Differential Expression Of Membrane Protein Complexes In A Neural Stem Cell Undergoing Differentiation Induced By OECs Conditioned Medium

Chapter one:OECs cultivation and supernatant on the differentiation of NSCsObjective:To study the differentiation of neural stem cells(NSCs) after neural differentiation cultured with olfactory ensheathing cells(OECs).Methods:In vitro NSCs derived from the cortex neonatal newborn mice were cultured in no-serum medium DMEM/F12with EGF(20ng/m1), bFGF2(20ng/ml) and2%B27,1%N2supplements,were passaged every5to7days,cultured2to3passages for reserve,Nestin immunostaining of NSCs.The OECs pooled tissue was digested at37℃in0.25%trypsin, OECs cultures were processed to an additional step of passaging cells from one flask to a new one. This step reduces contaminating cells because they adhered more readily to plastic than OECs. The culture was treated with cytosine arabinoside to minimize the population of fibroblasts. When the cells reached roughly80%confluence, the supernatant was removed and fresh medium (DMEM serum-free) was added. The supernatant was kept at-20℃as the conditioned medium (CM), with medium change every3to4days, nerve growth factor receptor p75(NGFR p75) immunostaining of OECs; Experimental group was supplemented with OECs conditioned medium and DMEM/F12 serum-free, control group was supplemented DMEM/F12serum-free, observed proliferation and differentiation of NSCs, neurofilament200(NF200) immunostaining of neurons, glial fibrillary acidic protein (GFAP) of glials.Results:NSCs were isolated and cultured from the cortex of neonatal KM mice. Some neuroshperes were formed and these neurospheres became active to the undifferentiation cell marker Nestin; OECs from the olfactory bulbs proliferated most on day7and these cells were immunostained NGFR p75; Control group NSCs barely attached, without proliferation and differentiation, gradually died on day12; Experimental group neuroshperes attached and differentiated on day2, appeared obviously on day7, differentiated neurons were identified.Conclusions:OECs can promote the neuronal differentiation of NSCs. Chapter two:Membrane protein complex research of C17.2neuralstem cellObjective:To study the molecular mechanisms during neural stem cells (NSCs) differentiation into neurons induced by supernatant of olfactory ensheathing cells(OECs), screen key proteins, provide experimental basis to clinical drug target for treatment of nerve injury.Methods:The OECs pooled tissue was digested at37℃in0.25%trypsin, OECs cultures were processed to an additional step of passaging cells from one flask to a new one. This step reduces contaminating cells because they adhered more readily to plastic than OECs. The culture was treated with cytosine arabinoside to minimize the population of fibroblasts. When the cells reached roughly80%confluence, the supernatant was removed and fresh medium (DMEM serum-free) was added. The supernatant was kept at-20℃as the conditioned medium (CM), with medium change every3to4days, nerve growth factor receptor p75(NGFR p75) immunostaining of OECs.C17.2neural stem cells unfreezed at37℃, and then centrifugation、disperse, cultured in H-DMEM (15%FBS), passaged every5to7days, cultured to3passages for reserve. Experimental group was supplemented with OECs conditioned medium and DMEM/F12serum-free, cultured in1/2、1and4hours, control group was supplemented DMEM serum-free. Apply ultracentrifugation extracted cellular membrane, BN-PAGE separation protein complexes, enzymolysis out-gel of differences protein point, and identified differences protein complexes by ESI-Q-TOF-MS at each time point. The data file generate by this instrument using Mascot software (www.matrixscience.com) the tandem mass spectrometry (MS) database Search (MS/MS Ion Search) function to Search.Results:OECs cultivate from newborn mice olfactory bulb, after seven days, NGFR p75dyeing see positive cells; C17.2NSCs recovery within one day after visible cell wall stick growth, cultivate4-5days,cells growth well, passaged2days later note the amount of cells, form with a spindle shape, refraction sex is strong; BN-PAGE to find out11protein differences points, each time point cut off this11differences points. ESI-Q-TOF-MS identified79difference peptides matching proteins, of which53significant difference expression, another26proteins have the same abundance expression, and in these proteins we detected5difference protein complexes expressions.Conclusions:There have difference membrane protein complexes expression at the early stage during OECs supernatant induced NSCs differentiation,we found5difference protein complexes expressions: mRNP granule complex、ATP synthase complex、eukaryotic translation initiation factor3(eIF-3) complex、BBS/CCT complex and Hsp90complex, Hsp90complexes may play a key activation, it may provide data for screening key proteins during NSCs differentiation.