The M2 Molecular Mechanisms Study Of The Macrophages Involved In The Glial Scar Formation Of Astrocytes

Objective: To study the M2 macrophages' secreting of the transforming growth factor ?(TGF-?),which is the underlying mechanism of astrocytosis and formation of glial scar,our findings may provide a theoretical and experimental basis for further exploring the mechanisms of formation of glial scar after central nervous system injuries and offer a new research strategy in treatment.Methods:(1)Methodology of separation,cultivation and identification of rat astrocytes: Ten new born SD rats(<3d)were anesthetized,and dorsal spinal cords dissected surgically under aseptic conditions.The dorsal spinal cords were cut into pieces and then incubated in 0.25% trypsin and digested into mono-cellular suspensions.The collected astrocytes were purified by differential adhesion method.Immunohistochemical staining was performed to detect the expression of glial fibrillary acidic protein(GFAP)in the third generation of astrocytes which is used for purity identification.The third generation of astrocytes can be used for further experiments.(2)Methodology of separation,cultivation of rat bone marrow macrophages and the macrophages were induced differentiating into M2 macrophages.Five SD rats(200-250 g)were anesthetized by 10% chloral hydrate(0.4 m L/100 g)and obtained the bone marrow cells from femurs and tibias under sterile conditions.The separated myelomonocyte was inoculated in the cell-conditioned medium which containing 20% L929 and the RPMI-1640 with 15%FBS for 7 days,then the purified M0 macrophages can be obtained.On the 8th day of cultivation,IL-4(20 ng/ml)and IL-10(20 ng/ml)were added in the cell-conditioned medium and after 24 hours' inducing cultivation,M2 macrophages can be obtained.Flow cytometry was used to identify the phenotypes of CD11b?CD206?Arg-1 and i NOS of M2 macrophages.(3)M2 macrophages' indirect co-culturing with astrocytes and relevant detection and analysis.The cells were co-cultured in Transwel 6-well plates with the diameters of 0.4 ?m.The cells in the upper chambers were macrophages,and in the lower chambers were astrocytes.The number of each kind inoculated was 1×105 / hole.The subjects were divided into four groups: the M2 macrophages + astrocytes co-cultured group,the M2 macrophages +TGF-? receptor inhibitor + astrocytes group,the M2 macrophages purely-cultured group and the astrocytes purely-cultured group.After 48hours' co-culturing,detected the expression of m RNAs of TGF-? and PDGF in M2 macrophages and GFAP,CSPG,IL-4 and IL-13 in astrocytes by using fluorescence quantitative PCR.Western blot was used to detect the expression of protein of astrocytes' GFAP and CSPG in each group.ELISA was used to detect the level of TGF-? in the culture supernatant in each group.Results:(1)Immunohistochemical staining results showed that the expression of GFAP in the cultured rat spinal cord astrocytes cytoplasm is high,the percentage of GFAP positive cells in third generation of astrocytes was(95.86 + 1.02)%.(2)Flow cytometry results indicated that the isolated induced M2 macrophages highly express CD11 b,CD206 and Arg-1 and lowly express i NOS,which were consistent with the phenotypic characteristics of M2.(3)q-PCR results showed that,compared with the astrocytes purely-cultured group,the expression of GFAP,CSPG and IL-13 in astrocytes were markedly enhanced in the M2 macrophages + astrocytes co-cultured group(P<0.05),while there is no significant difference in the expression of IL-4 between these two groups(P>0.05);compared with the M2 macrophages purely-cultured group,the expression of TGF-? and PDGF in M2 macrophages were markedly enhanced in the M2 macrophages + astrocytes co-cultured group(P<0.05);compared with the M2 macrophages + astrocytes co-cultured group,the expression of GFAP,CSPG and IL-13 in astrocytes and TGF-? as well as PDGF in the M2 macrophages were significantly suppressed in the M2 macrophages +TGF-? receptor inhibitor + astrocytes group(P<0.05).(4)Western blot results showed that,compared with the astrocytes purely-cultured group,the protein expression of GFAP,CSPG in astrocytes were markedly enhanced in the M2 macrophages + astrocytes co-cultured group and in the M2 macrophages +TGF-? receptor inhibitor + astrocytes group(P<0.05);while the protein expression of GFAP,CSPG in astrocytes in the M2 macrophages +TGF-? receptor inhibitor + astrocytes group was lower than that in the M2 macrophages + astrocytes co-cultured group(P<0.05).(5)ELISA results showed that,the ability of M2 macrophages to secrete TGF-? was significantly higher than that of AS;compared with the M2 macrophages group,the level of TGF-? in the culture supernatant was significantly increase in the M2 macrophages + astrocytes co-cultured group and in the M2 macrophages +TGF-? receptor inhibitor + astrocytes group;compared with the M2 macrophages + astrocytes co-cultured group,the level of TGF-? in the culture supernatant was significantly decreased in the M2 macrophages +TGF-? receptor inhibitor + astrocytes group(P<0.01).Conclusion: Our in vitro experiment focuses on the effect of rats' M2 macrophages on promoting fibrosis of astrocytes in spinal cords and preliminarily elucidates that M2 macrophages' secreting of TGF-? is the internal mechanism of astrocytes' activation,which leads to the formation of glial scar.It provides a basis for further research on the mechanisms of glial scar formation after central nervous system injuries and offers a new research strategy in glial scar therapy in the future.