关键词: 棘球蚴病, 糖组学技术, 唾液酸化修饰

Abstract:

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 50 μl serum using protein G agarose purification kit. Reductive alkylation reaction was performed for 50 μg purified IgG in 25 mmol/L dithiothreitol solution and 25 mmol/L dithiothreitol solution, respectively, followed by adding 1 μl 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 50 μl 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 di-sialylation 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 < 0.000 1). 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 < 0.000 1). 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 < 0.000 1). 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 echinococcosis from hepatic alveolar echinococcosis, and liver tumor from hepatic alveolar echinococcosis.

Key words: Echinococcosis, Glycomic techniques, Sialic acid