布鲁氏菌Omp10对巨噬细胞极化及STAT6信号通路影响的研究

Effects of Brucella outer membrane protein Omp10 on macrophage polarization and STAT6 signaling pathway

为探究布鲁氏菌(Brucella)外膜蛋白Omp10对小鼠骨髓源性巨噬细胞(BMDM)极化及STAT6信号通路的影响,本研究将重组菌p ET30a-Omp10/DE3经终浓度0.1 mmol/L的IPTG诱导6 h后,获得主要以包涵体形式表达的重组蛋白Omp10(r Omp10)。同时,通过巨噬细胞集落刺激因子(GM-CSF)刺激BALB/c小鼠胫骨和腓骨细胞获得本实验用的梭型、圆形或卵圆形“煎蛋样”形态的BMDM细胞。将获得的r Omp10纯化后刺激BMDM细胞,收集刺激后不同时间点细胞,提取基因组后通过q RT-PCR检测M1型巨噬细胞标志因子NOS2、IL-12,及M2型巨噬细胞标志因子JAK1、STAT6、ARG1、IL-10 m RNA的转录水平,结果显示,相对于对照组,r Omp10刺激细胞4 h和12 h后,NOS2和IL-12 m RNA转录水平显著上调,刺激12 h时JAK1 m RNA转录水平显著下调(P<0.05),刺激4 h、12 h和48 h时STAT6 m RNA转录水平均显著下调(P<0.05),ARG1和IL-10 m RNA转录水平逐渐升高而后降低。通过流式细胞术检测BMDM中M1型标记分子CD86和M2型标记分子CD206的表达情况,结果显示,在r Omp10刺激下CD86表达量极显著高于对照组(P<0.01),且随时间延长其表达量呈下降趋势;CD206在刺激12 h时显著低于对照组(P<0.05),但72 h时显著高于对照组,且随时间延长其表达量呈上升趋势(P<0.01)。进一步通过ELISA方法检测刺激后不同时间的BMDM中细胞因子TNF-α和IL-10的表达量,结果显示,在r Omp10刺激BMDM 4 h、12 h、24 h时TNF-α表达量极显著高于对照组(P<0.01);而在r Omp10刺激BMDM 48 h时,IL-10的表达量显著高于对照组(P<0.05)。上述结果首次证实布鲁氏菌外膜蛋白Omp10抑制巨噬细胞中STAT6信号通路的激活,且在外膜蛋白Omp10刺激前期主要诱导巨噬细胞极化为M1型,在感染后期诱导M2型细胞因子的表达,本研究为阐明布鲁氏菌胞内存活机制提供新的研究方向和实验基础。

To explore the effects of Brucella outer membrane protein Omp10 on polarization and STAT6 signaling pathway of mouse bone marrow derived macrophages(BMDM),in this study,Brucella recombinant pET30a-Omp10/DE3 was induced for6 hours to express by 0.1mmol/L final concentration of IPTG and purified to generate recombinant protein Omp10(rOmp10).BALB/c mice were stimulated by macrophage colony-stimulating factor(GM-CSF)to differentiate tibial and peroneal osteoblasts into mononuclear macrophages.rOmp10 was used to stimulate BMDM cells that prepared by our laboratory,and the transcriptional level of M1-type macrophage marker genes including NOS2 and IL-12 as well as M2-type macrophage marker genes including JAK1,STAT6,ARG1,and IL-10 were detected by qRT-PCR.In addition,the expression of M1-type marker molecule CD86 and M2-type marker molecule CD206 at protein level were measured by flow cytometry.The expression levels of Th1/Th2 cytokines TNF-αand IL-10 were detected by ELISA as well.qRT-PCR results showed that compared with the control group,r Omp10treatment could significantly increase m RNA transcription of NOS2 and IL-12 at 4 hours and 12 hours,but caused the downregulation of STAT6 mRNA transcription at 4 hours,12 hours and 48 hours.while the M2 type macrophage marker gene JAK1,was only significantly down-regulated at 12 hours(P<0.05),The m RNA transcription of ARGG1 and IL-10 was gradually increased until 48 hours post-treatment,and then declined to the normal level;Flow cytometry showed that the expression of CD86 in r Omp10 treated cells was significantly higher than that in control group(P<0.01),CD206 at 12 hours was significantly lower than the control group(P<0.05),but was extremely higher 72 hours post-treatment(P<0.01);ELISA results showed that TNF-αexpression level of r Omp10 at 4 hours,12hours and 24 hours was significantly higher than that of control group(P<0.01),the expression of IL-10 was significantly higher than that of the control group after rOmp10 stimulation for 48 hours(P<0.05).This study provided the evidence that outer membrane protein Omp10 could inhibit the activation of STAT6 signaling pathway,and modulate the macrophage M1/M2 ratio.