Experimental Study Onγ-Glutamyltransferase Isoenzymes,Sugar Chain Structured And Expression Of Genes In Human Hepatic Cancer

Objectives: (1) To estimate the diagnostic value of GOT activity and isoenzyme for primary hepatocellular carcinoma. (2) To study the relationship between sugar chains of GGT and primary hepatocellular carcinoma by comparing the sugar chains of sera GGT from nonnal individuals and from patients with primary hepatocellular carcinoma or with non-malignant liver diseases. (3) To make a further study on the relationship between sugar chains of GOT and primary hepatocellular carcinoma by comparing the sugar chains of human GOT extracted from normal liver tissue and from primary hepatocellular carcinomas, and GGT from liver tissues surrounding the hepatoma foci. (4) To testify if the GOT amino acid sequences in human GOT isoenzymes exist difference and to estimate the relationship between expression of GOT genes and primary hepatocellular carcinoma by analyzing the expression of all the seven human GOT genes in hepatoma tissues and hepatoma cell lines. Methods: (1) GOT activity was determined by kinetic methods in sera samples of patients with primary hepatocellular carcinoma or non-malignant liver diseases, and of healthy individuals. (2) GOT isoenzymes were separated by vertical slab electrophoresis on non-gradient polyacrylamide gel in sera samples of patients with primary hepatocellular carcinoma, or non-malignant liver diseases, and of healthy individuals. (3) Sera samples from patients with primary hepatocellular carcinoma, non-malignant liver diseases, and from healthy individuals were subjected to wheat germ agglutinin (WOA), Lens culinaris agglutinin (LCA), Concanavalin A (ConA) and Datura stramonium agglutinin (DSA) immobilized lectin affinity chromatography, the enzyme -5- activity specific for GOT was determined in the collected fractions. (4) GGT extracts were prepared from normal human liver, human hepatoma tissues and liver tissues surrounding hepatoma foci, and then were subjected to WGA, LCA, ConA and DSA immobilized affinity chromatography, followed which the GOT activity in collected fractions was determined. (5) Total RNA was extracted from human hepatoma tissues and cultured human hepatoma cell lines (HepG2, SMMC 7721), which was then subjected to reverse transcription-polymerase chain reaction (RT-PCR). After purified by agarose gel , these amplification products were then blotted and hybridized to seven oligonucleotide probes unique for seven GOT genes. Results: (1) The GOT activity was bellow 30U/L in normal human sera, but was over 30U/L in most sera samples of patients suffering from hepatobiliary diseases. The GOT decree of increasing in primary hepatocellular carcinoma was similar to that in acute hepatitis, and was lower than that in cholecystolithiasis. (2)The positive rate of serum GGT isoenzyme II (00Th) in patients with primary hepa- tocellular carcinoma was 78.7%, higher than that in patients with cholecystolithiasis, hepatocirrhosis, and hepatitis, which were 15.4%, 9.8% and 6.4%, respectively. 00111 was not detected in healthy control group. (3) In normal human mixed sera, 61.8% and 42.9% of GOT contained core fucose (Fuc) and 揵isecting?N-acetylglucosa.mine(G1cNAc) residues, respectively. 49.0% of OGT had monoantennary or 2,2-biantennary sugar chain, which did not bind with DSA. 51% of GOT had 2,4-biantennary or 2,...