Experimental Research On Immortalization Of Human Precartilaginous Stem Cells By Transfecting SV40Tag

【Objective】:To explore the feasibility of the cultivation of human precartilaginous stemcells(PSCs)in vitro and analyse their phenotypical properties.To study the biologicaleffect of pulsed electromagnetic fields(PEMFs)on the proliferation of Immunomagneticseparated human precartilaginous stem cells(PSCs)in vitro.To establish immortalizedhuman precartilaginous stem cells(PSCs)strain and provided stable cell resource to studythe molecular mechanism of gene targeting on differentiation of PSCs.【Method】:①The cells from the aborted fetus's metaphysis were digested by collagenase,the PSCs were isolated by magnetic-activated cell sorting(MACS),then were subculturedand amplified.Flow cytometry,immunohistochemistry,immunofluorescence and RT-PCRanalysis were performed to identify the purified PSCs.②PSCs were stimulated by PEMFsof 50 Hz frequency and 1 mT intensity,Cell proliferation was measured at different timepoints by using of MTI,and the growth curve was obtained.Flow cytometry was applied tomeasure the cellular cycle and apoptosis.③Plasmid pCMVSV40T/PUR containing simianvirus 40 large T antigen gene(SV40Tag)was transfected into human PSCs using lipofectintransfection method.Colonies were isolated by puromycin selection and expanded by manypassages.Immunohistochemistry,RT-PCR and Southern blot were used to identify thetransfected cells and to detect the expression and integration of SV40Tag in expanded celllines。【Result】:①PSCs from aborted fetus's metaphysis have been successfully cultured.There was fibroblast growth factor receptor-3(FGFR-3)on the surface of the PSCs.②Cellproliferation was promoted at 4 d and 6 d after stimulation(P＜0.05),The percentage ofcells at the S phase was increased compared with that of the control group(P＜0.01),theearly and total rates of apoptosis in experiment group were obviously decreased(P＜0.05).③The positive colonies were isolated and subcultured,named as immortalized precartilaginous stem cells(IPSCs),which was confirmed as fibroblast growth factorreceptor-3(FGFR-3)positive cells and was detected the existence of SV40Tag cDNA bySouthern blot and the expression of SV40Tag mRNA and protein by RT-PCR andImmunohistochemistry.【conclusion】:It is possible to cultivate the high density human PSCs in vitro.IPSCsstrain with SV40Tag is constructed successfully.