体外LPS预处理小胶质细胞炎性细胞因子分泌反应的探讨  

Studies of lipopolysaccride pretreatment on the release of proinflammatory cytokines from cerebral microglia in vitro

作　　者：谢泽锋[1] 辛岗[2] 李康生[2] 苏芸 

机构地区：[1]汕头大学医学院第一附属医院,广东汕头515041 [2]汕头大学医学院微生物学与免疫学教研室,广东汕头515041

出　　处：《中国热带医学》2015年第7期781-784,798,共5页China Tropical Medicine

基　　金：国家自然科学基金项目(No.81001340;No.81471622);广东省医学科研基金项目(No.B2010211);广东省优秀青年教师培训计划(No.Yq2013079);汕头市科技计划(No.2013-88)

摘　　要：目的通过革兰阴性菌LPS(脂多糖,Lipopolyssacride,LPS)预处理体外培养大脑皮质小胶质细胞,模拟轻型细菌感染,尝试建立体外LPS耐受模型,探讨小胶质细胞促炎细胞因子分泌反应的变化情况。方法体外培养与分离小鼠大脑皮质小胶质细胞,免疫荧光法进行纯度鉴定。纯化的小胶质细胞分为未刺激对照组、0.01μg/m L LPS单次刺激组、1μg/m L LPS刺激组、预处理组(先采用0.01μg/m L LPS刺激18h后,再用1μg/m L LPS刺激6、24、48h)。处理后收获各组细胞培养上清,采用ELISA法检测小胶质细胞TNF-α、IL-6分泌水平与变化趋势。结果经过LPS处理的小胶质细胞(0.01μg/m L LPS单次刺激组、1μg/m L LPS单次刺激组与预处理组)TNF-α与IL-6分泌增加,但预处理组在经过1μg/m L LPS再次刺激不同时间点(6h、24 h、48 h)分泌的总体水平变化不大(P>0.05)。在6 h,1μg/m L单次刺激组、预处理组的TNF-α与IL-6水平比对照组水平明显升高,差异均具有统计学意义(P<0.05);在48 h,TNF-α水平在1μg/m L单次刺激组、预刺激组中也较对照组水平明显升高(P<0.01)。另外,在LPS预处理24h与48h后,TNF-α水平较1μg/m L单次刺激组均有下降趋势,其中在48h,预刺激组较1μg/m L单次刺激组分泌减少,差异具有统计学意义(P<0.05),但IL-6水平预刺激组较1μg/m L单次刺激组分泌具有增加趋势。结论体外LPS预处理可在小胶质细胞中诱导部分耐受效应,表现为某些关键炎性细胞因子的分泌抑制,但并非是一种全面的抑制,这可能与细胞来源以及信号通路分子的重新调配有关。Objective To study the influence of lipopolysaccride pretreatmet on the release of proinflammatory cytokinesfrom cerebral microglia,so as to explore the phenomenon of LPS tolerance in microglia. Methods Mice cerebral icoglia wascultured and purified in vitro,then,microglia was identified by immunofluorescence. Thereafter,microglia was divided into 4groups： non-treated group,0.01μg /m L of LPS treated group,1μg /m L of LPS treated group and LPS-pretreated group. LPS-pretreated group was preconditioned with 0.01μg /m L of LPS for 18 hours. After the preconditioning,the cells received thesecond times of treatment with 1μg /m L of LPS for 6 hours,24 and 48 hours. Cultured supernatants were collected and theproinflammatory cytokines were determined by ELISA. Results The TNF-α and IL-6 levels in 0.01μg /m L of LPS treatedgroup,1μg /m L of LPS treated group and LPS-pretreated group 6 and 48 hours after pretreatment were increased（P〈0.05）.Moreover,TNF-α levels in LPS-pretreated group decreased 24 and 48 hours after LPS re-treatment,when compared with the1μg /m L-stimulated group respectively（P〈0.05）. The IL-6 level in LPS-pretreated group,although no significant differencein statistics,an enhancing trend was found 6,24 and 48 hours after LPS re-treatment. Conclusions LPS treatment couldinduce a partial tolerant effect on proinflammatory secretion by microglia in vitro. The diverse responses of various cytokinesmight result from the heterogeneity of cellular origin and the readjustment of signal pathways.

关 键 词：小胶质细胞 脂多糖 炎性细胞因子 预处理 

分 类 号：R-33[医药卫生]