A Study On The Mechanism Of B104Neuroblatoma Cells Conditioned Medium Inducing Oligodendrocyte Precursor Cells Proliferation

Objective To investigate key growth factors in B104neuroblatoma cell conditionedmedium (B104CM) which induces oligodendrocyte precursor cells (OPCs) toproliferate and explore molecular events involved in this proliferation progress.Methods (1) B104neuroblatoma cell line was cultured, and its conditioned mediumwas collected after filtration and centrifugation.(2) Embryonic (E) day16Sprague–Dawley (SD) rat spinal cords were dissected and dissociated, and OPCs wereisolated and purified by immunopanning method and cultured with oligodendrocyteprecursor cells conditioned medium.(3) Immunocytochemistry was used to labelspecific antibodies of OPCs.(4) B104CM of different concentrations (0,10,30,50and100%) was added into OPCs medium without any growth factor, and then BrdUincorporation assay combined with A2B5staining were used for observing proliferationof OPCs.(5) PDGF-AA, bFGF, and IGF-1mRNAs expression in B104neuroblatomacells were detected by reverse transcription PCR (RT-PCR), and the concentrations ofthese growth factors in B104CM was determined by enzyme linked immunosorbentassay (ELISA), and the expressions of PDGFR, FGFR2and IGFR in OPCs wereconfirmed by immunocytochemistry.(6) The specific inhibitors of growth factorreceptors were applied to observe their effects on B104CM-induced OPC proliferation..(7) Western Blot was used to detect activation (phosphorylation) of Erk1/2, PI3K, p38and JNK signal pathways which might plays a key role during B104CM induces OPCproliferation (8) IEGs and/or cyclins that involved in phosphorylation of signal pathwayin B104CM-induced OPCs proliferation was detected by RT-PCR and Real-time-PCR.Results (1) OPCs was collected efficaciously with purity of more than95%.(2) BrdUincorporation assay combined with A2B5staining showed that B104CM could induceOPCs to proliferate.(3) It was proved that B104expressed mRNA of PDGF-AA, bFGFand IGF-1, and the concentration of PDGF-AA, bFGF and IGF-1in B104CM were23.42±4.28,12.94±3.05and7.21±2.12ng/ml, respectively. Immunocytochemistrydisplayed that OPCs expressed PDGFR, FGFR and IGFR.(4) B104CM-stimulated OPC proliferation could be markedly decreased by both AG1295(an inhibitor of PDGFR)and PD173074(an inhibitor of FGFR). However, the inhibition of IGF-1R withAG1204did not affect the proliferation of OPCs.(5) Western Blot indicated B104CMinduced activation of Erk1/2and JNK in OPCs, and proliferation of OPCs induced byB104CM could be markly blocked by U0126, inhibitor of Erk1/2signal pathway, andSP600125, inhibitor of JNK.(6) RT-PCR showed B104CM could stimulatehigh-expression of IEGs c-fos, c-jun, Id-2and cyclin D1, D2, E in OPCs.High-expression of these molecules could be blocked by inhibitor of Erk1/2.(7)Real-time RT-PCR indicated that B104CM could stimulate IEGs c-jun, c-fos, c-mychigh-expressed and SMAD4, ATF-2to downregulate in OPCs. However, thesedownregulation could be reversed by inhibitor of JNK.Conclusion (1) PDGF-AA and bFGF played a key role in B104CM-induced OPCproliferation.(2) Activation of Erk1/2is a key molecular event in B104CM-inducedproliferation of OPCs, and gives rise to subsequent expression of IEGs c-fos, c-jun, Id-2and cyclins of D1, D2, E, which initiates cell cycle and thus OPC proliferation.(3)Activation of JNK is necessary for B104CM-induced proliferation of OPCs as well, andresults in subsequent expression of IEGs of c-fos, c-jun, and Id-2, which initiates cellcycle and thus OPC proliferation.