LPS预激神经胶质细胞培养上清对神经元的影响

Effects of neuroglia cultured supernatants with LPS-priming on neurons

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 study the effects of glia cultured supernatants with LPS-priming on neurons＇ viability and secretion activity.Methods Neurons and glia（mainly referring to astrocytes and microglia）were cultured in vitro and identified by immunofluorescence.Thereafter,these glial cells were divided into 4 groups：LPS-primed group,0.01μg/ml of LPS treated group,1μg/ml of LPS treated group and non-treated group.The LPS-primed group was preconditioned with 0.01μg/ml of LPS for 18 hours and re-retreated with a higher dose of LPS（1μg/ml LPS）for 24 hours.Then all cultured supernatants of 4 groups were collected and were used to treat the neurons for 24 hours in vitro.The cytotoxicity and secretion ability of proinflammatory cytokines were determined by CCK-8 kit and ELISA respectively.Results The cytotocixity of neurons was decreased by the treatment with the astrocyte supernatant of LPS-primed group,there was significant difference statistically if compared with 0.01μg/ml and 1ug/ml of LPS groups（P0.05;P0.05）.The cytotocixity showed no profound variation after the treatment with the microglia supernatants of 4 groups（P0.05）.As for TNF-α levels,it was increased by the treatment with the astrocyte supernatant of LPS-primed group,0.01μg/ml of LPS treated group,1μg/ml of LPS treated group,particularly in 1ug/ml of LPS treated group,statistical difference was found when compared with 0.01μg/ml of LPS treated group and non-treated group（P0.05;P0.05）;IL-6 levels were increased after the treatment with the astrocyte supernatants of LPS-primed group,0.01μg/ml of LPS treated group,1ug/ml of LPS treated group（P0.05）.Furthermore,after the treatment with the microglia supernatants of LPS-primed group,TNF-α level was increased profoundly when compared with non-treated group（P0.01）.Though IL-6 levels exhibited an increasing trend,no significant difference was found in statistically between groups（P0.05）.Conclusion There were diverse effects（ncluding protection or injury） of cultured supernatants from astrocytes and microglia on the cytotoxicity and secretion ability in the neurons,because of the readjustment of bioacitve molecules with LPS preconditioning in target cells.