| Background and Objective:Chronic hepatitis B is caused by hepatitis B virus (HBV), which not only have the inflammatory lesions mainly in liver,but also is an multiple organ damaged infectious disease.30%of these patients can converted to cirrhosis or liver cancer, so, Chronic hepatitis B became a serious threat to human health worldwide, which is also the most widely popular and most serious and dangerous infectious disease in our country. Until now, Traditional Chinese medicine treatment of chronic liver disease in our country has more than2,000years of history.Traditional Chinese medicine treatment of chronic hepatitis B is according to identify patterns and determine treatment and whether the syndrome of traditional chinese medicine is diagnosed correctly is the key to the effectiveness of traditional Chinese medicine. Syndromes objectif ication is always the hot point of research in the mordenization of traditional chinese medicine, some experts believe that syndromes of traditional chinese medicine in a certain level of structure or function must have their specific microscopic material basis. Syndromes objectif ication researchers begined to explore the syndromes microscopic material basis with application of systems biology technology following the overall concept of traditional Chinese medicine feature. Unitil today, there is only a few literatures about theproteomics Research of TCM syndrome., and not most of them used the latest international science quantitative proteomics analysis techniques. Here, we plan to use the experimental cytokine antibody array (AAH-CYT-G3) and itraq technology to do the proteomics research of the TCM syndromes in chronic hepatitis B. The objective of this study is to find the differential proteins between various syndromes of chronic hepatitis B, and identify the functions and pathways of these differential proteins.Method1. Use of protein chip technology---cytokine antibody array (AAH-CYT-G3), initially found the expression of differentialprotein. The microarray results were scaned by GenePix4000B scan, and the raw data was read and managed by the software GenePix Pro6.0.2. We using the relative and absolute quantification (iTRAQ-4plex) combined liquid chromatography and tandem mass spectrometry (LC-MS/MS) to do the difference proteomics comparative study of chronic hepatitis syndrome, then verified the result by westen-blot. The results were analyzed using bioinformatics analysis techniques such as Go functional classification and enrichment analysis, Pathway analysis to detect the characteristic of the differential protein expression s, functional classification and positioning.ResultCytokine antibody array result:MCP-1is the differential protein in spleen-stomach damp-heat group and normal group; IGF-I, MCSF, MCP-1, SCF, I-309are the the differential protein of liver and gallbladder damp-heat group and liver depression and spleen vacuity group; liver and gallbladder damp-heat group and group differences spleen-stomach damp-heat group protein IGF-I, SCF, MCP-1, SDF-1, there is a significant reduction compared to both MCP-1; thethe differential protein of liver and gallbladder damp-heat group and normal group in IGF-I, MCSF, MCP-1,1-309, MCP-1is significantly lower compared to both, among other groups showed no difference in protein.,Itraq:Combined LC-MS/MS (liquid chromatography and tandem mass spectrometry) and iTRAQ-4plex (based on relative and absolute quantitation of isotopically labeled) technology, we got the result below.1. Proteolytic cleavage effective, fully and uniform distribution of the protein continuously.2. Presence of proteins was identified by mass spectrometry quantification501groups,491groups can be quantified, the only peptide3920.3. There are59upregulated proteins and64down-regulated proteins in Liver depression and spleen vacuity group compared to liver and gallbladder damp-heat group;there are32upregulated proteins and188down-regulated proteins in liver depression and spleen vacuity group compared to spleen-stomach damp-heat group;, There are68up-regulated proteins and95down-regulated proteins in liver depression and spleen vacuity group compared to normal group,, up-regulated proteins189,21down-regulated proteins in spleen-stomach damp-heat group compared to liver and gallbladder damp-heat group,144up-regulated proteins in spleen-stomach damp-heat group compared to normal group,39down-regulated proteins and144up-regulated proteins in spleen-stomach damp-heat group compared to normal group,149up-regulated proteins72down-regulated proteins in. liver and gallbladder damp-heat group compared to normal group,4. By the KEGG pathway analysis, we obtained the differential pathway between each group, hsa04145(phagocytosis), hsa00480(glutathione metabolic pathway), hsa04514(cell adhesion molecule pathways)are part of the significant differential pathway.5. From the functional analysis of proteins, we found the proteins were different among groups with different functional characteristics,6. We verified three differential proteins in present study by Western blot, and we see the expression consistent trend in them.ConclusionIn TCM objectification study of chronic hepatitis B, using the new molecular biological methods such as microarray and differential proteomic method itraq is feasible, especially the latter method can analyzed the protein function and pathway characteristics between thedifferent TCM syndromes, which may become the basis to reveal themicroscopic material s of TCM syndrome... |
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