为研究布鲁菌侵染巨噬细胞过程中,对后者不同分化类型细胞中JAK-STAT6信号通路和细胞因子表达变化的影响,本研究利用小鼠巨噬细胞系RAW264.7,经40 ng/mL的IL-4体外作用24 h将其诱导为M2型巨噬细胞,采用细胞免疫荧光方法,检测M2巨噬细胞极化状态;通过荧光定量PCR (qRT-PCR)方法,检测布鲁菌疫苗株M5侵染不同类型(M0和M2型)巨噬细胞中JAK1、STAT6基因mRNA转录情况;采用ELISA方法检测M5侵染不同类型巨噬细胞上清中IL-10、INF-γ的表达情况.细胞免疫荧光检测结果显示,IL-4诱导24 h后,巨噬细胞表面标志CD206荧光表达量超过90％,表明巨噬细胞极化为M2型;qRT-PCR结果显示,M5侵染M0型巨噬细胞后能够引起JAK1、STAT6 mRNA转录水平持续降低,在24 h和48 h时其转录水平最低,二者差异极显著(p＜0.01);而M5侵染M2型巨噬细胞后,与未极化组(M0型)相比,JAK1、STAT6的mRNA转录水平增高,同时存在时间差异性,侵染8h时,二者mRNA转录水平最高,差异极显著(p＜0.01).ELISA结果显示,M5侵染巨噬细胞后,极化组(M2型)与未极化组(M0型)巨噬细胞相比,INF-γ的表达量差异不显著(p＞0.05),而IL-10的表达量上调且在侵染48 h达到最大值,差异显著(p＜0.05).上述结果表明,在1L-4的诱导下巨噬细胞可以从未极化的M0型极化为M2型;布鲁菌M5侵染M0型巨噬细胞中JAK1、STAT6的转录水平下调,但上调M2型巨噬细胞JAK1、STAT6的转录水平并促进下游细胞因子IL-10的分泌.本研究为探究布鲁菌引起持续感染的机制研究奠定了基础,同时为布鲁菌病药物靶标开拓了新的研究方向.%To investigate the effect of Brucella infection on the changes of JAK-STAT6 signaling pathway and cytokine expression in different differentiated cell types of macrophages,murine macrophage lineage RAW264.7 was induced to M2 type macrophage by 40 ng/mL IL-4 for 24 hours in vitro,and the polarization state of macrophages was detected by indirect immune fluorescence (IFA).The transcription levels of JAK1 and STAT6 were detected by qRT-PCR in different types macrophages infected by Brucella melitensis vaccine strain M5 and the expression levels of IL-10,INF-γ in infected macrophages supematant were detected by ELISA.The result of IFA showed that the expression of surface marker CD206 on the M2 type macrophage was over 90％ after induction by IL-4 for 24 h,which indicated that the M0 type macrophages were polarized to M2 type.The transcription levels of STAT6 and JAK1 were steadily decreased in M0 type macrophage,and their transcription levels decreased to a minimum at 24 h and 48 h post M5 infection (p＜0.01).The transcription levels of JAK1 and STAT6 were increased and reached the hightest lever at 8 h after the M2 macrophages infected with M5,and the difference was significant compared with the non-polarized group (M0 type macrophages) (p＜0.01).The results of ELISA showed that the expression of INF-γ was not significant compared with that of the non-polarized group (p＜0.05) after M5-infected M2 type macrophages.The expression of IL-10 was up-regulated in M5-infected M2 type macrophages and reached the maximum at 48 h post infection (p＜0.05).The above results showed that macrophages could be polarized to M2 type macrophages from M0 type induced by IL-4,and the transcription levels of JAK1 and STAT6 were down-regulated in M0 type macrophages infected byM5.However,the transcription levels of JAK1 and STAT6 were up-regulated in M2 type macrophages,which could promote the secretion of cytokine IL-10.This study laid the foundation for the research on the mechanism of persistently infected caused by Brucella,and opened up a new research direction for the brucellosis drug target.
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