| Objective:(1) To explore if recombinant adenovirus with cardiotrophin-1(Adv-CT-1)induce the neural differentiation of human umbilical cord blood mesenchymal stemcells(hUCB-MSCs) and look for a suitable cell cytokine induce hUCB-MSCs neuraldifferentiation.(2) To evaluate if the MAPK/ERK are modulated on neural differentiationof hUCB-MSCs transfected with Adv-CT-1.Methods:(1) Isolated, cultured and identifed hUCB-MSCs: Umbilical cord blood werecollected from full term normal delivery or cesearean section delivery infants under asepticcondition. By modified density gradient centrifugation with adherent culture, mononuclearcells (MNC) were harvested from human umbilical cord blood. The morphology of MNCand their growth characteristics were observed. The surface markers CD34, CD29, CD44and CD105were detected by flow cytometry.(2) hUCB-MSCs were transfected withAdv-CT-1: hUCB-MSCs were transfected with Adv-CT-1and recombinant adenoviruswith enhanced green fluorescence protein(Adv-EGFP) at different multiplicity of infection(MOI) ranging from50,100,200to300PFU/cell respectively. The transfection efficiencywas assessed by fluorescence microscope at24h,48h,72h and96h after transfectionadv-CT-1and Adv-EGFP. Meanwhile, the best transfection efficiency of Adv-CT-1wasdetermined by cell immunofluorescence at24h,48h and72h after transfection.(3)Detected the neural markers and apoptosis related proteins: The cell morphologicalchanges were observed by microscope and the expression of Nestin, NeuN and MBP weremeasured by cellular immunofluorescence at6h and4d after induction among controlgroup, EGFP group, CT-1group and neural induction medium(NIM) group respectively;Apoptosis related proteins bcl2, Bax, Bak, caspase3and caspase9were assessed byWestern Blot between hUCB-MSCs transfected with Adv-CT-1and untransfectedhUCB-MSCs.(4) The expression of gp130, LIFRβ, p-Raf-1, ERK1/2and p-ERK1/2were evaluated at24h,48h and72h after CT-1transfection hUCB-MSCs byimmunocoprecipitation method (CO-IP) and Western Blot. The changes of phosphorylationERK, MBP, Nestin and NeuN after delivered different concentration of MEK inhibitorPD98059.Results:(1) Under inversed microscope, polygonal cells, and a large number of large,round osteoclast cells were visible after5d, After12d a large number of spindle cells incolony were observed, about28d can be passaged. The morphology of third passage cellswas uniform.The cultured hUCB-MSCs showed high expression of mesenchymal relatedantigen CD105(89.31%), CD29(98.41%), CD44(99.20%), and cells were negative forlineage markers hematopoietic stem cells CD34.(2) hUCB-MSCs transfected withAdv-CT-1: With the increasing of time and MOI, transfection rates were graduallyincreased, when MOI100PFU/cell, the transfection efficiency was much higher and cellmorphology and growth characterics were not obvious changes between transfected anduninfected cells after72h transfection(87.60±1.36%).(3) The effect role of Adv-CT-1onthe neural differentiation of hUCB-MSCs: About6h after induction, the cells treated withNIM and Adv-CT-1exhibited stronger expression neural markers Nestin than cells treatedwith NIM and without Adv CT-1or control group or EGFP group. The positive rate ofNestin was the highest(86.82±4.29%) at6h after induction in CT-1group than other threegroups, the differences were statistically significant (P <0.05); The positive rate of Nestinwas fewer at4d after induction than that of at6h after induction, which was still higherthan that of other groups. the difference was statistically significant (P <0.05). Similarly,6h after induction, the expression of NeuN in CT-1group were14.26±2.20%, which werehigher than other groups. To4d after induction, the positive of NeuN was graduallyincreasing to48.33±2.53%. Among them, the positive of NeuN in CT-1groups at6h and4d after induction were higher than other groups which the difference were statisticallysignificant (P <0.05).6h after induction, the expression of MBP in CT-1group at6h(14.12±2.16%) and4d(45.63±2.78%) after induction were higher than other groupsrespectively, which difference were statistically significant (P <0.05). Western Blotresults show that the expression of bcl2was higher than that of control group and the expression of bax, bak, caspase3and caspase9(0.01±0.00，0.09±0.01，0.30±0.01，0.32±0.02) were lower than control group (0.16±0.01，1.14±0.02，0.58±0.01，0.72±0.01)at72h after transfection Adv-CT-1, the difference was statistically significant (P <0.05).(4)The expression of CT-1receptor at different time and phosphorylation of ERK1/2, Raf-1changes: At24h after hUCB-MSCs transfected with CT-1, p-Raf-1, p-ERK1/2and gp130were slight visible, while the expression of LiFRβ was almost undectable; At48h, LiFRβhas a slight expression, p-Raf-1, p-ERK1/2and gp130expression gradually increasing, thedifferences were statistically significant compared with24h respectively (P<0.05);72hafter transfecction, the levels of gp130/LiFRβ were no longer increase respectivelycomparing with48h and the differences between48h and72h were not as clear (P>0.05);while p-ERK1/2and the p-Raf-1expression still increase, the difference was statisticallysignificant compared with48h (P<0.05). The expression of MBP, NeuN, Nestin andp-ERK were slighted reduced after added10umol/l PD98059as compared with the controlgroup(P<0.05), while there were a dramatic reduction in the expression of MBP, NeuN,Nestin and p-ERK after added100umol/l PD98059and the differences were statisticallysignificant compared with10ummol/l group (P <0.05). There was statistically significantdifference compared with control group (P <0.05).Conclusion:(1) hUCB-MSCs can be cultured and expanded in vitro.(2) Adv-CT-1can beeffectively transfected to hUCB-MSCs.(3) hUCB-MSCs transfected with CT-1can bedifferentiated to neural-like cell.(4) ERK/MAPK signaling pathways may be participatedthe neural differentiation of hUCB-MSCs. MEK inhibitor PD98059have inhibitory effectson neural differentiation of CT-1induced hUCB-MSCs... |
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