Primary Culture Of Human Peritoneal Mesothelial Cells And Study The Impact Peritoneal Mesothelial Cells To Human Ovarian Carcinoma Cell Line SKOV3 In Invasion Adhesion And Migration

Objective:To establish a modified enzymatic disggregation method to isolate human peritoneal mesothelial cells in order to establish invasion , adhesion and migration model of peritoneal mesothelial cells to human ovarian carcinoma cell line SKOV3 in vitro.To verify the expression of some cell factors in human peritoneal mesothelial cells . To study the impact peritoneal mesothelial cells to human ovarian carcinoma cell line SKOV3 in invasion , adhesion and migration .Methods:human greater omenta was enzymatic disggregated with 0.25%trypsin-0.02% ethylene diaminetetraacetic acid(EDTA),and cultured after isolation from red blood cells.Morphologic changes were deteted through downward microscope during cellculture.Morphologic types were observed by HE staining and the purity was calculated.The ultrastructure was observed by scanning electron microscopy(SEM).Isolated cells were characterized by immunohistochemical analysis. To verify the expression of some cell factors in human peritoneal mesothelial cells by immunohistochemical analysis . Invasion , adhesion and migration of SKOV3 to HPMC were detected by adhension assay , Transwell Chamber invision assay and migration assay.Results:cultured cells were multipolar and presented a cobblestone-like appearance when they reached confluence,with a purity 95%.SEM verified the abundant microvilli on the surface of the cells.Immunohistochemical studies showed positive staining for cytokeratin and vimentin,but negative staining for white blood cell CD45 antigen and factorⅧassociated antigen.All the characters of the isolated cells were coincided with mesothelial cells.Hydrocortisone(0.5%) and insulin(20μg/ml) had a exact effect to stimulate the growth of mesothelial cells. Expression of CXCL12,MSLN in human peritoneal mesothelial cells were verified. The test also imformed CXCR4 is not impressed in human peritoneal mesothelial cells . Adhension assay was found human peritoneal mesothelial cells promote the adhension of human ovarian carcinoma cell line SKOV3 . Invision assay and migration assay were found human peritoneal mesothelial cells promote the invasion and migration of human ovarian carcinoma cell line SKOV3. .Conclusion:Trypsin-EDTA enzymatic disaggregation method is a simple,effective and repetitive protocol for isolation of human peritoneal mesothelial cells.RPMI-1640,calf serum(25%),hydrocortisone(0.5%) and insulin(20μg/ml) are effective and cheap medium ingredients to culture human peritoneal mesothelial cells. Expression of CXCL12,MSLN in human peritoneal mesothelial cells were verified . The test also imformed CXCR4 is not expressed in human peritoneal mesothelial cells . Adhension assay was found human peritoneal mesothelial cells promote the adhension of human ovarian carcinoma cell line SKOV3 . Invision assay and migration assay were found human peritoneal mesothelial cells promote the invasion and migration of human ovarian carcinoma cell line SKOV3. .