| Background:Spinal Cord Injury(SCI)after a variety of immune cell subtypes can invade the damage to the local microenvironment.Studies have shown that macrophages play an important role in the immune microenvironment after SCI.Previous studies confirmed that,in SCI local microenvironment,the nerve damaged M1 cells and neuroprotective M2 cells can be detected in the early stage,and mainly M1 cells,M2 cells in a small proportion.The result of RNA-Seq analysis showed that the level of Colony Stimulating Factor 2 Receptor(Beta,Low-Affinity,CSF2RB)in rat SCI model was significantly up-regulated in the injured spinal cords.This suggested that the increased M1 cells in the injured spinal cords may be related to the activation of CSF2RB-related signaling pathways.Therefore,this study was to first detect the expression of CSF2RB in the local spinal cord injury and to observe the polarization of CSF2RB-related signaling pathway on macrophages to explore whether it is predominantly with M1 cells in the injured spinal cord activation,and discuss its value in the treatment of SCI.Method:(1).Make a SD rat weight fall SCI model.(2).Spinal cord tissue was isolated from rats at different time points and sham operated after SCI.(3).The expression of CSF2RB in the spinal cord at different time points after SCI was detected by qRT-PCR.(4).The changes of CSF2RB expression at the protein level in rat spinal cord at different time points after SCI were detected by Western blotting.(5).Immunofluorescence staining was used to detect the distribution of CSF2RB in rat spinal cord at different time points after SCI.(6).The bone marrow of the femur was isolated,and GM-CSF,GM-CSF,IL-3 and IL-5 were respectively cultured in the medium for 7 days.Flow cytometry was used to identify the type M1 macrophages and M2 macrophages proportion.(7).The bone marrow of femur was isolated and cultured for GM-CSF,GM-CSF,IL-3 and IL-5in culture medium for 7 days.Immunofluorescence staining was used to identify the type M1 macrophages and M2 macrophages proportion.Results:(1)The results of qRT-PCR showed that the expression of CSF2RB in SCI group was significantly higher than that in sham operation group(P<0.05);(2)Western Blot results showed that compared with the sham group,the expression of CSF2RB was significantly increased after SCI,especially at 3dpi,7dpi and 14dpi(P<0.05).(3)The results of immunofluorescence staining showed that,in sham-operated group,CSF2RB was found in GFAP~+cells,CD11b~+cells,CNPase~+cells and NeuN~+cells,but not in CD45~+cells and CD68~+cells.However,CSF2RB was found in GFAP~+cells,CD11b~+cells,CNPase~+cells,NeuN~+cells,CD45~+cells and CD68~+cells after SCI,and increased at different stages of SCI.(4)The results of flow cytometry showed that the proportion of type M1macrophages cultured in combination with three kinds of cytokines was56.70%±3.90%,which was higher than that of type M1 macrophages in GM-CSF group(40.07%±4.11%,respectively)(P<0.05).In macrophages cultured with GM-CSF,IL-3 and IL-5,the proportion of M2 macrophages was 24%±5.09%,which was much lower than that of GM-CSF group(49.77%±5.61%).The results were statistically significant(P<0.05).(5)The results of immunofluorescence staining showed that the proportion of type M1 macrophages cultured in combination with three kinds of cytokines was 69.33%±4.51%,the percentage of type M1 macrophages in combination with GM-CSF alone was 58.07±5.09%),but the difference was not statistically significant(P>0.05).However,the ratio of M2 macrophages in macrophages cultured with GM-CSF,IL-3 and IL-5cytokines was 29.00%±2.30%,which was much lower than that of GM-CSF group(42.33%±5.21%).The results were statistically significant(P<0.05).Conclusion:1.The findings of this study indicate that CSF2RB expression is significantly elevated after SCI.2.The quantitative analysis proves that neurons and oligodendrocytes are not involved in SCI-induced CSF2RB increases.However,infiltrated leukocytes,activated microglia/macrophages,and astrocytes are the major sources of increased CSF2RB.3.In vitro culture of macrophages in combination with GM-CSF,IL-3 and IL-5 allows them to be transformed into M1 macrophages. |
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