AFT024-SCF construction and functional identification / 中国病理生理杂志

ABSTRACT

AIM:To construct AFT024-SCF cell line and HPC-Lhx2 cell line for confirming the biological function of AFT024-SCF.METHODS:The HPC-Lhx2 cell line, AFT024-SCF cell line and AFT024-GFP cell line were constructed by retro-viral infection.The expression of stem cell factor(SCF) in AFT024-SCF cells was detected by real-time PCR and Western blot.SCF in the supernatant of AFT024-SCF was detected with ELISA.The supernatant of AFT024-SCF and AFT024-GFP were collected and then diluted (110) with basic IMDM medium.So we made 4 culture mediumAFT024-SCF medium was used for experiment group, AFT024-GFP medium was used for endogenous negative control, IM-DM basic medium was used for exogenous negative control, and IMDM basic medium with SCF was used for positive con-trol.SCF-dependent HPC-Lhx2 cell line was cultured in these 4 different medium for 72 h.According to MTT method and colony forming experiment, the biological function of AFT024-SCF was confirmed by the proliferation ability of SCF-depend-ent HPC-Lhx2 cell line.RESULTS:SCF was highly expressed in AFT024-SCF cells.After cultured for 72 h, neither IM-DM basic medium nor GFP-AFT024 medium support HPC-Lhx2 cell line proliferation.However, AFT024-SCF medium supported HPC-Lhx2 cell line expansion as well as the positive control medium.CONCLUSION:AFT024-SCF cells ex-press SCF successfully and recombinant SCF can be replaced by the supernatant of AFT024-SCF culture medium for expan-ding HPC-Lhx2 cell line in vitro.