抗膜突蛋白抗体与系统性硬化病相关肺间质病变的临床相关性研究

Objective To identify a novel auto-antibody in sera of systemic sclerosis (SSc) patients and to analyze its relevance with SSc-associated interstitial lung disease (ILD). Methods The anti-moesin antibody in the sera of 62 SSc patients, who had participated the European League Against Rheumatism's Scl eroderma Trial and Research Group (EUSTAR), were tested by enzyme linked immunosorbent assay (ELJSA). Patients were grouped by high resolution computerized tomography (HRCT) features, pulmonary function test (PFT) abnormalities, inflammatory markers and disease course. The prevalence and titer (Optical density value) of anti-moesin antibody were compared between groups with t and χ2 test. Results The titer of anti-moesin antibody was significantly higher in the SSc-ILD group than non-ILD group (0.156±0.062 vs 0.107± 0.026, P=0.005). Among SSc patients, the diagnostic sensitivity and specificity of the anti-moesin antibody for ILD was 44.0% and 91.7% respectively (Kappa=0.2, P=0.022). Anti-rnoesin antibody was more prevalent in SSc patients with HRCT features of honeycomb-like lesion, lobular septal thickening and mediastinal lymphadenopathy (P<0.05). SSc patients with deteriorated total lung volume (TLC %) had higher titer of anti-moesin antibody significantly (0.172±0.067 vs 0.133±0.039, P=0.011), as the same tendency in patients with decreased diffusing capacity of the lung for carbon monoxide (DLco% ) but without statistical significant difference (0.153±0.580 vs 0.120±0.340, P=0.089). The anti-moesin antibody was equally prevalent between abnormal ESR, C reactive protein, immunoglobulin and complements groups and their normal controls (P> 0.05). Group of patients who had SSc courses more than or less than 5 years demonstrated similar anti-moesin antibody titers (0.146±0.047 vs 0.164±0.077, P=0.272). However, patients with ILD courses less than 12 months had higher liter of the antibody than controls (0.182±0.073 vs 0.138±0.049, P=0.040). Conclusion This study suggests that the novel anti-moesin antibody has comparatively high specificity for SSc-associated ILD patients, which may contribute to further understanding the pathogenesis of ILD in SSc patients. Further investigations are deserved to evaluate the application of anti-moesin antibody in facilitating early screening and evaluation of ILD.     

原发性胆汁性肝硬化患者共刺激分子B7-H4的检测及临床意义

目的 探讨原发性胆汁性肝硬化(primary biliary cirrhosis,PBC)患者共刺激因子B7-1t4的表达及其与疾病发病机制的关系.方法 分别采用荧光实时定量PCR法(real-time PCR)、酶联免疫吸附试验(ELISA)及流式细胞术(FCM)检测65名PBC患者外周血单个核细胞(PBMC)B7-H4mRNA表达水平、血清IL-2水平以及CD4+、CD8+ T细胞亚群和T细胞表面B7-H4表达百分率,同时监测抗线粒体抗体(anti-mitochondrial antibody,AMA)及临床各项生化指标的关系.结果 (1)PBC患者PBMC B7-144 mRNA水平及T细胞表面B7-H4表达百分率显著低于非PBC肝硬化组及健康对照组(P<0.01).(2)活化72 h后各实验组及对照组IL-2含量及CD4+、CD8+、CD4+ CD8+T淋巴细胞表达水平均低于活化前,以非PBC肝硬化组与健康对照组降低显著(P<0.05);PBC组IL-2含量及CD4+、CD4+ CD8+ T淋巴细胞表达水平高于非PBC肝硬化组与健康对照组(P<0.01).(3)AMA-M2阳性患者血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)及γ-谷氨酰转肽酶(GGT)水平升高,其中ALP及GGT升高显著(P<0.05);AMA-M2阳性患者与阴性患者T细胞表面B7-H4表达百分率差异无统计学意义(P>0.05).结论 共刺激因子B7-H4对PBC患者体内T细胞活化增殖及细胞因子分泌的抑制作用减弱,为研究PBC的发生发展和阐明PBC的发病机制提供了试验资料.     

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.     

系统性红斑狼疮患者外周血单个核细胞中MBD4 mRNA表达水平的研究

目的探讨系统性红斑狼疮（SEE）患者外周血单个核细胞中MBD4 mRNA表达及其与疾病发病机制、疾病活动性的关系。方法以pactin为内参照,建立逆转录实时荧光定量聚合酶链反应（FQ-RT-PCR）方法,在LightCycler RealtimePCR检测仪上检测70例SLE患者（活动期32例、缓解期38例）、其他结缔组织病（CTD）患者30例（疾病对照组）、健康体检者30例（正常对照组）外周血单个核细胞（PBMCs）中MBD4 mRNA的表达水平和含量。以△Ct=Ct（待测基因）-Ct（内参基因）来比较基因表达水平的高低。结果SLE患者PBMC中MBD4mRNA表达水平高于正常对照组与疾病对照组（P〈0．05）,且疾病不同病期（活动期与缓解期）MBD4 mRNA表达水平差别有统计学意义（P〈0．01）。结论结果显示SLE患者PBMC中MBD4 mRNA表达水平显著高于正常人、疾病对照组,提示MBD4 的表达与SLE的发生发展存在一定关联性,可能参与了SEE的发病过程并与疾病的活动性有关。     

溃疡性结肠炎患者外周血单个核细胞中TREM-lmRNA 的表达水平

目的 通过比较髓系细胞触发受体-1(TREM-1)mRNA在活动期和缓解期溃疡性结肠炎(UC)患者以及健康体检者外周血单个核细胞(PBMC)中的表达水平,探讨TREM-1与UC疾病活动性的关系.方法 依据结肠镜检和病理活检结果,将UC患者分为活动期和缓解期2组,采用实时荧光定量逆转录(FQ-RT)-PCR检测70例UC患者(活动期患者38例,缓解期患者32例)和20名健康体检者PBMC中TREM-1 mRNA的表达水平,采用相对定量ACt值比较基因表达水平的高低,并比较TREM-1 mRNA表达水平与ESR和C反应蛋白(CRP)的相关性.结果 UC活动期患者PBMC中TREM-1 mRNA的表达水平(4.19±1.86)显著高于uC缓解期患者(5.29±1.71,P=0.007)和健康体检者(5.19±1.04,P=0.032),而UC缓解期患者与健康对照组差异无统计学意义(P=0.892);TREM-1 mRNA表达的受试者操作特性曲线(ROC)下面积为0.763(P<0.001),表明TREM-1 mRNA表达水平对UC疾病活动性具有诊断价值;当△Ct值为4.63时,是判断UC活动性的最佳临界点,其诊断的敏感度和特异度分别为73.7%和68.7%;UC活动期患者中TREM-1 mRNA的表达与ESR显著相关(r=0.582,P=0.03),而与血清中CRP水平不相关(r=0.447,P=0.055).结论 TREM-1mRNA的表达与UC的活动程度明显相关,TREM-1可能参与了UC的炎症进程.TREM-1 mRNA的表达水平可能与ESR相关,而与血清中CRP水平不相关.     

原发性胆汁性肝硬化患者血清自身免疫性肝病相关自身抗体谱的检测及临床意义

目的 通过检测PBC患者血清自身免疫性肝病相关自身抗体,探讨这些抗体在PBC患者中的阳性状况及临床意义.方法 采用IIF法检测247例肝病患者(173例PBC、37例AIH和37例LDC)血清AMA,采用ELISA检测血清自身免疫性肝病相关自身抗体(AMA-M2、抗GP210抗体、抗SP100抗体、抗SLA抗体、抗LC1抗体、抗LKM-1抗体).结果 AMA、AMA-M2、抗GP210抗体、抗SP100抗体、抗LC1抗体、抗SLA抗体和抗LKM-1抗体在173例PBC组的阳性率分别为92.5%(160/173)、86.7%(150/173)、35.8%(62/173)、24.3%(42/173)、0.6%(1/173)、O%(0/173)、0.6%(1/173),在37例AIH组的阳性率分别为18.9%(7/37)、5.4%(2/37)、8.1%(3/37)、13.5%(5/37)、0%(0/37)、5.4%(2/37)、2.7%(1/37),在37例LDC组的阳性率分别为5.4%(2/37)、2.7%(1/37)、5.4%(2/37)、10.8%(4/37)、0%(0/37)、0%(0/37)、0%(0/37).AMA、AMA-M2和抗GP210抗体在PBC患者的阳性率显著高于AIH患者x~2值分别为101.3,100.8和11.0,P均<0.01).而抗SLA抗体在AIH患者阳性率高于PBC患者(x~2=9.4,P<0.01).抗GP210抗体阳性PBC患者ALT、TBIL、DBIL、0GT和ALP水平显著高于阴性患者(U值分别为1212.0、1199.0、1218.0、1074.0、1030.0,P均<0.01).AMA阳性PBC患者IgM水平显著高于阴性患者(U=94.0,P<0.05).结论 抗LC1抗体、抗SIA抗体和抗LkM-1抗体在PBC和AIH中阳性率较低,筛查这3种抗体的临床意义可能不大.抗GP210抗体与PBC患者的肝功能损伤有关,AMA与PBC患者的免疫功能相关,临床筛查抗GP210抗体和AMA对PBC诊断有重要意义.     

IRF7/KIAA154和STAT4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 分析IRF7/KIAA1542(rs4963128,rs2246614)和STAT4 (rs7574865)基因多态位点与中国汉族人群SLE易感性的关系,探讨这些基因多态性与SLE患者狼疮肾炎和自身抗体产生及各种临床症状的相关性.方法 采用MALDI-TOF-MS基因分型法对748例SLE患者(SLE组)和750名健康人(健康对照组)进行关联分析.同时采用IIF法检测抗dsDNA抗体,采用免疫双扩散法检测抗SSA抗体、抗SSB抗体、抗Sm抗体和抗RNP抗体,并分析这些抗体与SLE的关系.结果 在健康对照组中rs574865(STAT4)T/T、T/G、G/G基因型频率和T、G等位基因频率分别为9.4%、45.6%、45.0%、32.2%、67.8%,SLE组为17.0%、48.1%、34.9%,41.0%59.0%,两组基因型频率和等位基因频率比较,差异有统计学意义(x2=26.30,P<0.01).另外,与健康对照组比较,SLE组中T/T基因型频率和T等位基因频率明显升高,而且在3个遗传模型(加性模型、显性模型、隐性模型)下基因型频率差异都有统计学意义(P均<0.01).rs4963128和rs2246614(IRF7/KIAA1542)2个多态位点的基因型频率和等位基因频率在SLE组和健康对照组中的差异均无统计学意义(x2值分别为4.49、5.32,P均>0.05),但rs2246614位点基因型频率在隐性遗传模型下的差异具有统计学意义(P<0.05),而rs4963128位点基因型频率在3个遗传模型下的差异均无统计学意义(P均>0.05).临床症状分析结果显示,rs4963128(IRF7/KIAA1542)在狼疮肾炎组(OR=2.69,95% CI=1.89～3.82,P<0.01)、抗SSA抗体组(OR=0.61,95%C/=0.43～0.87,P<0.05)和抗SSB抗体组(OR=0.36,95% CI=0.23～0.56,P<0.01)的差异都有统计学意义.同时,rs2246614在关节症状阳性组与阴性组中的差异有统计学意义(OR=1.34,95% CI:1.06～1.69,P<0.05).结论 rs7574865(STAT4)与中国汉族人群SLE易感性相关.rs4963125,rs2246614(IRF7/KIAA1542)虽然与SLE易感性无明确相关性,但却与SLE患者的多种临床症状(如狼疮肾炎、关节症状)和抗SSA抗体、抗SSB抗体的产生有相关性.     

原发性胆汁性肝硬化患者外周血单个核细胞中糖皮质激素诱导的肿瘤坏死因子受体mRNA表达水平的研究

目的 探讨原发性胆汁性肝硬化(PBC)患者外周血单个核细胞(PBMCs)中肿瘤坏死因子受体(GITR)mRNA表达及其与疾病发病机制、疾病活动性的关系.方法 采用反转录实时荧光定量聚合酶链反应(FQ-RT-PCR)的方法 ,在Light Cycler Real time PCR检测仪上检测78例PBC患者(活动期44例、缓解期34例)、肝癌患者30例(疾病对照组)、健康体检者30名(健康对照组)PBMCs中GITR mRNA的表达水平和含量.以△Ct=Ct(待测基因)-Ct(内参基因)来比较基因表达水平的高低.结果 活动期PBC患者GITR mRNA表达水平(△Ct=10.5±2.8)明显低于缓解期PBC患者(△Ct=7.2±2.3,P<0.01);活动期PBC患者GITR mRNA表达水平(△Ct=10.5±2.8)明显低于健康对照组(△Ct=7.4±1.1)与肝癌组(△Ct=5.9±1.3,P<0.01);缓解期PBC患者GITR mRNA表达水平与健康对照组差异无统计学意义(P>0.05);肝癌患者GITRmRNA表达水平明显高于健康对照组及PBC患者(P<0.01).结论 GITR的表达与PBC的发生发展存在定关联性,可能参与了PBC的发病过程并与疾病的活动性有关.     

检验仪器自动报警通知系统应用效果评价

目的评价检验仪器自动报警通知系统在临床检验工作中的应用效果。方法建立检验仪器自动报警通知系统,当仪器出现报警信息时可实时通过短信和微信将报警信息定向传输给指定操作人员,提醒相关人员及时处理。以监控7台仪器7 d时间进行对比分析,比较该系统使用前后检验标本完成时间及操作人员的满意度。结果未使用报警通知系统时,36次需处理的报警信息每次使仪器待机,较使用该系统延迟(7.39±3.84)min,差异有统计学意义(P<0.05),平均每天延迟38 min;对于“检测完成”提醒信息中共20次需要立即准备下一批次实验,较使用该系统延迟(8.95±5.85)min,差异有统计学意义(P<0.05),平均每天延迟25 min。未使用该系统会造成发现仪器待机不及时和实验中断时间过长而导致实验失败和试剂浪费。结论检验仪器自动报警通知系统可显著节约人力配置,提高工作效率,避免试剂浪费,亦可用于评价仪器故障率,为实验室仪器智能化集中管理和仪器性能评价提供依据。     

抗线粒体抗体阳性患者的AMA-M2亚型检测及ANA核型分析

目的了解不同滴度抗线粒体抗体(AMA)阳性患者AMA M2亚型抗体(AMA-M2)浓度分布状况及在原发性胆汁性肝硬化(PBC)患者中的阳性率及相伴ANA核型。方法采用间接免疫荧光法检测自身抗体,采用酶联免疫吸附试验检测502例AMA阳性标本中的AMA-M2,并对已明确诊断的71例PBC患者进行AMA-M2亚型及ANA核型分析。结果在502例AMA阳性患者中AMA-M2阳性率为76.1%;71例PBC中95.8%AMA抗体效价大于1:160、90.1%AMA抗体效价大于1:320、73.2%AMA抗体效价大于1:640。95.8%AMA-M2阳性:67.6%M2200 RU/ML、84.5%M2100 RU/ML,并有56.3%存在ANA。结论 PBC中AMA、AMA-M2呈高滴度、高浓度集中,高效价AMA、AMA-M2及ANA的部分核型有助于PBC的诊断。