The Composition And Structure Alterations Of Glycosaminoglycan In Primary Hepatic Carcinoma

Glycosaminoglycans (GAGs) are a kind of important biological macromolecules found mainly in extracellular matrices and cell membrane. Great differences in glycosaminoglycans have been found in a variety of neoplasms as compared with the normal ones in terms of content, composition, molecular size of sugar chain and disaccharide composition, and parts of the differences can be regarded as prognostic indices in many cancers. But whether differences exit among different histological types or different differentiation statuses of the carcinoma is still unknown. Therefore, the aim of this study is to investigate the content, relative molecular mass and disaccharides composition alterations of glycosaminoglycans in different hepatic carcinomas.Chapter one is to set up a new method, which can be used to isolate and purify trace amount of GAGs from a small amount of liver tissue. The glycosaminoglycans were extracted by trypsin and neutrase in series, and purified by dialysis, cation exchange chromatography, gel filtration chromatography and anion exchange chromatography. The method can extract about 100-200μg heparin sulfate from about 1g fresh porcine liver. The product has been examined by thin layer chromatography and cellulose acetate membrane electrophoresis, which illustrated that no protein or peptide and no other glycosaminoglycans have been found.Chapter two is to set up a microanalysis method of glycosaminoglycan. The glycosaminoglycans have been analyzed by both chemical and enzymatic method. The two methods have been analyzed and compared. The different disaccharides produced by enzymatic method have been separated by high performance liquid chromatography. The lowest analysis limits of disaccharides with and without 2-aminobenzamide (2-AB) labeled have been compared and found that less than 100pg glycosaminoglycan can be analyzed by fluorescent labeling method. The method was then used to analyze the purified porcine liver heparin sulfate which have found has a quite different disaccharides composition to porcine intestine heparin sulfate. This method is suitable for the fine structure study of glycosaminoglycans from different tissues.Chapter three is to diagnose the primary hepatic carcinoma by using histological method in accordance with the Edmondson pathological diagnosis standards. All samples have been divided into four group: Intrahepatic cholangiocarcinoma, well-differentiated hepatocellular carcinoma, moderately differentiated hepatocellular carcinoma and poorly differentiated hepatocellular carcinoma. This method made it possible for the structural comparison of GAGs from different carcinomas.Chapter four is to compare the difference of glycosaminoglycans isolated from different primary hepatic carcinomas. A stepwise increase of chondroitin sulfate, low molecular mass glycosaminoglycans proportion and non-sulfated disaccharides, and gradual decrease of heparin sulfate proportion have been found as the differentiation status of hepatocellular carcinoma become poorer. Intrahepatic cholangiocarcinoma has been found a significant increase of hyaluronic acid, which only slightly increased in hepatocellular carcinoma. The study can further conclude that the cancer can exhibit different glycosaminoglycan characteristics among its different histological type, and even in the same histological type, differences of glycosaminoglycan can still exit, and these differences are closely associated with the different differentiation status of the tumors.