Study on primary culture and secreted proteoglycan profiles of hCAFs

Objective: To study the primary culture of human hepatocellular carcinoma associated fibroblasts(hCAFs) and their secreted proteoglycan profiles.Methods: The primary culture of hCAFs and normal fibroblasts (NFs) was performed by tissue block adhesion method and enzyme digestion combined with tissue block adhesion method and the effect of the two methods on the number of days of cell emergence around the tissue was observed.Fibroblasts were purified by differential digestion and repeated adherence.The fibroblasts were identified by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR), western blotting and indirect immunofluorescence combined with morphological observation.The secreted proteoglycan profiles of hCAFs and NFs were detected by lectin microarray.Results: The number of days of cell emergence was reduced by enzyme digestion combined with tissue block adherence method compared with tissue block adherence method (P＜0.01).After purification, hCAFs had larger cell bodies and more cell processes, while NFs had smaller cell bodies and fewer cell processes.The protein and mRNA expressions of FSP, FAP, α-SMA and Vimentin were found in both types of fibroblasts, and those in hCAFs were higher(P＜0.05).The affinity of secreted proteins of hCAFs to five lectins increased, including CALSEPA, JAC, MPL, SSA and STL, while the affinity of secreted proteins of HCAFs to IRA, IAA, PSA, CSA and GSL-IA4 decreased.Conclusion: Enzyme digestion combined with tissue block adhesion method can accelerate cell emergence, and is more suitable for primary culture to form fiber cells.By comparing the secreted proteoglycan profiles of hCAFs and NFs, it shows that T/Tn antigen, sialic acid and N-acetylglucosamine structure increase in the secreted proteins of hCAFs while the structure of N-acetylgalactosamine and α-mannose decrease.