脑缺血/再灌注损伤中产生IL-17A的神经细胞类型鉴定

Identification of neural cell-type as a source of IL-17A during cerebral ischemia/reperfusion injuries

目的在离体细胞水平鉴定脑缺血/再灌注损伤中产生白细胞介素-17A（IL-17A）的脑固有神经细胞类型。方法利用原代培养小鼠脑皮层神经元、小胶质细胞和星形胶质细胞1-4 h氧-糖剥夺/24 h复糖复氧（OGD/R）模拟细胞离体缺血/再灌注损伤模型,用免疫荧光双标、实时定量聚合酶链式反应（RT-qPCR）和蛋白免疫印迹（Western blot）等技术,确定产生IL-17A的神经细胞类型。结果三种神经细胞中,除NeuN阳性神经元外,小胶质细胞和星形胶质细胞在OGD/R处理后,均可表达IL-17A蛋白,且分别与其特定标志物Iba-1和GFAP存在共定位现象。星形胶质细胞经过1-6 hOGD/24 h R处理后,IL-17A mRNA表达水平随OGD时间延长显著增高,且在4 hOGD/R时达高峰[（2.74±2.48）,P〈0.001,每组n=5]。此外,OGD/R可促进星形胶质细胞表达IL-17A蛋白[（3.17±0.91）,P〈0.05,每组n=5]。结论脑缺血/再灌注损伤中星形胶质细胞可能是产生IL-17A的主要来源。

Objective To identify the neural cell-type as a source of IL-17 A during cerebral ischemia/reperfusion injuries in vitro. Methods Primary cultured mouse cortical neurons,microglia and astrocytes were subjected to 1 - 4 h oxygen-glucose deprivation/24 h reoxygenation（ OGD/R） simulating cell ischemia/reperfusion injury model in vitro,and then the double immunofluorescence staining with IL-17 A and their specific markers of NeuN,Iba-1 and GFAP,RT-qPCR and Western blot were performed to determine the neural cell-type of IL-17 A production. Results The primary cultured microglia and astrocytes（ but not NeuN ＋ neurons） expressed IL-17 A and respectively co-localized with their specific markers Iba-1 and GFAP after OGD/R treatment. The mRNA expression level of IL-17 A increased withOGD duration and reached the peak at 4 hOGD [（ 2. 74 ± 2. 48）,P 0. 001,n = 5 per group]in astrocytes after1 - 6 hOGD/24 h R treatment. In addition,4 hOGD/R treatment could promote IL-17 A protein expression in primary cultured astrocytes [（ 3. 17 ± 0. 91）,P〈 0. 05,n = 5 per group]. Conclusions Astrocytes is a potential source of IL-17 A production during cerebral ischemia/reperfusion injuries.