摘要
目的探索血清IgG唾液酸化修饰在肝棘球蚴病诊断及鉴别中的应用价值。方法采集西藏自治区、甘肃省、四川省、青海省2014—2016年的98例肝细粒棘球蚴病患者和29例肝多房棘球蚴病患者血样,广西医科大学附属医院2017年1—12月的30例肝癌住院患者血样,广西医科大学体检科22份健康体检者血样。提取各组血样的血清,每份50μl,采用基于荧光色谱的糖组学技术对血清中IgG的唾液酸化修饰进行定量分析。使用Protein G琼脂糖纯化试剂盒分离纯化血清中的IgG。取50μg纯化后的IgG样品,在25 mmol/L二硫苏糖醇溶液和50 mmol/L碘乙酰胺溶液中进行还原烷基化反应后加入1μl N‑糖苷酶缓冲液,酶解后采用石墨化碳小柱进行糖链的富集并将其冷冻干燥后溶于二甲基亚砜和冰醋酸的7∶3(v/v)混合溶液中,快速加入5 mmol/L的2‑氨基苯酰胺溶液和10 mmol/L氰基硼氢化钠溶液各50μl,反应2 h后,使用石墨碳小柱纯化。将标记后的糖链在Waters Acquity UPLC(H‑Class)系统上进行亲水色谱柱分离,荧光检测器设置的激发和发射波长分别为330 nm和420 nm,分析样品的单唾液酸化修饰、双唾液酸化修饰以及总唾液酸化修饰。采用Empower 3软件进行数据收集和处理,SPSS 25进行数据统计分析,数据的比较采用D’Agostino&Pearson omnibus正态分布检验(P1值)与单因素方差分析检验(P2值),两两之间的比较采用Tukey’s multiple comparisons test分析(P3值)。结果IgG糖链的色谱图共检测到21个IgG糖链峰,其中单唾液酸化修饰糖链5种,分别为FA2BG1S1(17.79 min)、A2G2S1(19.02 min)、A2BG2S1(20.02 min)、FA2G2S1(20.27 min)和FA2BG2S1(21.13 min);双唾液酸化修饰糖链4种,分别为A2G2S2(22.70 min)、A2BG2S2(23.29 min)、FA2G2S2(23.78 min)和FA2BG2S2(24.26 min)。各组血清样品各类唾液酸化修饰的含量均具有正态分布特性(P1>0.1)。肝细粒棘球蚴病组、肝多房棘球蚴病组血清IgG单唾液酸化修饰含量分别为(14.15±2.89)%和(10.24±2.97)%,双唾液酸化修饰含量分别为(3.02±0.98)%和(1.92±0.65)%,总唾液酸化修饰含量分别为(17.25±3.69)%和(12.27±3.65)%,均低于健康对照组的(16.55±2.95)%、(3.71±1.04)%和(20.26±3.79)%(F=23.70、11.14、22.98,均P2<0.0001)。其中肝细粒棘球蚴病组与肝多房棘球蚴病组相比,单唾液酸化修饰、双唾液酸化修饰和总唾液酸化修饰含量差异有统计学意义(t=6.352、4.701、6.392,P3<0.0001)。利用受试者工作曲线(ROC)进行了IgG唾液酸化修饰潜在诊断性能的评估,肝细粒棘球蚴病组与肝多房棘球蚴病组间AUC为0.829(95%CI:0.738~0.921),灵敏度和特异性分别为82.7%和87.7%。肝癌组的血清IgG的单唾液酸化修饰、双唾液酸化修饰和总唾液酸化修饰含量分别为(15.09±2.68)%、(3.28±1.30)%和(18.37±3.69)%,与肝多房棘球蚴病组的差异有统计学意义(t=6.587、4.318、6.372,P3<0.0001),肝多房棘球蚴病组和肝癌组间IgG总唾液酸化修饰ROC的AUC为0.881(95%CI:0.793~0.963),灵敏度和特异性分别为86.7%和93.1%。结论本研究发现了血清IgG唾液酸化修饰可能与肝棘球蚴病的发病机制有关,在肝棘球蚴病诊断,肝细粒棘球蚴病与肝多房棘球蚴病、肝癌与肝多房棘球蚴病的鉴别研究中具有一定的应用价值。
Objective To assess the application value of serum IgG sialylation in diagnosis and type‑differention of echinococcosis.Methods Blood samples of 98 hepatic cystic echinococcosis patients and 29 hepatic alveolar echinococcosis patients were collected from Tibet Autonomous Region,Gansu Province,Sichuan Province and Qinghai Province from 2014 to 2016,in addition,30 blood samples were from hepatocellular carcinoma patients hospitalized in the Affiliated Hospital of Guangxi Medical University during January to December 2017,and 22 from examinees of the Physical Examination Department of Guangxi Medical University collected.With the serum samples collected,sialylation modification of IgG was quantitatively analyzed using fluorescence chromatography‑based glycomic technology.IgG was isolated and purified from 50μl serum using protein G agarose purification kit.Reductive alkylation reaction was performed for 50μg purified IgG in 25 mmol/L dithiothreitol solution and 25 mmol/L dithiothreitol solution,respectively,followed by adding 1μl N‑glycosidase buffer for enzymolysis at 37℃for 18 h.Following enrichment with graphitized carbon column the glycans were freeze‑dried,and then dissolved in a mixed solution of dimethyl sulfoxide and glacial acetic acid at a proportion ratio 7∶3(v/v),in which 50μl each of 5 mmol/L 2‑aminobenzamide solution and 10 mmol/L sodium cyanoborohydride was quickly added,respectively,to carry on the labelling reaction at 65℃for 2 h,and purification of the reactants afterwards with graphitized carbon column.Upon the final step,the labelled N‑glycans were analyzed by fluorescence chromatography to define monosialylation,bisialylation and total sialylation modification using a Waters Acquity UPLC(H‑Class)system for hydrophilic chromatographic column separation,and the resultants were detected by fluorescence detector with the setting of excitation and emission wavelengths at 330 and 420 nm,respectively.Empower 3 software was used for data collection and processing,while SPSS 25 for statistical analysis.Inter‑group data were compared by D’Agostino&Pearson omnibus normal distribution test(P1 value)and one‑way ANOVA test(P2 value).Pairwise comparison was performed using Tukey’s multiple comparisons test(P3 value).Results A total of 21 peaks were detected in the chromatogram of IgG glycans,including five kinds of mono‑sialylation modified glycans:FA2BG1S1(17.79 min),A2G2S1(19.02 min),A2BG2S1(20.02 min),FA2G2S1(20.27 min)and FA2BG2S1(21.13 min),and four kinds of di‑sialylation modified glycans,namely A2G2S2(22.70 min),A2BG2S2(23.29 min),FA2G2S2(23.78 min)and FA2BG2S2(24.26 min).All kinds of sialylation modifications in serum samples of each group had Gaussian distribution characteristics(P1>0.1).The content of mono‑sialylation was(14.15±2.89)%and(10.24±2.97)%,and the content of disialylation was(3.02±0.98)%and(1.92±0.65)%,respectively,in hepatic cystic echinococcosis and hepatic alveolar echinococcosis groups.The content of total sialylation in hepatic cystic echinococcosis and hepatic alveolar echinococcosis groups was(17.25±3.69)%and(12.27±3.65)%,respectively.The above sialylation of IgG glycan from hepatic cystic echinococcosis and hepatic alveolar echinococcosis group were lower than those of the healthy control group with(16.55±2.95)%,(3.71±1.04)%and(20.26±3.79)%(F=23.70,11.14,22.98,P2<0.0001).There was a statistically significant difference in the content of the mono‑sialylation,di‑sialylation and total sialylation between the hepatic cystic echinococcosis group and the hepatic alveolar echinococcosis group(t=6.352,4.701,6.392,P3<0.0001).The potential diagnostic performance of the sialylation of IgG glycans was also evaluated using ROC curves.Between hepatic cystic echinococcosis and hepatic alveolar echinococcosis groups,it demonstrated an AUC value of 0.829(95%CI:0.738 to 0.921)with a sensitivity of 82.7%and a specificity of 87.7%.The mono‑sialylation,di‑sialylation and total sialylation of IgG glycans in hepatocellular carcinoma group is(15.09±2.68)%,(3.28±1.30)%and(18.37±3.69)%,respectively,which shown statistically significant difference with hepatocellular carcinoma group(t=6.587,4.318,6.372,P3<0.0001).ROC analysis showed the model has a differential diagnosis of hepatic alveolar echinococcosis group and hepatocellular carcinoma group response(AUC=0.840)with a sensitivity of 64.00%and a specificity of 100%.Conclusion The present study found that the serum IgG sialylation modification may be related to the pathogenesis of echinococcosis,and possess certain application value in diagnosis of liver echinococcosis,and in the study on differentiation of hepatic cystic echino‑coccosis from hepatic alveolar echinococcosis,and liver tumor from hepatic alveolar echinococcosis.
作者
封晓晓
白玛央金
张颋
陆豪杰
魏黎明
FENG Xiaoxiao;BAIMA Yangjin;ZHANG Ting;LU Haojie;WEI Liming(National Health Commission Key Laboratory of Echinococcosis Prevention and Control,Tibet Center for Disease Control and Prevention,Lhasa 850000,China;Institutes of Biomedical Sciences,Depart-ment of Chemistry&NHC Key Laboratory of Glycoconjugates Research,Fudan University,Shanghai 200032,China;National Institute of Parasitic Diseases,Chinese Center for Disease Control and Pre-vention(Chinese Center for Tropical Diseases Research),NHC Key Laboratory of Parasite and Vector Biology(National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention),Shanghai 200025,China)
出处
《中国寄生虫学与寄生虫病杂志》
CSCD
北大核心
2023年第4期434-439,445,共7页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家卫生健康委棘球蚴病防治研究重点实验室开放课题(2020WZK2006)。
