| Background:Chronic hepatitis B is a common liver inflammatory disease that induced by Hepatitis B Virus?HBV?.In China,chronic hepatitis,especially the HBV-induced chronic hepatitis?HB?is highly popular,nearly 2%10%of the patients with HB could transfer to Hepatic Cirrhosis?HC?.Currently,due to the technological limits,some deficiencies like low specificity,inconvenient sample collection and late diagnosis are recpectively included in the diagnosis methods towards HC.Our laboratory has analyzed 491 HBV-induced HB,HC,HCC?Hepatocellular Carcinoma?and 156 Healthy Volunteer?HV?salivary samples during early researches using lectin microarrays and the results showed that the glycopatterns which specifically recognized by 14 lectins?such as MAL-II,UEA-I and DSA?altered significantly during the development of liver diseases,and we designed a small-scale?14 lectins?lectin microarrays and constructed mathematic formula for the detection of HBV-induced liver diseases.In this study,firstly,we evaluated the accuracy of the constructed mathematic formula using small-scale lectin microarrays through double-blind test,then,we verified the alteration of salivary glycan structures using saliva microarrays and DSA lectin blotting,finally,we further analyzed the?-D-GlcNAc glycosylated N-/O-glycans which recognized by DSA using MALDI-TOF/TOF-MS.Method:Firstly,104 salivary samples were double-blind tested by small-scale lectin microarrays to evaluate the constructed HC/HCC mathematic formulas,then,the high expression level of DSA-recognized salivary glycan structures in HC saliva were validated using saliva microarrays,salivary glycoproteins were isolated from HV,HB,HC and HCC patients using DSA-magnetic particle compounds and further verified by SDS-PAGE silver staining and lectin blotting,finally,N-/O-glycans were released using enzyme digestion of PNGase-F and oxidation of NaClO and further analyzed by MALDI-TOF/TOF-MS.Results:Based on the results of 104 salivary samples double-blind test,we found that 48cases of 52 HC were correctly identified by Model HC with a positive rate of 92.3%?48/52?,9 cases of 50 HCC and 2 cases of 2 HB were incorrectly identified as HC with a false-positive rate of 21.2%?11/52?,so the ultimate accuracy for Model HC was 85.6%?89/104?;41 cases of 50 HCC were correctly identified by Model HCC with a positive rate of 82.0%?41/50?,4 cases of 52 HC were incorrectly identified as HCC with a false-positive rate of7.41%?4/54?,so the ultimate accuracy for Model HCC was 87.5%?91/104?;the normalized fluorescent intensity value of HV,HB,HC and HCC in saliva microarrays were analyzed by ordinary one-way ANOVA analysis,we found that DSA-recognized glycopatterns were significantly highly expressed in HB?p=0.0021?,HC?p<0.0001?and HCC?p=0.0320?compared with HV,It was noteworthy that the expression level of DSA-recognized glycopatterns in HC were highest compared with HV?p<0.0001?,HB?p=0.0216?and HCC?p=0.0013?;SDS-PAGE silver staining and lectin blotting validated that DSA-recognized glycan structures were highly expressed in HC;MALDI-TOF/TOF-MS showed that there were total 61/46 and respectively 24/24,16/26,36/25 and 27/22 N-/O-glycans in HV,HB,HC and HCC,what's more,there were 9/7,7/8,15/6,13/8 DSA-recognized N-/O-glycans in each group and 9 N-glycans were only found in HC?m/z 1752.618?Fuc?2?Gal?1?GlcNAc?1?Man?1+?Man?3?GlcNAc?2,m/z 2006.732?Gal?3?GlcNAc?3+?Man?3?GlcNAc?2,m/z 2136.795?Fuc?2?GalNAc?1?Gal?2?GlcNAc?2+?Man?3?GlcNAc?2,m/z2371.864?Gal?4?GlcNAc?4+?Man?3?GlcNAc?2,m/z 2402.858?NeuAc?1?Fuc?1?Gal?2?GlcNAc?2?Man?2+?Man?3?GlcNAc?2,m/z 2424.840?Fuc?1?NeuAc?1?Gal?2?GlcNAc?2?Man?2+?Man?3?GlcNAc?2,m/z 2574.943?Gal?4?GlcNAc?5+?Man?3?GlcNAc?2,m/z2705.006?Fuc?2?Gal?3?GalNAc?1?GlcNAc?4+?Man?3?GlcNAc?2,m/z 2993.052?Fuc?1?NeuAc?1?Gal?5?GlcNAc?4+?Man?3?GlcNAc?2?.In conclusion,with the development of liver diseases,salivary proteins were highly?-D-GlcNAc glycosylated in HC,and these findings could become the potential methods for HC screening,early diagnosis,risk assessment,drug selection and/or efficacy assessment. |
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