胆绿素还原酶在巨噬细胞分化模型中表达情况

Expression of Biliverdin Reductase During Macrophage Differentiation

目的探讨巨噬细胞分化模型中BVR的表达情况方法体外培养L929细胞2 d,收集L929细胞上清,用此培养基来体外培养小鼠骨髓来源巨噬细胞7 d,分别用LPS 100 ng/mL和IFN-γ100 ng/mL、IL-410 ng/mL和IL-1310 ng/mL刺激24 h后收集细胞.用Western印迹检测INOS和ARG-1的表达;用流式细胞术检测CD45、F4/80、CD11b、CD86和DECTIN-1的表达;用RT-PCR检测INOS、ARG-1、FN和FIZZ1的表达来判断M1和M2的模型是否建立成功,然后在巨噬细胞模型中检测BVR的表达.同时在RAW264.7细胞系构建的巨噬细胞分化模型中检测BVR的表达.结果①Western印迹发现LPS和IFN-γ组(M1巨噬细胞)INOS表达增加,IL-4和IL-13组(M2巨噬细胞)ARG-1表达增加;②流式细胞术发现LPS和IFN-γ组巨噬细胞中CD86表达增加,IL-4和IL-13组巨噬细胞中DECTIN-1表达增加;③RT-PCR发现LPS和IFN-γ组巨噬细胞中INOS和TNF-α表达增加;IL-4和IL-13组巨噬细胞中ARG-1、FN和FIZZ1表达增加;④RT-PCR发现在小鼠骨髓来源的巨噬细胞和RAW264.7细胞系巨噬细胞分化模型中BVR均在M1表达降低,在M2中表达增加.结论①LPS和IFN-γ,IL-4和IL-13刺激小鼠骨髓来源巨噬细胞构建M1和M2模型成功;②BVR在小鼠骨髓来源巨噬细胞和小鼠单核巨噬细胞系构建的巨噬细胞分化模型中均在M1中表达降低,在M2中表达增加.

Objective To investigate the expression of biliverdin reductase(BVR)in bone marrow derived macrophages and RAW264.7 cells.Methods L929 cells were cultured for 2 days,the supernatant was then collected.The murine bone marrow derived macrophage cells were isolated and were cultured with the above supernatant for 7 days.Cells were then divided into three groups:control group,LPS+IFN-group,IL-4+IL-13 group.Cells in the LPS+IFN-group and the IL-4+IL-13 group were stimulated by 100 ng/mL LPS and IFN-or 10 ng/mL IL-4 and 10 ng/mL IL-13 for 24 hours respectively.Western blotting was used to detect the expression levels of INOS and ARG-1 proteins.RT-PCR was used to detect the RNA expression levels of INOS,ARG-1,FN and FIZZ1.Flow cytometry was used to detect the expression of CD45,F4/80,CD11b,CD86 and DECTIN-1 on cells..The expression level of BVR in the macrophage polarization model established in BMDM cells and RAW264.7 cells was detected by RT-PCR.Results①The protein expression level of INOS was increased in the LPS+IFN-group,and the protein expression level of ARG-1 was increased in the IL-4+IL-13 group.②The expression of CD86 on cells was increased in the LPS+IFN-group,and the expression of DECTIN-1 on cells was increased in the IL-4+IL-13 group.The RNA expression levels of INOS and TNF-αwere increased in the LPS+IFN-group,the RNA expression levels of ARG-1,FN and FIZZ1 were increased in IL-4+IL-13 group.①The expression level of BVR was increased in the M2-shift macrophages,but decreased in the M1-shift macrophages in BMDM and RAW264.7 cell models.Conclusion①The macrophage polarization models are successfully established by stimulation of LPS and IFN-or IL-4 and IL-13 in vitro.②The expression level of BVR is increased in the M2-shift macrophages,but decreased in the M1-shift macrophages in vitro.