Preparation Of Monoclonal Antibodies Against Human MMP-2 And Research About Expression And Clinical Significance Of MMP-2 And P185 Protein In Ovarian Cancer By Tissue Chip Technology

Objective Prepare monoclonal and polyclonal antibodies against Matrix Metalloproteinase-2(MMP-2) and identified its biology characteristics.To detect the expression of MMP-2 and P185 in ovarian cancer tissue,to explore their relationship and the role in ovarian cancer formation,and the correlation with clinic pathologic characteristic in ovarian cancer.The study hence provide theory supportings for invasion of ovarion cancer,mechanism study of infiltration and produce new path way for nonsurgical targeted therapy.Methods MMP-2 is a small molecule compound and has immunogenicity binding with macromolecular carriers.MMP-2 were linked to the carrier protein(Bovine serumalbumin(BSA)and keyhole limpet hemocyanin (KLH))respectively with glutaraldehyde method,and immunogens and coat antigens were obtained.The successful coupling were proved by indirect ELISA and SDS-PAGE.Balb/c mice were immunized with the artificial antigen MMP-2-BSA protein. The anti-MMP-2 monoclonal antibody was obtained by using hybridoma technique.Characterization of the antibodies was performed by indirect enzyme-linked immunosorbent assay and Western blot analysis.Rabbits were immunized with the artificial antigen MMP-2-BSA protein to prepare polyclonal antibodies against MMP-2.Immunohistochemical EnVision method was used to detect the expression of MMP-2 and P185 in transplanted tumor,after building the mouse ovrian tumor model.To collect 80 cases of ovarian cancer and 30 cases of normal tissue in the Affiliated Hospital of Anhui Medical University from1999.1 to 2008.12 at the same time.To detect the expression of MMP-2 and P185 in ovarian cancer by using tissue microarray and immununohistochemistry EnVision methhod and to analyze the relationship with clinicopathologic characteristics in ovarian cancer.Results1.The artificial antigen MMP-2-BSA ,MMP-2-KLH protein and the hybridoma cell line 3G10 secreting monoclonal antibodies (McAbs) against MMP-2 were obtained. The subclass of McAbs was found to be IgG1. The titer of peritoneal exudates was l:1×106.2.The expression of MMP-2 and P185 in transplanted tumor of the nude mouse were all strong.The positive rate of MMP-2 expression in ovarian cancer tissue was73.75%(59/80) and in normal tissue was 13.3%(4/30),there is significant difference between them(P<0.01).The expressions of MMP-2 were closely correlated with histological grade, FIGO stage,lymph node (P<0.05～P<0.01),but no association was observed with the histological types(P>0.05). The positive rate of P185 expression in ovarian cancer tissue was 77.5%(62/80) and in normal tissue was 20%(6/30),there is significant difference between them(P<0.01).The expressions of P185 were closely correlated with histological grade, FIGO stage,lymph node (P<0.05～P<0.01),but no association was observed with the histological types(P>0.05). MMP-2 is positive correlated with P185 in ovarian cancer (r=0.38,P<0.01).Conclusion1.We synthetized the artificial antigen MMP-2-BSA ,MMP-2-KLH protein and prepared monoclonal antibody of MMP-2.2.The expression of MMP-2 and P185 have relationship with tumor grade,tumor stage and Lymph node metastasis,While have no relationship with histological types.The high expression of MMP-2 and P185 may play an important role in the process of carcinogenesis,differentiation and progression of ovarian cancer,these may be used to determine the biological behavior,the indicator of prognosis and the ideal targets for molecular biology treatment.3.There was significant positive correlation between MMP-2 and P185 in ovarian cancer,these may be cooperation in the process of carcinogenesis,differentiation and progression of ovarian cancer.