Effect Of Soluble Substance Secreted By BV-2 Microglia On GL261 Glioma Cell

Glioma is a primary intracranial tumor with high mortality.As the innate immune cells of the central nervous system（CNS）,microglia can secrete immunosuppressive cytokinesand promote the development of glioma under the influence of glioma cells.Therefore,exploring the cytokines secreted by microglia is essential for the development of glioma.There have been some reports on the mechanism of glioma-induced microglia regulating the development of glioma.However,the effect of soluble substances secreted by microglia on glioma cells were unclear.In this study,we first established an orthotopic transplantation model of glioma using CX₃CR1^GFP/+transgenic mice and GL261-RFP glioma cells.Bytaking advantage of laser confocal fluorescence microscopy,we observe the changes of microglia in the glioma regaion.The results showed that microglia in the brain infiltrated into the glioma regaion,and formed an aggregation zone on the peritumoral at 7 days after inplantation of GL261 glioma cells.The branch length of the microglia in this regaion were shortened,and microglia mostly exists in the form of amoeba-like structures.We then constructed an orthotopic transplantation animal model of glioma by transplanting GL261-RFP cells into C57BL/6 mouse brain,and examined the dynamic changes of GL261-RFP glioma cells by using a two-photon microscorpe.It was found that the morphology of GL261-RFP glioma cells changed from the initial round to elongated spindle shape.In addition,we used the conditioned medium of BV-2 microglia to conditionally culture GL261 glioma cells.Combining with experimental methods such as flow cytometry,MTT cell viability detection,Brd U immunofluorescence staining,cell scratching and RT-q PCR,the effect of the conditioned medium of BV-2 cells on GL261glioma cells was explored.The results of MTT experiment showed that the viability of GL261 cells were increased after conditioned culture for 24 h with the conditioned medium of BV-2 microglia.The results of Brd U immunofluorescence staining showed that the density and proliferation ability of GL261 cells have not been altered by conditioned medium of BV-2 microglia.By analyzing the aspect ratio of GL261 cells,it was found that the aspect ratio of conditioned cultured GL261 cells were significantly increased compared with control group.The result of cell scratching showed that BV-2microglia conditioned medium promoted the migration of GL261 cells.Compared with the control group,the m RNA expression levels of MMP-8,MMP-9,MMP-14 and IL-1βin the conditioned culture group were not significantly changed at 24 h and 48 h;the m RNA expression levels of TGF-β1,MMP-2 and i NOS were significantly up-regulated at 48 h;the m RNA expression levels of IL-1αwere significantly up-regulated at 24 h and 48 h.The m RNA expression levels of MMP-10 and IL-18 were significantly down-regulated at 24 h and 48 h,and the m RNA expression levels of Arg-1 were significantly down-regulated at 24 h,but had no significant change at 48 h.The results of flow cytometry showed that BV-2 microglia conditioned medium promoted the apoptosis of GL261 glioma cells.Finally,we used the conditioned medium of GL261 cells to conditionally culture BV-2 cells.The results showed that the phagocytic ability of conditioned BV-2 cells were significantly increased from 3 h to 6 h after conditionally culture,but the phagocytic ability began to weaken after 6 h of conditional culture.The results of this study showed that the morphology and migration of GL261glioma cells can be effected by the soluble substances secreted by BV-2 microglia.Meanwhile,the phagocytic function of BV-2 microglia can be changed by the soluble substances secreted by GL261 glioma cells.This study provides a reference basis for further study of the mechanism of microglia on glioma cells.