Effect Of Neurotrophic Growth Factor On The Differentiation Of NSCs And The Formation Of Neuronal Axon

ObjectiveTo observe the effect of NGF on the differentiation of NSCs isolated from young SD rat,and on outgrowth of axons.MethodsAll SD-rats are obtained from the Laboratory Animal Center of China Medical University.The main reagents include DMEM/F12(1:1),B27,EGF,bFGF,NGF,as well as Nestin,NSE,GFAP immunochemical reagent box.NSCs were isolated from 3-day SD rat and cultured in serum-free medium.Nestin, NSE and GFAP immunochemical staining was used to identify NSCs.NSCs were classified 2 groups during differentiation(Control group:10%FBS;NGF group: 10%FBS+NGF).After 6-day differentiation,immunocytochemical staining was used to identify the expression of neuro-specific enlase(NSE)in order to study the situation of differentiation of NSCs into neurons.The number of neurons was obtained through accounting NSE-positive cells with microscope.The number of axons is measured through a concentric circle attached to the neurons.Statistic software,SPSS,was used to analyze the figures.ResultsAll Neurospheres were Nestin-positive and could differentiate into the NSE-positive or GFAP-positive cells.The mean number of neurons in the NGF group is 12.93±2.4 and that in the control group is 5.83±1.7.the mean number of axons in the NGF group is 8.9±0.9,and 3.9±0.2 in the control group.The number of neurons and the number of axons in the NGF group were significantly more than those in the control group(P＜0.01).DiscussionNSCs were characterized by self-reproduce,diverse differentiation potential,low immunity and migration so on.It is promised to treat many nervous system diseases.It is showed by our study that the neurospheres were observed in the culture medium at the second day and its volume and the number increased continuously until the subculture.Mono-clone culture was used after the forth subculture.All of the mono-clone neurospheres were stained by Nestin immunocytochemical staining.After differentiation,the cells can be stained by NSE or GFAP.These evidences collectively show that the cultured cells are NSCs which can differentiate into glial cells or neurons and self-reproduce.The NSE-positive cells are accounted.The results show that the number of neurons in the NGF group is remarkably more than that in the control group. It proves that NGF can induce NSCs to differentiate into neurons.The experiment also examined the effect of NGF on the outgrowth of neuronal axons.The results show that the number of axons in the NGF group is apparently more than that in the control group.It demonstrates that NGF can induce the primary neurons to polarize and the growth cone to initiate,eventually increase the number of neuronal axons.Correlating the experiment with previous studies which show NGF can increase regeneration,sprouting,and branching of axons.It is predicted that the mechanism underlying the outgrowth of the growth cone and process of primary neurons is possibly similar with that underlying regeneration,sprouting and branching of axons. Even if both two mechanisms were different with each other,It is beyond doubt that they can be triggered by NGF.Some further studies should be conducted to determine how signal are delivered and what signal pathway is triggered during NGF affecting the outgrowth of axons.It will be critical for NSCs to be used in clinical treatment.ConclusionNGF can induce NSCs to differentiate into neurons,and increase the number of the axons of the neurons differentiated from NSCs.