论文部分内容阅读
目的探讨神经胶质细胞经过脂多糖(Lipopolyssacride,LPS)预激后,其培养上清对神经元功能的影响。方法体外培养神经元与神经胶质细胞,免疫荧光法进行纯度鉴定。神经胶质细胞分为4组:未刺激对照组、0.01μg/ml LPS单次刺激组、1μg/ml LPS单次刺激组、预刺激组(先采用0.01μg/ml LPS刺激18h后,再用1μg/mlLPS刺激24h)。经过相应处理后收获各组条件培养上清,分别加入到神经元中,与神经元共同孵育24h后收集细胞与培养上清。采用CCK-8试剂盒与ELISA法检测神经元的存活率和分泌TNF-α、IL-6水平。结果星形胶质细胞预刺激组条件培养上清作用于神经元后,细胞存活率下降,与0.01μg/ml、1μg/ml LPS单次刺激组比差异均具有统计学意义(P<0.05);小胶质细胞各组条件培养上清作用于神经元后,细胞存活率变化不明显(P均>0.05);在星形胶质细胞各刺激组上清作用下,神经元产生TNF-α的水平,各LPS处理组培养上清刺激下水平升高,其中1μg/ml LPS单次刺激组升高明显,与对照组和0.01μg/ml LPS单次刺激组比差异具有统计学意义(P均<0.05);产生IL-6的水平亦升高,与对照组比亦均具有统计学差异(P<0.05或P<0.01);并且,预刺激组较1μg/mlLPS单次刺激组上清作用后,产生IL-6水平较低(P<0.05)。各组小胶质细胞条件培养上清作用于神经元后,产生TNF-α的水平,预刺激组水平升高明显,与对照组比差异具有统计学意义(P<0.01);对于IL-6水平,变化趋势同TNF-α,但各组间差异无统计学意义(P>0.0.5)。结论 LPS预激参与重新调配了神经胶质细胞分泌活性介质的作用,培养上清中这些分子相互制约或是协同,对神经元产生有可能是保护或是损伤效应。
Objective To investigate the effects of cultured supernatant on neuronal function after glial cells pre-stimulation with lipopolysaccharide (LPS). Methods Neurons and glial cells were cultured in vitro, and their purity was identified by immunofluorescence. Glial cells were divided into 4 groups: unstimulated control group, 0.01μg / ml LPS single stimulation group, 1μg / ml LPS single stimulation group and pre-stimulation group (stimulated with 0.01μg / ml LPS for 18h, 1μg / ml LPS stimulation 24h). After corresponding treatment, the conditioned culture supernatants of each group were harvested and added to the neurons separately. After incubation with neurons for 24 hours, the cells and culture supernatants were collected. The survival rate of neurons and secretion of TNF-α and IL-6 were detected by CCK-8 kit and ELISA. Results Astrocytes preconditioned with astrocytes could decrease the survival rate of neurons, which was significantly different from that of 0.01μg / ml and 1μg / ml LPS single stimulation groups (P <0.05) ; The survival rate of neuron was not significantly changed by the conditioned medium supernatant of each group of microglia (P> 0.05). Under the action of supernatant of astrocyte stimulation group, the production of TNF-α (P <0.05). The level of LPS in the LPS-treated group was significantly higher than that in the LPS-treated group (P <0.01) (P <0.05 or P <0.01). Compared with the control group, the level of IL-6 production in the pre-stimulation group was significantly higher than that in the 1 μg / ml LPS single stimulation group After treatment, the level of IL-6 produced was lower (P <0.05). Compared with the control group, the level of TNF-α produced by conditioned medium supernatant of each group was significantly higher than that of the control group (P <0.01). The levels of IL-6 Level and trend of change with TNF-α, but there was no significant difference among the groups (P> 0.0.5). Conclusion LPS preconditioning is involved in reprogramming the mediators of glial cell secretory activity. These molecules in the culture supernatant are restricted or synergistic with each other and may exert protective or damaging effects on neurons.