Role of astrocyte CCL2 in microglial activation: an in vitro experiment / 中华麻醉学杂志

ABSTRACT

Objective To evaluate the role of astrocyte chemokine (C-C motif) ligand 2 (CCL2) in microglial activation in an in vitro experiment.Methods Primary astrocytes and microglias were isolated from the brain tissues of C57BL/6J mice at postnatal day 1-2.The experiment was performed in two parts.Experiment Ⅰ Astrocytes were inoculated in 6-well culture plates at a density of 3 × 104 cells/well (2 ml/well) and divided into 5 groups (n=3 each) using a random number tablecontrol group (group C),tumor necrosis factor-alpha (TNF-cα) group,1 μg/ml CCL2 small interference RNA (siRNA) group (group CCL2-siRNA1),2 μg/ml CCL2-siRNA (group CCL2-siRNA2) and negative control siRNA group (group NC-siRNA).Astrocytes were cultured routiuely in group C,and 10 ng/ml TNF-α was added and astrocytes were incubated for 15 min followed by washout with phosphate buffer solution (PBS),and then astrocytes were incubated for 3 h in the other 4 groups.At 24 h before TNF-α was added,CCL2-siR-NA 1 and 2 μg/ml were added in CCL2-siRNA1 and CCL2-siRNA2 groups,respectively,and NC-siRNA 2 μg/ml was added in group NC-siRNA.The concentrations of CCL2 were determined by enzyme-linked immunosorbent assay.Experiment Ⅱ Microglias were inoculated in 6-well culture plates at a density of 3×104 cells/well (2 ml/well) and divided into 3 groups (n=3 each) using a random number tablecontrol group (group C),TNF-α group and CCL2-siRNA group.Microglias were cultured routinely in group C.In group TNF-α,10 ng/ml TNF-α was added to astrocytes which were incubated for 15 min followed by washout with PBS,astrocytes were then incubated for 3 h,and the supernatant was collected and added to microglias which were incubated for 24 h.In group CCL2-siRNA,2 μg/ml CCL2-siRNA was added to astrocytes which were incubated for 24 h,10 ng/ml TNF-α was also added to astrocytes which were incubated for 15 min followed by washout with PBS,astrocytes were then incubated for 3 h,and the supernatant was collected and added to microglias which were incubated for 24 h.The activity of microglias was measured by immunofluorescence,and the migration of microglias was evaluated by Transwell migration assay.Results Experiment Ⅰ The concentrations of CCL2 were significantly higher in TNF-α,CCL2-siRNA1,CCL2-siRNA2 and NC-siRNA groups than in group C (P＜0.05).The concentrations of CCL2 were significantly lower in CCL2-siRNA1 and CCL2-siRNA2 groups than in TNF-α and NC-siRNA groups (P＜0.05).There was no significant difference in CCL2 concentrations between group TNF-α and group NC-siRNA (P＞0.05).Experiment 1Ⅱ Compared with group C,the activity of microglias was significantly increased,and the migration of microglias was enhanced in TNF-α and CCL2-siRNA groups (P＜0.05).Compared with group TNF-α,the activity of microglias was significantly decreased,and the migration of microglias was weakened in group CCL2-siRNA (P＜0.05).Conclusion Astrocyte CCL2 is involved in mieroglial activation in an in vitro experiment.