Exprimental Study On Hepatoma Associated Antigen HAb18G/CD147 Expression On Tumor Cells And Stromal Cells And Anti-hepatoma Metastasis Of Antagonistic Peptides Targeting HAb18G

Aims: Primary hepatocellular carcinoma is a kind of malignant disease that seriously threatens mankind's health. Dissemination in liver and distant metastasis are easily happened, so long-term curative effect of primary hepatocellular carcinoma is worse. Therefore, it is necessary to find an effective therapy method to inhibit hepatocellular carcinoma metastasis. HAbl8G, a hepatoma associated antigen, is a heavily glycosylated membrane protein. The cDNA sequence of HAbl8G from the cDNA library of hepatoma has been cloned by using the monoclonal antibody HAblS prepared by our lab. The open reading frame sequence of HAbl8G is identical to that of CD 147 molecule, a member of immunuglobulin super family (IgSF). CD 147 was also named as extracellular matrix metalloproteinase inducer (EMMPRIN), Basigin and Nureothelin and it was expressed in different tissues or cells. HAbl8G stimulates fibroblast to produce Matrix Metalloproteinase (MMPs), which mayplay an importmant role in tumor invasion and metastasis. Antagonistic peptides of HAbl8G were obtained by panning a phage displayed randoml2-mer peptide library. Therefore, the first purpose of the study is to investigate the expression of HAbl8G on tumor cells and stromal cells to provide some evidences for learning the relationship between HAbl8G and tumor high metastasis and high recurrence, and further studying the functions of HAbl8G. The second purpose of the study is to identify the sequence similarities of HAbl8G/CD147 antagonistic peptides (APs) with those of known proteins by bioinformatics methods to provide some cues for deeply studying the functions of APs. The third purpose of the study is to detect the affinities of APs with HAbl8G on hepatocellular carcinoma cells. The fourth purpose of the study is to determine the anti-hepatocellular carcinoma metastasis functions of APs in vitro and the last one is to study the MAPK signal transduction pathway involved in the production of the gelatinase induced by HAbl8G/CD147.Methods: the study was made of five parts.1 . Study of HAb 1 8G expression on tumor cells and stromal cellsIndirect immunofluroscence method was used to analyze the expression of HAbl8G antigen on hepatoma cell lines including HHCC, SMMC-7721 and BEL7402, colon cell line SW1 116, vascular endothelial cell line ECV304 and human embryonic lung fibroblast cell line (HELP). Immnohistochemistry method was employed to detect the expression of HAbl8G antigen on hepatoma cell lines including HHCC, SMMC7721, HCC9204, HCC9724, BEL7402 and normal liver cell line QZG. Fluorescence-activated cell sorting analysis (FACS) was adopted to detect the expression of HAbl8G antigen on hepatocellular carcinoma cell HHCC, SMMC7721, MHCC97-H and MHCC97-L. 2. Study on the characteristics of 9 high affinity peptides of HAb 1 8G/CD 1 47Integrated bioinformatics database ExPASy was used to analyze and predict APs' molecular weight (Mr), isoelectric point (pI), hydrophobicity and half-lives. BLASTP , PDB_ISL and SCOP database were employed to compare the sequence similarities of APs with those of other known proteins.3. Study on the affinities of antagonistic peptides with HAb 18G/ CD 147Antagonistic peptides AP-1, AP-2 and AP-6 were labelled with biotin at their N-terminals respectively. The affinity abilities of APs to HAblSG on HHCC or SMMC7721 cells were determined by fluorescence-activated cell sorting analysis (FACS) and laser confocal microscop. The abilities of HHCC cells adhering to the peptides immobilized on the plastic surfaces of microplates at different concentration were compared. The HHCC cells were cultured in APs-coated 96-well micro-plates (0.8 g/well, 4.0 g/well, 20.0 u g/ well and 100.0 u g/ well). After 60-min incubation at 37, the attached cells were stained with crystal violet and determined by absorbance at 570 nm.4. Study on the inhibitory effects of HAbl8G/CD147 antagonistic peptides on invasion and metastasis of hepatocellular carcinoma in vitroThe anti-metastasis effects of 9 high affinity pep...