| More than90%of primary liver cancers are HCC (hepatocellular carcinoma HCC) in China, and approximately75%to80%of worldwide HCC is associated with the liver chronic viral infection. The present studys showed that alteration of protein glycosylation plays an important role in the process of HCC, by sequentially inducing hepatitis, fibrosis, cirrhosis, and ultimatedly, HCC. Protein glycosylation is one of the most important post-translation modifications of proteins, and the result of glycosylation makes the proteins with different markers, changes the polypeptide conformation, increases the stability of the proteins, and influences the interaction of proteins and other biological macromolecules. Carbohydrates are one of four major classes of biological molecules, along with nucleic acids, lipids, and proteins, which are the continuation of the genetic information. With the development of molecular biology and cell biology, glycans are being constantly recognized to be involved in nearly every process of life and the major life activities. Due to the complex structure and diverse functions of glycans, aberrant protein glycosylation is associated with various diseases, for instance, the defects of the activeties of relevant enzymes of carbohydrate metabolism resulting in the abnormal glycosylation levels of glycoprotein lead to cell dysfunction, and even malignant development. This topic mainly study the alteration of protein glycosylation in cells, tissues, serum and saliva associated with hepatitis B, liver fibrosis, cirrhosis and HCC through glycomics technology, and characterize the differentially expressed glycans related with the development of HCC.Hear chapter1overviewed the research progress of glycomics, the alteration of protein glycosylation in liver diseases, human humoral glycoproteomics research, lectins and lectin microarray technique, and the future prospects in this research area.Chapter2studied the alteration of protein glycosylation in human hepatic stellate cells (HSCs) activated with transforming growth factor-β1. The human HSCs, LX-2were activated by TGF-β1. The lectin microarrays were used to probe the alteration of protein glycosylation in the activated HSCs compared with the quiescent HSCs. Lectin histochemistry was used to further validate the lectin binding profiles and assess the distribution of glycosidic residues in cells. As a result,14lectins (e. g. AAL, and ConA) showed increased signal while7lectins (e. g. UEA-I and GNA) showed decreased signal in the activated LX-2compared with the quiescent LX-2. Meanwhile, AAL, PHA-E and ECA staining showed moderate binding to the cytoplasma membrane in the quiescent LX-2, and the binding intensified in the same regions of the activated LX-2. In conclusion, the precision alteration of protein glycosylation related to the activation of the HSCs may provide useful information to find new molecular mechanism of HSC activation and antifibrotic therapeutic strategies.Chapter3studied the alteration of protein glycosylation in tissue with development of cirrhosis and HCC. The lectin microarrays were used to probe the alteration of protein glycosylation in cirrhosis tissue and HCC tissue compared with normal para-carcinoma tissue. Lectin histochemistry was used to further validate the lectin binding profiles and assess the distribution of glycosidic residues in liver tissues. As a result,6lectin (e. g. Jacalin and AAL) showed increasingly signals from normal tissues to cirrhosis tissues, and to HCC tissues. Besides,4lectins (e. g. SNA and NPA) showed increased signals, while7lectins showed decreased signals only in HCC tissues. Meanwhile, AAL staining showed moderate binding to the membrane of hepatocytes and to membrane and cytoplasm of sinusoidal cells in the para-cancerous tissues, and the binding gradually intensified from the same regions of cirrhosis tissues to HCC tissues; LTL staining showed moderate binding to cytoplasm of hepatocytes in the para-cancerous tissues, strong binding to cytoplasm of hepatocytes as well as moderate binding to cytoplasm of sinusoidal cells in cirrhosis tissues, and very strong binding to cytoplasm of hepatocytes as well as strong binding to cytoplasm of sinusoidal cells in HCC tissues.Chapter4studied the alteration of protein glycosylation in serum with development of hepatitis B, cirrhosis and HCC. The lectin microarrays were used to probe the alteration of protein glycosylation in serum from patients with hepatitis B, cirrhosis after hepatitis B and HCC compared serum from healthy volenteers. Lectin blot was used to further validate the lectin binding to the protein bands of pathological liver tissues. As a result,17lectins (e. g. Jacalin and MAL-Ⅱ) showed altered signal intensity in all serum groups (hepatitis B, cirrhosis after hepatitis B and HCC), while12lectins (e. g. GSL-Ⅱ) showed altered signal intensity in only one or two groups. Meanwhile, a high mannose and Manal-3Man binder GNA showed stronger binding at a band approximately50kDa (a5), a band approximately35kDa (a7), and a band approximately lOkDa (a8) for cirrhosis serum and HCC serum than normal serum, and a band approximately140kDa (b3), a band between100kDa and140kDa (b4), and a band approximately40kDa (b7) for cirrhosis tissue and HCC tissue than normal para-carcinoma tissue.Chapter5studied the the alteration of protein glycosylation in saliva according to age and sex and with development of hepatitis B, cirrhosis and HCC. The lectin microarrays were used to probe the alteration of protein glycosylation in saliva from healthy volunteers according to age and sex as well as the alteration of protein glycosylation in saliva from patients with hepatitis B, cirrhosis after hepatitis B and HCC. Here we also fabricated saliva microarrays to validate the terminal carbohydrate moieties of glycoproteins in individual saliva samples. Lectin blot was used to further validate the lectin binding to the protein bands of saliva samples. As a result,7lectins (e.g. MAL-Ⅱ and SNA) showed significant age differences in both females and males, and7lectins (e.g. WFA and STL) showed significant sex differences in children, adults and elderly people. Interestingly, we observe that saliva from elderly individuals has strongest resistance to IVA mainly by presenting more terminal a2-3/6-linked sialic acid residues that bind with the influenza viral hemagglutinations. Futhermore,12lectins,14lectins and16lectins showed altered signal intensity in males with hepatitis B, cirrhosis and HCC respectively, while14lectins,13lectins and16lectins showed altered signal intensity in females with hepatitis B, cirrhosis and HCC respectively.Chapter6analyzed the relationship among the alteration of protein glycosylation in cells, tissues, serum and saliva with the development of hepatitis B, cirrhosis and HCC. Compared the differentially expressed glycans in cells, tissue, serum and saliva related with various stages of liver diseases, we found that some of the proteins or their glycosylation closely related with liver disease stages altered in cells, tissues, serum and saliva in different ways. For instance, some of the changes in the glycosylation that can not be detected in pathological tissues can be identified in serum or saliva. Moreover, the alteration of protein glycosylation patterns in serum and in saliva is not identical. These results demonstrated that the alteration of protein glycosylation in cells and tissues can help people to understand the molecular mechanism of liver disease stage, while the alteration of protein glycosylation in serum and saliva are conducive to distinguish the different stages of the liver diseases and the early diagnosis of HCC. |
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