The Effects Of Aβ On Astrocytes Proliferation And Activiton In Vitro

Alzheimer's disease is caused primarily by neurotoxic deposition of beta-amyloid(Aβ)within specific areas of the brain including the septum.Aβprotein derives from the hydrolysate of amyloid precursor protein(APP)and is a 40-42 amino acid peptide that principally constitutes senile plaques.In AD patient's brain,reactive astrocytes are basal components of neuritic plaques and contain Aβfragments. Astrocytes are the most abundant cells in the central nervous system(CNS)and play an important role in the maintenance of normal physiological function in the brain. The dysfunction of astrocytes may promote neurodegeneration and loss of neuronal synapses.However,the exact relationship between the activated astrocyte and AD remains unclear.It has been previously demonstrated that oligomeric Aβ_1-42is more toxic than the fibrillar assembly.Therefore,this research investigated the effects of Aβon astrocytes proliferation and activation in vitro.It can provide a theoretical basis for the process of AD and a better understanding of pathogenesis of AD in vivo.Primary astrocyte cultures were prepared from 1 day neonatal ICR mouse.The brain cortical tissue was mechanically separated,and astrocytes were further isolated and obtained.After 3-5 weeks in culture,the primary cells matured.The morphology of passaged cells changed from protoplasmic to fibrous astrocyte.The purity of primary cell is more than 95%after identifying by immunofluorescence.The passaged astrocytes were more pure than primary cells.In vitro,we exposed astrocytes to 0.1μM,1μM,3μM,10μM,30μM oligomeric Aβ_1-42for 3 h,8 h,16 h,24 h,48 h respectively.We observed the morphology changes from protoplasmic to fibrous astrocyte and proliferation decline in a dose and time dependence.Acording to cell proliferation,we exposed astrocytes to different concentration of oligomeric Aβ_1-42(0.1μM,1μM,3μM,10μM,30μM)at 8 h and 48 h respectively,and detected the release of BDNF by ELISA and expression of GFAP and IL-1βby RT-PCR.The result demonstrated that release of BDNF increased at 30μM dose and 8h.While the expression of GFAP and IL-1βwas not time-dependent but concentration-dependent.At the same time,we investigated the neutralizing effect of anti-Aβantibody A8(our laboratory prepared)for Aβ_1-42.The result showed that the antibody A8 could inhibit the cytotoxicity of Aβon astrocytes,and that A8 play a protective role on astrocytes in certain period.