星形胶质细胞CCL2在小胶质细胞激活中的作用：离体实验

Role of astrocyte CCL2 in microglial activation： an in vitro experiment

目的 评价星形胶质细胞趋化因子C-C型配体2（CCL2）在小胶质细胞激活中的作用。方法 出生后1～2 d的C57BL/6J小鼠，分离脑组织的星形胶质细胞和小胶质细胞。实验Ⅰ 星形胶质细胞以3×104个/孔的密度接种于6孔板中（2 ml/孔），采用随机数字分为5组（n＝3）：对照组（C组）、TNF-α组、1 μg/ml CCL2小干扰RNA（siRNA）组（CCL2-siRNA1组）、2 μg/ml CCL2-siRNA（CCL2-siRNA2组）和阴性对照siRNA组（NC-siRNA组）。C组常规培养，其它4组加入10 ng/ml TNF-α孵育15 min，PBS洗脱后培养3 h。加入TNF-α前24 h时，CCL2-siRNA1组和CCL2-siRNA2组加入CCL2-siRNA 1或2 μg/ml，NC-siRNA组加入NC-siRNA 2 μg/ml。采用ELISA法测定CCL2浓度。实验Ⅱ 将小胶质细胞以3×104个/孔的密度接种于6孔板中（2 ml/孔），采用随机数字表法分为3组（n＝3）：对照组（C组）、TNF-α组和CCL2-siRNA组。C组常规培养，TNF-α组取星形胶质细胞加入10 ng/ml TNF-ɑ孵育15 min，PBS洗脱后培养3 h，取其上清，加入小胶质细胞中孵育24 h；CCL2-siRNA组取星形胶质细胞加入2 μg/ml CCL2-siRNA孵育24 h，再加入10 ng/ml TNF-ɑ孵育15 min，PBS洗脱后培养3 h，取其上清，加入小胶质细胞中孵育24 h。采用免疫荧光法检测小胶质细胞活性，Transwell迁移实验测定小胶质细胞迁移能力。结果 实验Ⅰ 与C组比较，TNF-α组、CCL2-siRNA1组、CCL2-siRNA2组和NC-siRNA组CCL2浓度升高（P〈0.05）；与TNF-α组和NC-siRNA组比较，CCL2-siRNA1组和CCL2-siRNA2组CCL2浓度降低（P〈0.05）；TNF-α组和NC-siRNA组CCL2浓度比较差异无统计学意义（P〉0.05）。实验Ⅱ 与C组比较，TNF-α组和CCL2-siRNA组小胶质细胞活性升高，迁移能力增强（P〈0.05）；与TNF-α组比较，CCL2-siRNA组小胶质细胞活性降低，迁移能力减弱（P〈0.05）。结论星形胶质细胞CCL2参与了小胶质细胞的激活。

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 table： control group（group C）, tumor necrosis factor-alpha（TNF-α）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 routinely 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-siRNA 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 table： control 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-ɑ 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-ɑ 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 Ⅱ 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 microglial activation in an in vitro experiment.