血清ANCA和GAB不同类型抗体检测在溃疡性结肠炎中的临床应用

目的:探讨血清中抗中性粒细胞胞浆抗体(ANCA)和抗小肠杯状细胞抗体(GAB)lgA、lgG对溃疡性结肠炎(UC)诊断的临床价值。方法:收集2018年4月到2019年4月在北京协和医院门诊或住院就诊的患者为研究对象,分为UC组93人,其他肠道疾病患者组93人,分别用间接免疫荧光法检测两组患者的ANCA-IgA、ANCA-IgG、GAB-IgA、GAB-IgG共4种自身抗体,对IgA、IgG的阳性率进行比较。结果:UC组ANCA-IgG、ANCA-IgA和GAB-IgG、GAB-IgA的阳性率分别为43.0%、26.8%、38.7%和5.3%,高于其他肠道疾病组的2.1%、0%、5.3%、0%,两组间ANCA-IgA、ANCA-IgG、GAB-IgG阳性率差异有统计学意义(P<0.05),GAB-IgA差异无统计学意义(P>0.05)。ANCA-IgA、GAB-IgA的特异性(100%、100%)高于ANCA-IgG、GAB-IgG(97.8%、94.6%),但敏感性(26.8%、5.3%)低于ANCA-IgG、GAB-IgG(43.0%、38.7%)。联合ANCA、GAB 4种抗体检测的阳性率为70.9%,但特异性为92.5%。结论:ANCA、GAB-IgA比IgG特异性好,IgG辅之IgA同时检测有助于提高UC的诊断水平,ANCA-IgA/IgG和GAB-IgA/IgG 4种抗体联合可提高UC的检出率,减少漏检率。     

HLA-DQB1基因多态性与中国汉族人群系统性红斑狼疮的相关性

目的 探讨HLA-DQB1基因单核苷酸多态性(single nucleotide polymorphisms,SNP)与汉族人群系统性红斑狼疮(systemic lupus erthematosus,SLE)遗传易感性的相关性.方法 通过聚合酶链式反应-连接酶检测反应(polymerase chain reaction-ligase detection reaction,PCR-LDR)技术对908例SLE患者和961例健康对照rs3129716(HLA-DQB1)位点进行基因分型,同时结合临床表现分型,分析该位点与疾病及临床表型的相关性.分型结果用PLINK1.07软件进行统计分析.结果 rs3129716(HLA-DQB1)位点等位基因频率和基因型频率在SLE疾病组和对照组的分布差异无统计学意义(P＞0.05).三种遗传模型下的分析显示,两组间差异无统计学意义(P＞0.05).将SLE患者按血清学指标及临床表现分型,未发现相关性.结论 rs3129716(HLA-DQB1)与中国汉族人群SLE患者遗传易感性不相关.     

抗酿酒酵母细胞抗体在原发性胆汁性肝硬化患者中的临床意义

目的 检测原发性胆汁性肝硬化(PBC)患者血清抗酿酒酵母细胞抗体(ASCA),探讨其在PBC中的阳性状况和临床意义.方法 采用酶联免疫吸附试验(ELISA)检测162例PBC患者、44例自身免疫性肝炎(AIH)患者、41例肝病对照组(LDC)患者、144例炎症性肠病(IBD)患者及35名健康体检者血清ASCA的IgA和IgG亚型.采用χ2检验和非参数U检验进行分析.结果 ASCA-IgA在PBC患者中的阳性率为24.1%,高于溃疡性结肠炎(UC)组11.6%(χ2=5.5,P＜0.05)和健康对照组0(χ2=10.5,P＜0.01),与AIH组(20.5%)、LDC组(14.6%)和克罗恩病(CD)组(34.5%)比较,差异无统计学意义(P＞0.05);ASCA-IgG在PBC患者中的阳性率为11.1%,明显低于CD组27.6%(χ2=8.9,P＜0.01),高于健康对照组0(χ2=10.5,P＜0.01),与AIH组(15.9%)、LDC组(7.3%)和UC组(8.1%)比较,差异无统计学意义(P＞0.05);ASCA-IgA和IgG同时阳性的PBC患者仅占6.2%,显著低于CD组17.2%(χ2=6.3,P＜0.05);ASCA-IgA或IgG阳性的PBC患者占29.0%,显著低于CD组44.8%(χ2=4.8,P＜0.05),高于UC组(χ2=5.9,P＜0.05)和健康对照组(χ2=13.3,P＜0.01).抗GP210抗体阳性的PBC患者ASCA阳性率高于抗GP210抗体阴性PBC患者(38.6%和23.8%,χ2=3.9,P＜0.05);抗线粒体抗体(AMA)、抗SP100抗体阳性和阴性PBC患者间ASCA的阳性率差异无统计学意义.ASCA-IgA阳性PBC患者总胆红素、直接胆红素、总胆汁酸、乳酸脱氢酶(LD)、IgA、IgM、红细胞沉降率(ESR)高于ASCA-IgA阴性PBC患者,而白蛋白(ALB)、白蛋白/球蛋白和胆碱酯酶低于ASCA-IgA阴性PBC患者(P均＜0.05).ASCA-IgG阳性PBC患者与阴性患者间肝功能损伤指标和免疫功能指标差异均无统计学意义.结论 ASCA并不是IBD特异性自身抗体,在PBC患者有较高的阳性率,且以IgA亚型为主.ASCA-IgA与PBC患者肝功能损伤指标和免疫活动指标改变有关,而ASCA-IgG与PBC患者肝功能损伤指标和免疫活动指标改变无关.     

去唾液酸糖蛋白受体H1亚单位基因片段的克隆表达及其检测自身免疫性肝炎的价值

Objective To clone and express the human asialoglycoprotein receptor(ASGPR) H1 subunit, purify and identify the immunoreactivity of the recombinant protein, and establish the enzyme linked immunosorbent assay (ELISA) to detect anti-ASGPR antibodies in diagnosis of autoimmune hepatitis. Methods The CRDHI cDNA (435 bp) was subcloned into eukaryotic vector PEGH, and the recombinant protein expression was induced by D (+)-Galactose. The recombinant CRDH1 was purified with Glutathione Sepharose 4B, and its immunoreactivity was identified by SDS-PAGE and western blot as well as MALDI-TOF. ELISA was established to detect the anti-ASGPR antibodies in serum samples of 45 patients with AIH, 30 patients with SLE, 30 patients with RA, 10 patients with SS and 30 normal controls. Results The sequencing of recombinant plasmid showed the CRDH1 gene was successfully inserted to the eukaryotic expression vector with correct sequence and open reading frame. The fusion protein showed a molecular weight of 42 500 Da on SDS-PAGE gel and confirmed to be the human ASGPR by MALDI-MS through peptide mass fingerprint analysis with Mascot in human protein database. It shared 98. 34% homology with ASGPR H1 subunit. Western blot analysis showed that the fusion protein had the same immunoreactivity as human ASGPR. The results of ELISA indicated that the positive rate of anti-ASGPR was 35.6% ( 16/45 ), but the ELISA was negative in other control. There was significant difference of positivity of the autoantibodies between AIH and non-AIH controls (χ2 = 31.85,P < 0. 01 ). Conclusions The human plasmid containing ASGPR is successfully clone into Saccharomyces cerevisiae Y258. The recombinant autoantigen owns good antigenicity and specificity. ELISA established with the purified protein shows good specificity for diagnosis of AIH.     

核包膜蛋白gp210、p62和LBR自身抗原基因克隆表达及其抗体诊断原发性胆汁性肝硬化的价值初探

目的克隆人核包膜蛋白自身抗原gp210、p62和LBR基因，构建重组表达质粒，获得具免疫学活性的纯化重组蛋白，建立酶联免疫吸附法（ELISA），初探抗核包膜蛋白gp210、p62和LBR自身抗体在原发性胆汁性肝硬化（PBC）诊断中的意义。方法构建重组表达载体，在大肠杆菌BL21、M15中表达；融合蛋白经Ni—NAT树脂柱进行亲和层析纯化，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）及免疫印迹法（WB）进行免疫活性鉴定；应用表达蛋白建立间接ELISA，检测60名健康体检者、68例原发性胆汁性肝硬化（PBC）、60例病毒性肝炎、60例结缔组织病患者血清中抗gp210、p62和LBR抗体。结果经重组质粒测序和酶切结果证实，gp210、p62和LBR目的基因已正确插入原核表达载体中，基因序列正确，符合表达框架；SDS-PAGE检测表达产物分别在69000、62000和27000处有一明显的蛋白表达条带，WB分析表明重组蛋白具有人gp210、p62和LBR抗原反应性。ELISA检测标本血清结果显示，PBC中，抗gp210抗体、抗p62抗体和抗LBR抗体阳性率分别为36．8％、30．9％和5．9％；而病毒性肝炎组、结缔组织病组和健康体检组中，除抗gp210抗体在结缔组织病患者中阳性率为1．7％外，3种自身抗体检测结果均为阴性。PBC组3种自身抗体阳性率与疾病对照组及正常对照组比较，差异均有统计学意义（P〈0．05和P〈0．01）。结论本研究成功克隆人核包膜蛋白自身抗原gp210、p62和LBR基因，并将其在大肠杆菌中成功表达。应用纯化融合蛋白建立的ELISA检测抗gp210抗体、抗p62抗体和抗LBR抗体，对PBC具有较好特异性，为PBC的特异性临床诊断提供了有效的工具。     

原发性胆汁性肝硬化患者调节性T细胞及其与肝功能损伤的研究

目的检测原发性胆汁性肝硬化（primary biliary cirrhosis，PBC）患者外周血CD4^＋ CD8^＋ T、CD4^＋ CD25^＋调节性T细胞亚群，探讨其与肝功能损伤、抗AMA—M2抗体产生的关系。方法采用流式细胞术检测北京协和医院住院和门诊PBC患者（n=27）外周血CD4^＋ CD8^＋ T细胞、CD4^＋ CD25^＋ T细胞群比例，以26例其他肝脏疾病患者作为疾病对照组，30例健康体检者作为正常对照，观察调节性T细胞亚群与患者的肝功能指标及自身抗体如AMA—M2、ANA的关系。结果PBC患者外周血CD4^＋ CD25^＋ T细胞比例与正常对照组比较结果差异无统计学意义（P〉0．05），但显著高于其他肝脏疾病组（P〈0．05）。CD4^＋ CD8^＋细胞群与正常人相比结果差异无统计学意义，其他肝病组明显高于正常对照组（P〈0．05）。PBC患者外周血NK细胞比例显著低于正常对照组（P〈0．05）。肝功严重损伤的PBC患者外周血CD4^＋ CD25^＋ T细胞比例明显高于肝功能轻度损伤者（P〈0．05）。PBC患者外周血CD4^＋ CD25^＋ T细胞数量与肝功能损伤指标ALB呈负相关（r=－0．338，P〈0．05），与TBIL、DBIL呈正相关（r分别为0．362，0．386，P〈0．05）。CD4^＋ CD25^＋ ／CD4^＋比例与GGT呈负相关（r=－0．335，P〈0．05），与TBIL、DBIL呈正相关（r分别为0．333，0．339，P〈0．05）。抗AMA—M2抗体阳性患者外周血CD4^＋ CD25^＋细胞比例明显高于AMA—M2阴性患者（P〈0．01）。未发现CD4^＋ CD25^＋ T、CD4^＋ CD8^＋T细胞比例在ANA^＋和ANA—PBC患者之间差异有统计学意义。结论PBC患者外周血CD4^＋ CD25^＋T细胞群比例与肝功能损害和抗AMA—M2抗体的产生密切相关，因此推测CD4^＋ CD25^＋T细胞的变化可能是导致病情发展以及肝脏损伤的关键环节之一。     

系统性硬皮病抗核抗体谱的检测分析

目的 通过对系统性硬皮病（SSc）患者抗核抗体谱（ANAs）检测结果的分析,探讨这些抗核抗体（ANA）对系统性硬皮病的诊断价值。方法 分别采用间接免疫荧光法（IIF）和免疫印迹法（IB）对62例SSc患者和20例健康对照者血清中15种抗核抗体进行测定。结果 IIF检测ANA结果显示,SSc患者中ANA的阳性率为98.4%（61/62）,其荧光染色模型主要为均质核仁型;IB检测15种ANA结果显示,SSc患者中的ANA以抗Scl-70抗体、抗SS-A抗体和抗Ro-52抗体为主,在46例弥漫性硬皮病（dSSc）患者中抗Scl-70抗体的阳性率高达67%,特异性为100%。结论 多种自身抗体同时检出,可能提示SSc患者同时伴随有其他的自身免疫性疾病,或者有发生其他自身免疫性疾病的危险;此外,欧蒙免疫印迹试剂盒,对ANAs检测的阳性率和特异性均较高,对SSc的诊断帮助很大。     

去唾液酸糖蛋白受体H1亚单位基因片段的克隆表达及其检测自身免疫性肝炎的价值

Objective To clone and express the human asialoglycoprotein receptor(ASGPR) H1 subunit, purify and identify the immunoreactivity of the recombinant protein, and establish the enzyme linked immunosorbent assay (ELISA) to detect anti-ASGPR antibodies in diagnosis of autoimmune hepatitis. Methods The CRDHI cDNA (435 bp) was subcloned into eukaryotic vector PEGH, and the recombinant protein expression was induced by D (+)-Galactose. The recombinant CRDH1 was purified with Glutathione Sepharose 4B, and its immunoreactivity was identified by SDS-PAGE and western blot as well as MALDI-TOF. ELISA was established to detect the anti-ASGPR antibodies in serum samples of 45 patients with AIH, 30 patients with SLE, 30 patients with RA, 10 patients with SS and 30 normal controls. Results The sequencing of recombinant plasmid showed the CRDH1 gene was successfully inserted to the eukaryotic expression vector with correct sequence and open reading frame. The fusion protein showed a molecular weight of 42 500 Da on SDS-PAGE gel and confirmed to be the human ASGPR by MALDI-MS through peptide mass fingerprint analysis with Mascot in human protein database. It shared 98. 34% homology with ASGPR H1 subunit. Western blot analysis showed that the fusion protein had the same immunoreactivity as human ASGPR. The results of ELISA indicated that the positive rate of anti-ASGPR was 35.6% ( 16/45 ), but the ELISA was negative in other control. There was significant difference of positivity of the autoantibodies between AIH and non-AIH controls (χ2 = 31.85,P < 0. 01 ). Conclusions The human plasmid containing ASGPR is successfully clone into Saccharomyces cerevisiae Y258. The recombinant autoantigen owns good antigenicity and specificity. ELISA established with the purified protein shows good specificity for diagnosis of AIH.     

类风湿关节炎相关肺间质病变生物标志物

肺间质病变(ILD)是类风湿关节炎(RA)最常见的关节外表现,死亡率和发病率较高.目前,RA-ILD的诊断基于患者临床表现和影像学检查,但缺乏有效的生物标志物.本文对近年来RA-ILD生物标志物的研究进行综述,寻找与RA-ILD早期诊断,预后评估以及致病机制相关的基因和自身抗体,为临床上诊断RA-ILD提供新思路.