氧糖剥夺损伤诱导星形胶质细胞分泌IL-10的机制初探  

Oxygen glucose deprivation injury induces IL-10 secretion from astrocytes and its mechanism

作　　者：吕泽宇 何慕兰 石霞 张赟[1] 杨亭[1] 李廷玉[1] 陈洁[1] 

机构地区：[1]重庆医科大学附属儿童医院儿童营养研究中心儿童发育疾病研究教育部重点实验室儿童发育重大疾病国家国际科技合作基地,重庆400014

出　　处：《重庆医科大学学报》2017年第10期1227-1233,共7页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81300522;81271385)

摘　　要：目的:探讨氧糖剥夺(oxygen glucose deprivation,OGD)损伤诱导星形胶质细胞分泌白细胞介素-10(interleukin-10,IL-10)的可能机制。方法:培养并鉴定原代星形胶质细胞,体外建立OGD损伤模型,分别利用si TLR2(Toll-like receptor-2,TLR2)腺病毒、NF-κB(NF-kappa B)抑制剂PDTC干预,q RT-PCR和ELISA分别检测损伤细胞IL-10的表达水平,Western blot检测损伤细胞TLR2、磷酸化NF-κB p65(p-NF-κB p65)蛋白表达水平。免疫荧光检测OGD损伤细胞中NF-κB p65的亚细胞定位。结果:免疫荧光观察发现分离培养的原代星形胶质细胞纯度可达90%以上。经体外OGD损伤处理后,OGD损伤组TLR2、pNF-κB p65的蛋白表达水平[(1.16±0.10),(1.01±0.08)]与IL-10的m RNA及蛋白表达水平[(0.00±0.00),(513.01±34.47)]较正常对照组[(0.49±0.08),(0.23±0.09),(0.00±0.00),(381.89±22.26)]均明显升高(P=0.001,P=0.000,P=0.026,P=0.005),且p65蛋白在细胞核内表达增加。si TLR2腺病毒感染组OGD损伤的星形胶质细胞中TLR2、p-NF-κB p65的蛋白表达水平[(0.36±0.13),(0.38±0.10)]与IL-10的m RNA及蛋白表达水平[(0.00±0.00),(567.05±50.57)]较对照组[(1.33±0.42),(0.92±0.28),(0.00±0.00),(703.60±12.97)]明显降低(P=0.019,P=0.034,P=0.004,P=0.011)。与OGD处理组[(1.24±0.23),(0.00±0.00),(769.23±23.37)]相比,PDTC干预可有效降低p-NF-κB p65蛋白(0.26±0.07)与IL-10的m RNA及蛋白表达水平[(0.00±0.00),(598.92±42.73)](P=0.002,P=0.000,P=0.046),但并未影响TLR2的表达水平变化[(1.11±0.24),(1.09±0.94)](P=0.949)。结论:OGD损伤可激活TLR2/NF-κB信号通路诱导星形胶质细胞分泌IL-10。Objective ：To investigate the potential mechanism involved in intedeukin （IL-10） secretion from astrocytes when being treated with oxygen glucose deprivation（OGD）. Methods：Primary astrocytes isolated from neonatal Sprague Dawley rats were cultivated and identified as usual methods. After being suffered from OGD injury,the cultured astrocytes were treated with either vehicle, siTLR2 （Toll-like receptor-2,TLR2） or PDTC （a NF-KB inhibitor）, respectively. First, real-time PC R and ELISA techniques were employed to examine the levels of IL-10 transcription and expression in the OGD-injured astroeytes. Next,the protein levels of TLR2 and p- NF-KB p65 among these OGD-injured astroeytes were detected using Western blot. In addition,the subcellular distribution of NF-KB p65 in OGD-injured astrocytes was tested with immunofluorescence. Results：Immunofluoreseence revealed that the isolated primary cultured astrocytes reached a high purity more than ninety percent. Compared with the control group[（0.49 ± 0.08）, （0.23 ±0.09）,（0.00 ±0.00）, （381.89 ±22.26）],OGD treatment not only upregulated the TLR2,p-NF-KB p65 and IL-10 expression levels [（1.16±0.10）, （1.01 ±0.08）, （0.00 ±0.00）, （513.01 ±34.47） ] among these astrocytes （P=0.001,P=0.OOO,P=0.026, P=0.005） ,but also increased the protein expression level of NF-KB p65 in the nucleus. However,TLR2 siRNA effectivelyreduced the TLR2 expression level and prevented OGD-induced p-NF-KB p65 and IL-10 upregulation[（0.36 ±0.13）, （0.38 ± 0.10）, （0.00 ± 0.00）, （567.05 ± 50.57）] when compared with those in siTLR2-untreatment group [（ 1.33 ±0.42）, （0.92 ±0.28）, （0.00 ± 0.00）, （703.60 ±12.97）]. Additionally,PDTC also dramatically decreased p-NF-KB p65 and IL-10expression levels[（0.26 ±0.07）, （0.00 ± 0.00）, （598.92 ± 42.73）] without affecting TLR2 expression in OGD-injured astrocytes（ 1.09 ± 0.94） comparing with those in OGD group[（ 1.24 ± 0.23）, （0.00 ± 0.00）,

关 键 词：星形胶质细胞 白细胞介素-10 氧糖剥夺 TOLL样受体2 核因子-ΚB 

分 类 号：R741.02[医药卫生—神经病学与精神病学] R392.6[医药卫生—临床医学]