Objective To investigate the effects of acidic microenvironment on the expression of periodic bioclock 1 and M2 type polarization in macrophages.Method Twenty C57/6J female mice aged 6 to 8 weeks were injected intraperitoneally with normal saline containing 5％starch for 3 consecutive days and killed under anesthesia.The mice were irrigated intraperitoneally and the irrigated liquid was recovered and centrifuged at 800 r/min for 5 min,then inoculated into a six-well plate for 2 hours,and the adherent cells were known as mouse abdominal macrophages.pH7.3 medium(pH7.3 group),pH6.5 medium(pH6.5 group)and 0.02 mol/L lactic acid medium(0.02 mol/L lactic acid group)were added respectively and cultured for 24 h.Real-time PCR was used to detect interleukin-1β,vascular endothelial growth factor,arginase-1,hypoxia inducible factor-1α and period 1 messenger RNA expression in 3 groups of mouse peritoneal macrophages.The levels of interleukin-10,interleukin 12,VEGF,and tumor necrosis factor α protein in peritoneal macrophages of three groups of mice were detected by enzyme-linked immunosorbent assay.DOTAP,DOPE,cholesterol,DSPE-PEG2000-NHS(molar ratio was 7∶7∶5∶1)were fully dissolved in chloroform solution and the liposome films were formed by swirl evaporation at 60 ℃ and 120 r/min for 1 h using a rotary evaporator.A single layer of liposome was prepared by adding nuclease-free double water in ultrasonic water bath and small extruder.The PER1 small interfering RNA was dissolved in enzyme-free water and mixed with the liposome solution at room temperature according to the mass ratio of 1∶10.Allophycocyanin-anti-mannose receptor(APC-CD206)antibody was then added according to the volume ratio of 1∶1 000.The liposome-PER1 complex(Liposome-per1,Lipo-PER1)cationic liposomes were prepared by incubation at room temperature for 30 min without light.Transmission electron microscopy was used to observe the morphology and structure of liposome-periodic bioclock 1 complex.B16-F10 melanoma cells and RAW264.7 macrophages were co-cultured at a 1∶1 ratio,and the uptake of Lipo-PER1 by B16-F10 melanoma cells and RAW264.7 macrophages was observed by confocal microscopy.RAW264.7 macrophages with logarithmic growth were cultured for 6 h with Lipo-PER1 and liposome negative control(Lipo-NC)complexes.The expression of IL-1β,antigen-differentiated cluster 86(CD86),ARG-1,IL-10 mRNA and TNF-α,transforming growth factor-β(TGF-β)protein in RAW264.7 macrophages of the 2 groups were detected by RT-PCR and ELISA.Results Compared with pH7.3 group,the mRNA expression of IL-1β in peritoneal macrophages of mice in pH6.5 group and 0.02 mol/L lactic acid group was down-regulated(P＜0.05),and the mRNA expressions of ARG-1,VEGF,HIF-1α,and PER1 were up-regulated(P＜0.05).The levels of IL-12 and TNF-α were decreased(P＜0.000 1)while the levels of IL-10 and VEGF were increased(P＜0.01).The results of TEM and confocal microscopy showed that Lipo-PER1 was successfully constructed and could be successfully taken up by macrophages.Compared with Lipo-NC group,mRNA expressions of IL-1β and CD86 in RAW264.7 macrophages of Lipo-PER1 group were up-regulated(P＜0.05 or P＜0.001)while mRNA expressions of ARG-1 and IL-10 were down-regulated(P＜0.01 or P＜0.05).The expression of TGF-β was increased(P＜0.0001)and the expression of TNF-α was decreased(P＜0.05).Conclusions The acidic microenvironment upregulates the expression of PER1 in macrophages,thereby promoting the M2-type polarization of macrophages.
维普
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