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

作　　者：何明枫[1] 方印[1] 陈静[1] 董洪权[1] 金文杰[1] 

机构地区：[1]南京医科大学第一附属医院麻醉科,210029

出　　处：《中华麻醉学杂志》2017年第5期565-568,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金青年基金（81400889）

摘　　要：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

关 键 词：趋化因子CCL2 星形细胞 小神经胶质细胞 

分 类 号：R614[医药卫生—麻醉学]