摘要
研究背景
原发性胆汁性肝硬化(PBC)是一种以多见于女性的慢性自身免疫性肝脏疾病,其发病机制至今仍没有完全阐明,PBC的血清标志物在PBC的诊断中具有无可替代的作用,但目前使用的PBC血清标志物并不理想,因此用新的研究手段进行PBC血清标志物的研究显得非常重要。
研究方法
以人类cDNA文库为模板,设计目的基因的特异引物,将PCR扩增的目的基因产物和线性化的酵母表达质粒pEGH共转染酵母Y258。利用半乳糖诱导含有目的基因的酵母克隆表达重组蛋白。通过谷胱甘肽亲和层析柱纯化含GST标签目的重组蛋白点制高通量芯片。采用人类基因组编码蛋白高通量芯片筛选原发性胆汁性肝硬化血清,寻找PBC特异性血清标志物。
研究结果
制备的高通量蛋白芯片共计38 400个蛋白点,含17 718个人类基因编码蛋白,该芯片蛋白点的检出率为97.6%,蛋白复点间检测信号强度的相关系数为0.98。该芯片在PBC组与对照组间筛选出18个具有统计学意义(P<0.01)标志物。在最好的5个标志物中,针对PDHA1、DBT和DLAT的抗体为现在已经使用的PBC标志物AMA-M2组成部分,针对HKl和ZNF364的抗体为新发现的PBC标志物,在PBC中的阳性率分别为52.38%(11／21)和38.10%(8／21),在对照组中的阳性率均为0.00%(0／30)。AMA-M2阳性与AMA—M2阴性PBC患者间没发现具有统计学意义的血清标志物。ACA阳性与ACA阴性PBC患者间仅针对CENPB的抗体具有统计学意义(P<0.01)。
研究结论
人类基因组编码蛋白高通量芯片是一种快速全面筛选PBC新标志物的技术。针对HKl和ZNF364的抗体具有较好的敏感性和特异性,可以作为PBC新标志物。AMA-M2阳性与AMA-M2阴性PBC患者间没发现具有意义的血清标志物,所谓的AMA-M2阴性患者可能是由于检测方法所致的假阴性。PBC患者的ACA以B亚型为主,而针对CENPB的抗体可以作为ACA阳性与ACA阴性PBC患者间的标志物。
研究目的
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中的阳性状况以及与其他PBC自身抗体的关系和临床意义。研究方法
采用ELISA检测198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、169例炎症性肠病(IBD)患者及167例其他疾病对照组患者血清ASCA的IgA和IgG亚型,分析该抗体与各种指标的相互关系。
研究结果
ASCA-IgA在PBC患者中的阳性率为24.2%,高于溃疡性结肠炎(ulcerative colitis,UC)组11.3%(X2=6.8,P<0.01)和其他疾病对照组组12.6%(X2=8.0,P<0.01),与AIH组(20.5%)、LDC组(14.6%)和克罗恩病(Crohn's disease,CD)组(33.9%)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13.6%,明显低于CD组27.4%(X2=6.4,P<0.05)和其他疾病对照组24.6%(X2=7.1,P<0.01),与AIH组(15.9%)、LDC组(7.3%)和UC组(7.2%)比较,差异无统计学意义(P>0.05)；ASCA-IgA或IgG阳性的PBC患者占29.8%,显著低于CD组45.2%(X2=5.0,P<0.05),高于UC组14.4%(X2=8.3,P<0.01)和其他肝病对照组9.8%(X2=7.0,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(39.7%&24.6%,X2=4.9,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TBIL、DBIL、ALP、AST、TBA、LD、IgA、IgM、ESR高于ASCA-IgA阴性PBC患者,而ALB和CHE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均无统计学差异。
研究结论
ASCA并不是炎症性肠病特异性自身抗体,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。
Screening serum markers of primary biliary cirrhosis with high-throughput protein chip encoded by the human gene
Background
Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease of unknown etiology. It predominantly affects middle-aged women. Serum autoantibodies are crucial tools for diagnosis of PBC, but currently used serum markers are not perfect. it is very important to study the serum markers of PBC with newly research methods.
Methods
Human cDNA library was used as template to designed gene-specific primers. The PCR amplification of the target gene products were co-transfected into yeast (Y258) with the linearized yeast expression vector pEGH. Galactose was use to induced yeast containing the target gene to clone and express recombinant protein. All the target recombinant proteins were tagged with GST, and could be purified by glutathione-affinity chromatography material. High-throughput protein chip was fabricated and used to screen serum markers of primary biliary cirrhosis. Results
The fabricated high-throughput protein chip contains a total of 38 400 protein spots, includingl7 718 human genes encoding proteins. The detection rate of protein spots on the chip was 97.6%, and the signal intensity correlation coefficient of double protein spots was 0.98.18 serum markers were screened between PBC group and the control group with a statistically significant (P<0.01). Of the best of five markers, antibodies to PDHAl、DBT and DLAT were the component of AMA-M2 which has been used as the marker of PBC; antibodies to HK1 and ZNF364 were identified as new marker of PBC, whose positive rates were 52.38%(11/21) and 38.10%(8/21) respectively in PBC, and 0.00%(0/30) in the control group. There is no serum marker were screened between the AMA-M2 positive and negative PBC patients. Only antibody to CENPB was identified as marker between the ACA positive and negative PBC patients (P<0.01) Conclusions
High-throughput protein chip encoded by the human gene is a technology for screening of new markers of PBC quickly and comprehensively. Antibodies to HK1 and ZNF364 can be used as a new marker for PBC with highly sensitivity and specificity. No serum marker was found between the AMA-M2 positive and negative PBC patients whereas only antibody to CENPB was identified as marker between the ACA positive and negative PBC patients.
Objective
To explore the prevalence of the anti-Saccharomyces cerevisiae antibody (ASCA) in patients with primary biliary cirrhoses and evaluate it's clinical significance.
Methods
The subtypes of ASCA including IgA and IgG in specimens from 198 patients with PBC, 44 patients with AIH,41 patients with other non-autoimmune liver diseases controls (LDC), 169 patients with inflammatory bowel disease (IBD) and 167 other diseases controls were measured by ELISA.
Results
The positive rate of ASCA-IgA in PBC is 24.2%, which is higher than in ulcerative colitis (UC) group (11.3%, x2=6.8 P<0.01) and other diseases controls (12.6%, x2=8.0, P<0.01). Compared with the AIH group (20.5%) or LDC group (14.6%) or Crohn's disease (CD) (33.9%), there is no difference statistically significant (P>0.05). The prevalence of ASCA-IgG in PBC is 13.6%, lower than the CD group (27.4%,x2=6.4, P<0.05), and other diseases controls (24.6%, x2=7.1, P<0.01). There is no statistically significant difference (P>0.05) between PBC and the AIH group (15.9%) or LDC group (7.3%) or UC group (7.2%). The prevalence of ASCA-IgA or ASCA-IgG in PBC is 29.8%, which is statistically lower than that of CD group (45.2%, x2=5.0, P<0.05), but higher than that of UC group (14.4%,x2=8.3,P<0.01) and other non-autoimmune liver diseases controls (9.8%,x2=7.0, P<0.01). ASCA was detected more frequently in PBC patients with positive anti-GP210 antibody than in anti-gp210 antibody negative PBC patients (39.7%&24.6%, x2=4.9, P<0.05). The positive rate of ASCA between AMA positive and negative patients with PBC or anti-SP100 antibodies positive and negative patients with PBC was not significant different. PBC patients with positive ASCA-IgA have higher level of TBIL, DBIL, ALP, AST, TBA, LD, IgA, IgM, ES Rand lower level of ALB, CHE than patients with negative ASCA-IgA. There is no statistically significant difference of liver injury indicators and immune function parameters between patients with positive ASCA-IgG and negative ASCA-IgG.
Conclusions
ASCA is not an IBD-specific antibody. There is a high prevalence of ASCA in patients with PBC, especially the subtype of ASCA-IgA. ASCA-IgA was found to be associated with the severity of liver damage and immune activity whereas ASCA-IgG was not found to be associated with.
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