Objective To investigate the primary culture and identification of human colon carcinoma-associated fibroblasts (CAFs) and normal fibroblasts (NFs),and analyze their differences in biological characteristics and protein expression patterns.Methods The primary human colon CAFs and NFs were obtained by tissue culture and digestion methods.The CAFs and NFs at third generation were identified by morphological observation and immunocytochemistry (ICC) staining.Cell counting kit-8 (CCK-8) assay was used to detect the proliferative activity and a growth curve during seven days was drawn.Osteopontin (OPN),transforming growth factor-β (TGF-β),vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2 were measured by using enzyme linked immunosorbent assay (ELISA).Results The cytoplasmic processes of CAFs were decreased,CAFs were arranged haphazardly and their shape was irregular,with conjugate nuclei or more nuclei.ICC staining revealed negative cytokeratin (CK),and positive vimentin and α-smooth muscle actin (α-SMA) in CAFs.The proliferative activity of CAFs was significantly enhanced as compared with NFs.The expression levels of OPN in the culture supernatants of CAFs and NFs were (2.106 ±0.137) and (1.499 ±0.151) μg/L (P<0.O1),those of TGF-β (331.203 ±12.203) and (315.391 ±12.998) ng/L (P<O.01),those of VEGF (17.216±0.632) and (14.887 ±0.661) ng/L (P < 0.01),and those of MMP-2 (1.830 ± 0.192) and (1.436 ± 0.102) μg/L (P < 0.01),respectively.Conclusion The purified human colon CAFs and NFs can be obtained successfully by both tissue culture and digestion methods.There are significant differences in morphological characteristics,proliferative activity and expression perterns of certain proteins between the CAFs and NFs,which may be a biological basis for human colon CAFs to play their role in promoting the development of colon cancer. Key words: Colon carcinoma; Carcinoma-associated fibroblasts ; Primary culture ; Biological characteristics