体外LPS预处理小胶质细胞炎性细胞因子分泌反应的探讨

发布时间：2019-01-19 09:31

【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(P0.05)。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(P0.05);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(P0.01)。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(P0.05),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。
[Abstract]:Objective to culture cerebral cortex microglia by LPS (lipopolysaccharide (Lipopolyssacride,LPS), to simulate mild bacterial infection, and to establish a model of LPS tolerance in vitro. To investigate the changes of inflammatory cytokine secretion in microglia. Methods Mouse cerebral cortex microglia cells were cultured and isolated in vitro and purified by immunofluorescence. The purified microglia were divided into three groups: unstimulated control group, 0. 01 渭 g / mL LPS single stimulation group, 1 渭 g / mL LPS stimulation group, pretreatment group (0. 01 渭 g / mL LPS for 18 h, then 1 渭 g / mL LPS for 6? 24 h). After treatment, the supernatants of each group were harvested and the levels and trends of TNF- 伪 and IL-6 secretion in microglia cells were detected by ELISA assay. Results the secretion of TNF- 伪 and IL-6 was increased in LPS treated microglia (0. 01 渭 g / mL LPS, 1 渭 g / mL LPS single stimulation and pretreatment group). But in pretreatment group, the total level of secretion was not changed at different time points (6 h ~ 24 h ~ 48 h) after 1 渭 g / mL LPS stimulation (P0.05). The levels of TNF- 伪 and IL-6 in preconditioning group were significantly higher than those in control group at 6 h after 1 渭 g / mL single stimulation (P0.05). The level of TNF- 伪 in 1 渭 g / mL single stimulation group and prestimulation group was significantly higher than that in control group at 48 h (P0.01). In addition, after pretreatment with LPS for 24 h and 48 h, the level of TNF- 伪 decreased compared with that of 1 渭 g / mL single stimulation group, and at 48 h, the secretion of TNF- 伪 in prestimulation group was significantly lower than that in 1 渭 g / mL single stimulation group (P0.05). However, the secretion of IL-6 in prestimulation group was higher than that in 1 渭 g / mL prestimulation group. Conclusion LPS pretreatment in vitro can induce partial tolerance in microglia, which is shown by the inhibition of the secretion of some key inflammatory cytokines, but it is not a comprehensive inhibition. This may be related to cell sources and signal pathway molecules reassignment.
【作者单位】：汕头大学医学院第一附属医院;汕头大学医学院微生物学与免疫学教研室;
【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)
【分类号】：R329.2

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