Effects Of Human Colon Carcinoma-associated Fibroblasts On Biological Behavior Of Colon Carcinoma LoVo Cells

Part1Primary culture and biological characteristics of human coloncarcinoma-associated fibroblastsObjective To investigate the method of primary culture and identification of humancolon carcinoma-associated fibroblasts (CAFs) and normal fibroblasts (NFs), then analysistheir differences in biological characteristics and protein expression. Methods The primaryhuman colon CAFs and NFs were obtained by tissue culture and digestion methods. Identifiedthe third generation cells by morphological observation and immunocytochemistry (ICC)staining. CCK-8assay was used to detect their proliferative activity and draw a growth curveduring seven days. Osteopontin (OPN), TGF-β, VEGF and MMP-2were measured by ELISAassay. Results The cytoplasmic processes of CAFs decrease, the shape of CAFs isirregular and arranged haphazardly, with conjugate nuclei or more nuclei, ICC showscytokeratin (CK) is negative, while vimentin and α-Smooth muscle actin (α-SMA) positive.The proliferative activity of CAFs is significantly enhanced compared with NFs. Theexpression levels of OPN in CAFs and NFs supernates are (2.106±0.137) ng/mL and (1.499±0.151) ng/mL (P＜0.01), TGF-β (331.203±12.203) pg/mL and (315.391±12.998) pg/mL(P＜0.01), VEGF (17.216±0.632) pg/mL and (14.887±0.661) pg/mL（P＜0.01）, MMP-2(1.830±0.092) ng/mL and (1.436±0.102) ng/mL (P＜0.01). Conclusion The purifiedhuman colon CAFs and NFs can be performed successfully by both tissue culture anddigestion methods. There are significant differences in morphological characteristics,proliferative activity and expression of certain protein between the CAFs and NFs, Thesedifferences may be a biological basis for human colon CAFs to play its role in promoting thedevelopment of colon cancer. Part2Effects of human colon carcinoma-associated fibroblasts on biologicalbehavior of colon carcinoma LoVo cellsObjective To investigate the effects of human colon carcinoma-associated fibroblasts(CAFs) on proliferation, adhesion, migration and invasion of colon carcinoma LoVo cells.Methods To collect conditioned medium (CM) of human colon CAFs and colon normalfibroblasts (NFs), the co-culture models between colon CAFs, NFs and LoVo cell wasestablished, LoVo cells in the blank control group was dealt with serum-free culture medium.The effects of human colon CAFs on proliferation, adhesion, migration and invasion of coloncarcinoma LoVo cells were detected by cell proliferation assay, adhesion assay, migrationassay and Transwell invasion assay. Results After LoVo cells being co-culture with colonCAFs, the absorbance (A) value of LoVo cells was (0.667±0.059) in48h and (0.709±0.030) in72h, the A value of LoVo cells adhere to Fibronectin was (0.588±0.067), the mobility rate ofcells is (35.2±8.7)%in12h and (64.6±7.1)%in24h; the number of invasive cells was (56.2±4.8), which were increased compared with those in the blank control group and NFs group (P＜0.05). Conclusion Human colon CAFs may be characteristics of promoting coloncarcinoma LoVo cells proliferation, adhesion, migration and invasion.