Effects Of Microglia And Macrophages On The Migration Of Rat C6 Glioma Cell Lines

Glioma is the most common primary malignant tumors in central nervous system.Although various treatment methods have made some progress,the treatment is still unsatisfactory.The mortality rate is high.It is a difficult problem in the field of Department of neurosurgery.There are a large number of glioma infiltrating immune cells.In different literatures,some of these cells called microglia,some called macrophages,some called glioma associated microglia / macrophages(GMAs).In combination with the literatures,the three names refer to the same group of cells.During the growth of glioma,microglia,the specific macrophages in the brain,are recruited by a variety of chemokines released by the tumor.At the same time,because of the damage of blood-brain barrier,the peripheral macrophages were infiltrated into the glioma.Macrophages and microglia together form glioma associated microglia / macrophages(GMAs).Related research suggest that although glioma tissues accompanied by a large number of GAMs,but the antitumor activity of GAMs,which had been domesticated in the tumor microenvironment,was inhibited.GAMs provides favorable conditions for glioma growth,invasion and immunosuppression.In the process of interaction between glioma cells and GAMs,IL-10,IL-18,MMP-9 and other cytokines produced by GAMs play an important role in immune suppression and tumor metastasis.At the same time,the expression of TGF-? and FAS ligand which produced by glioma cells also induce and acclimate GAMs,which played a role in the transformation from anti-tumor effect to tumor promoting effect.Microglia and macrophages from peripheral blood are both macrophage group.They are specific differentiation macrophages in different tissues and different periods.They have basically the same molecular markers.It is difficult to making two cells in glioma.Whether these two kinds of microglia / macrophages have the same biological effect in glioma,it is a worthy question.In this study,we isolated and cultured microglia and peripheral blood macrop hages in vitro.Than microglia and macrophages were co-cultured with C6 glioma cells.Scratch test were done at 0h,24 h and 48 h after co-culture.The co-culture of fibroblasts and glioma cells was positive,while that of glioma cells was negative.The resu lts were observed and the healing rates were calculated at 6h after each scratch test.Flow cytometry were used to separate microglia,macrophages and C6 glioma cells when co-cultured 24 h and 48 h.The obtained cells in each group made western blot test(WB)and compared with three cells without co-culture.The changes of IL-10,IL-18 and MMP-9 expression in microglia and macrophages before and after co-culture were observed.Alao the expression of TGF-? and FAS ligand in the C6 glioma cell line before and after co-culture were observed.ResultsThe scratch test results showed that in the early stage of co-culture,microglia and macrophages co-cultured with C6 glioma cells(0h),both microglia and macrophagea showed antitumor effect and inhibited tumor migration(P<0.05).However,after a period of time(24h,48h),the anti-tumor effect disappeared,and the two kinds of cells were domesticated,all of them showed tumor migration and the effect of tumor metastasis were consistent(P>0.05).Microglia and macrophages were co-cultured with C6 glioma cells.At different time points(24h,48h)the expression of IL-10,IL-18 and MMP-9 protein in microglia and macrophages were higher than those at 0h,but at the same time the two kind cells protein expression level were the same(P>0.05).The expression of cytokines in microglia and macrophages that dociled by glioma were consistent.The expression levels of TGF-? and FAS ligand in glioma cells that co-cultured with two kind of cells at 24 h and 48 h were consistent(P>0.05)and higher than that before co culture(0h).The results showed that co-cultured C6 cells have consistent influence by microglia and macrophages.ConclutionCombined with the scratch test and WB test results,it can be considered that microglia and macrophages were acclimated by glioma cells and transformed into the same GAMs.That is to say,two kinds of cells can replace each other in vitro.