Study On The Effects Of Nerve Growth Factor On Astrocyte Differentiation

Astrocytes are the most abundant glia cells of the central nervous system（CNS）,they play a great role in CNS physiology and pathology.Injury to the CNS causes reactive astrogliosis leading to the formation of the glial scar.This process is important for protecting the surrounding healthy tissue from the spread of injury.One of the consequences of astrocyte activation is the production of nerve growth factor（NGF）which has traditionally been considered important for their ability to protect neurons.Recent studies have suggested that NGF may serve as a negative modulator to restrict astrocyte proliferation and limit the degree of glial scarring.According to“two glial cell lineages theory”of Raff,there are two types of astrocytes:type 1 astrocytes（T1As）and type 2astrocytes（T2As）.T2As derived from oligodendrocyte-type 2 astrocyte progenitor cell（OPCs）can be induced by basic fibroblast growth factor（bFGF）to convert to multipotent neural stem-like cells（NSLCs）,which can then generate both neurons and glials called neural stem-like cell differentiated cells（NSLDCs）.Thus our study intends to evaluate the effects of NGF on the differentiation of astrocytes.Objective:The aim of this research is to investigate the effect of NGF on the morphology and growth related proteins of two type astrocytes in vitro.And whether NGF could promote NSLCs differentiation into neurons is further studied to explore the influence of NGF on astrocyte differentiation.Methods:Two type astrocytes were purified and treated with NGF（100 ng/ml）in vitro,the morphology of which were observed after 72 h treatment.Cells were then harvested and the expression of NGF receptor p75 neurotrophine receptor(p75^NTR),glial fibrillary acidic protein（GFAP）,fasciculation and elongation protein zeta-1（FEZ1）was detected by Western blot.OPCs,T2As,NSLCs and NSLDCs were purified and cultured in vitro and each cells were divided into two groups,NGF（100 ng/ml）treatment group and non NGF group.Regulation of p75^NTR,Tropomyosin receptor kinase A（TrkA）and induction of protein protein kinase B（PKB or Akt）and extracellular regulated protein kinase 1/2（Erk1/2）was detected by Western blot after 72 h of treatment.NSLDCs antigen phenotype was identified after 3 days of NGF（100 ng/ml）treatment by detecting choline acetyl transferase（ChAT）,FEZ1 and growth associated protein 43（GAP43）expression using Immunofluorescence,ChAT was also detected by Western blot.Results:（1）The cell body became small and no branch processes were observed on T1As after NGF treated,and also the cell number was less than control group.The expression of p75^NTR was up-regulated while,GFAP and FEZ1was down-regulated when T1As were under NGF treatment.The processes of T2As increased and prolonged,FEZ1upregulated while p75^NTR and GFAP had no change after NGF treatment.（2）TrkA began to express after T2As dedifferentiated.After treated with NGF,the expression of Trk A on NSLDCs was significantly up-regulated,and Akt and Erk1/2 were phosphorylated.（4）The ChAT positive rate and expression level of NSLDCs was increased after NGF treatment,and the cells were GAP43 and FEZ1 positive.Conclusions:Our results indicate that NGF appear to bind on p75^NTR expressed on the membrance of T1As and inhibit T1As growth.NGF show more predominant binding to TrkA rather than p75^NTR expressed on NSLCs and lead to TrkA downsteam targets Akt and Erk1/2 phosphorylation,the consequence of which is cholinergic neuron differentiation of NSLCs.NSLDCs also labelled with GAP43 and FEZ1 indicated that these cells may participate in nerve regeneration and repair.