体外弓形虫感染小鼠小胶质细胞及对其极化的影响

摘要

To investigate the effect of T.gondii infected on mice microglia in vitro.PLK strain of T.gondii was inoculated in primary microglia cells and culture supematants were collected at 6,12,24,36 and 48 hrs after infection with T.gondii.The mRNA transcriptional and expression levels of M1 type and M2 type marker molecules of microglia were detected by real-time PCR and westem blot,respectively.Induced nitric oxide synthase (iNOS) and Arginase 1 enzyme activity were detected by Griess method and Arginase activity test.The results showed that the mRNA transcription and protein expression levels of iNOS and IL-6 in M1 type microglia were significantly up-regulated (p＜0.001) in the cells after infection with T.gondii while the mRNA transcription and the protein expression of Arginase-1 and IL-10 didn't change significantly (p＞0.05).In addition,the enzyme activity tests showed that expression level of iNOS was significantly different from that of the control group at 6 h to 48 h post infection (p＜0.001),while the expression level of Arginase-1 was not significant at these time.The results of this study revealed that the resting state of microglia could be significantly polarized to M1 type after infection with T.gondii,which could change the phenotype and biological functions of microglia at the same time.This study provides an useful reference for further elucidating the interaction between T.gondii and the immune system of the body.%为探究体外弓形虫(T.gondii)感染小鼠小胶质细胞(Microglia)后对其极化的影响,将PLK株弓形虫接种原代小胶质细胞,分别在弓形虫感染细胞后6h、12h、24 h、36 h以及48 h收集细胞及培养上清,利用荧光定量PCR、western blot分别检测小胶质细胞M1型以及M2型标记分子的表达水平.采用Griess法和精氨酸酶活性试验分别检测细胞中诱导型一氧化氮合酶(iNOS)和精氨酸酶-1(Arginase-1)的酶活性.结果显示,弓形虫感染细胞后M1型标记分子iNOS与IL-6的mRNA转录水平与蛋白水平均显著上调(p＜0.001),而M2型标记分子Arginase-1与IL-10无明显变化(p＞0.05).此外,酶活性检测结果显示iNOS在弓形虫感染6 h～48 h与对照组相比其表达水平有显著差异(p＜0.001),而Arginase-1在该时间段表达差异并不显著.本研究结果表明,弓形虫感染小胶质细胞后能够明显诱导静息状态的小胶质细胞向M1型极化,从而改变小胶质细胞的表型和生物学功能,本研究为进一步阐明弓形虫与机体免疫系统相互作用提供了依据.