| Objective:To explore the protective roles of aFGF in the injury of gentamicin on astrocytes of hippocampus of SD rats.Method:After hippocampuses were separated, sheared and digested with trypsin, astrocytes were cultured. The cell types were identified by Rabbit Anti-GFAP immunocytochemistry, The cells with third generation were inoculated in96or6well plates:(1) in96-well plates:After24h cultivation, aFGF with different concentrations(0.85,2.55,4.25,5.95μg/L)was added into the cultures, and MTT assay was performed to test the cell viability after24h.(2) in96-well plates:After24h cultivation, GM with different concentrations (1.2,1.6,2.0,2.4,2.8,3.2,3.6,4.0g/L)was added into the cultures, and MTT assay was performed to test the cell viability after24h; After24h cultivation, aFGF with certain concentrations and GM with different concentrations were added into the cultures at the same time, and MTT assay was used to test the cell viability; And the inhibition rate and the corresponding IC50values of GM were calculated.(3) The cells were cultured for48h in a96-well culture plate and divided into groups for the experiment randomly:control group:normal culture; GM group:2g/L GM was added for24h; GM plus aFGF (Ⅰ、Ⅱ、Ⅲ、Ⅳ) intervention:after adding aFGF with different concentrations (0.85,2.55,4.25,5.95μg/L) aFGF,24h, then added GM,24h.(4) The cells were cultured for3days in a24-well culture plate and divided into3groups for the experiment:①control group:normal culture;②GM group:2g/L GM was added for24h;③GM plus aFGF intervention group(aFGF+GM):after adding4.25μg/L aFGF,24h, then added GM,24h. Each group contained6-hole complex and the experiment repeated3times.Cell morphology and the changes of MDA、NO、 SOD、GSH-Px were observed.Result:(1) Morphological observation:Control group:cell morphology was various, three-pole or multi-pole, exhibited strong refraction, processes were intertwined and formed a network; GM group:large number of cells exhibited shrinkage, fusing into a mass, the network and the process connection was destroyed,and much particles and vacuoles existed in cytoplasm, and under transmission electron microscopy, results appeared relatively typical features of apoptosis, such as cytoplasm concentration, nucleolemma introcession margination of chromatin to the nuclear membrane and mitochondria with enlarged inner space obviously; aFGF+GM group:the extent of cell shrinkage and cell fusion were relieved, the damage of process connection was not obvious, and few particles and vacuoles existed in cytoplasm, and under transmission electron microscopy, results appeared essentially normal density of cytoplasm, nucleolemma introcession, margination of chromatin to the nuclear membrane and mitochondria with enlarged inner space less obviously.(2) aFGF with concentrations followed by0.85μg/L,2.55μg/L,4.25μg/L,5.95μg/L could promote the growth and generation of astrocytes.(3) The concentrations of GM that inhibited50%cell growth(IC50) increased because of the effects of aFGF.(4) After adding aFGF with a certain concentration for24h, the inhibition rate of astrocytes by GM could be reduced;(5) Biochemical and enzymatic observation:Comparing GM group with control group, SOD, GSH-Px enzyme activities decreased, while MDA, NO level increased(P<0.01) in GM group, indicating that the cultures of astrocytes and the injured models of GM were successful. The differences of biochemistry and enzymology of the culture contents between GM group and aFGF+GM group were significant (P<0.01).Conclusion:aFGF had certain proliferative effect on astrocytes. aFGF could protect the injury of astrocytes of hippocampus in some degree. The protective effect of aFGF was related to antagonize free radicals indirectly or directly, enhanced the activity of antioxidant enzymes which could scaven free radicals, and protect antioxidant enzymes. |
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