E5#=0.008 and RR=0.72, 95% CI=0.53–0.98, #E5/E5#=0.037, respectively). We concluded that, the CTLA4 49 A-G polymorphism is associated with RA in Chinese patients from Taiwan.     

Corticosteroid prescribing in rheumatoid arthritis and psoriatic arthritis

Corticosteroid usage was assessed in rheumatoid arthritis (RA) and psoriatic arthritis (PA) patients in Italy. A multicentre, observational study was undertaken in 10 Italian rheumatological centres from 1990 to 1992 using a computerised clinical data bank. Nine hundred and seven RA patients and 180 PA patients were studied; 510 (56.2%) RA patients and 44 (24.4%) PA patients were using corticosteroids. The percentage of patients taking corticosteroids ranged from 20.5 to 85.4% for RA patients and from 0 to 55% for PA patients for the different centres. Methylprednisolone was the most prescribed corticosteroid, both in RA patients (63.2%) and in PA patients (65.9%). The average methylprednisolone daily dose was 5.7±3.6 mg in RA patients and 4.5±1.4 mg in PA patients. The data provide evidence that corticosteroids are taken in an unexpectedly high percentage of patients with RA and PA in Italy.     

中国大陆汉族人群类风湿关节炎与HLA-DRB1*0401基因的相关性分析

目的 探讨中国大陆汉族人群类风湿关节炎(RA)与人类白细胞抗原(HLA)-DRB1*0401基因的关联情况。方法 检索已发表的有关中国大陆汉族人群RA患者和HLA-DRB1*0401的文献，进行Meta分析。结果 国内7项研究共578例RA患者和743例正常对照，DRB1*0401基因亚型是中国大陆汉族人群RA的关联基因(OR=2．66，P=0．004)。结论 Meta分析证实DRB1*0401与中国大陆汉族人群RA有相关性，样本大小对基因关联分析有重要影响。