The Influence Of AFGF To The Apoptosis Of Rats’ Hippocampal Astrocytes Induced By Gentamicin

Objective:To establish the cellular model of hippocampus astrocytes (AS) in vitro and explore the protective roles of acidic Fibroblast Growth Factor(aFGF) in the apoptosis of astrocytes induced by gentamicin(GM).Method:After hippocampal astrocytes of SD newborn rats were isolated, cultivation and purified,The third generation of astrocytes were identified by Rabbit Anti-GFAP immunofluorescence, The purity of astrocyte achieved to 95% was considered to be used in the study. The cells were cultured in a 24-well culture plate and divided into 3 groups for the experiment:① control group: normal culture;② injury group (GM group):2g/L GM was added for 24h;③ GM plus aFGF intervention group(aFGF+GM):after adding 4.25μg/L aFGF,24h, then added GM,24h.Using TUNEL method combined with DAPI nuclear staining and Annexin V-FITC/PI staining reflect the status of the expression of apoptosis.Result:(1) Morphological observation:Under light microscope,the cell outline of control group were clear,exhibited strong refraction,stretched out long branch protrusions from their somas, appears the vital interweaving of network structure with their somases showing spindle and polygonal;,in the GM group cellular morphology have changed:some cells exhibiting shrinkage,the branch connection interrupting and even,losing the integrity of the cell structure, there were many granules and vacuoles in the cytoplasm; in the aFGF+GM group, cellular shrinkage were reduced, the somas morphological structure,cells fusion and lose were relieved, and few particles and vacuoles existed in cytoplasm. (2) in order to induce AS apoptosis,2g/L GM was added to the cultures and incubated for 24h. But in the aFGF+GM group,4.25μg/L aFGF were given 24 h ahead, AS apoptosis rate showed an decreasing trend.The TUNEL positive rate of the injury group were higher than in normal control(p<0.01);The protection group with TUNEL staining positivity was obviously less than in injury group (p<0.01); The comparition between protection group and the normal group showed no obvious difference (P>0.05); but in theAnnexin V-FITC/PI staining:The total apoptosis rate of the normal group,the injury group and the protection group, accounting for 8.37%,28.66% and 25.55% respectively.The early apoptosis rate of the protection group and the injury group showed no obvious difference, but the late apoptosis rate of the protection group and the injury group showed reduced. It showed that aFGF could hinder the early apoptosis astrocytes to the further development by GM.Conclusion:(1)Astrocyte cells were cultured with "method of trypsinization + subculture of purification process "in vitro.This method belongs to the optimizated cultivation method and could provided enough astrocytes.(2) aFGFcould relieve the astrocyte apoptosis induced by GM and its mainly influence on hinder the early apoptosis astrocytes to the further development.(3) aFGF could protect the injury of astrocyts of hippocampus in some degree.