Establishment Of Culture Method Of NG2 Glial Cells In Vitro And Preliminary Investigation Of Their Function

Recent studies suggest that a variety of the central nervous system(CNS)diseases such as Parkinson's disease,Alzheimer's disease and multiple sclerosis are closely associated with neuroinflammation.Investigation of the mechanism of neuroinflammation is important for developing new therapies for the treatment of these diseases.Microglias and astrocytes are known as immune cells in brain,the aberrant interaction between these two types of glial cells contributes to the development of the neuroinflammation.NG2 glial cells were the fourth kind of glial cells found in brain,and they are widely distributed throughout the mammalian brain.NG2 glial cells have been found activated in injury and degenerative disease of CNS,these diseases model is accompanied by neuroinflammation,suggesting that NG2 glial cells may participate in the neuroinflammation.However,there roles in the maintenance of immune hemostasis in the brain is unclear.We established a culture procedure in vitro to get NG2 glial cells with high purity.We also investigated the function of NG2 glial cells in vitro model.We found that the NG2 glial cells showed distinct response to LPS stimulation from microglial cells.NG2 glial cells may produce and release a souble factor(s)to elevate the CX3CL1 protein expression level in neurons thereby suppressing inflammation.Our data provide useful information for understanding the role of NG2 glial cells in the diseases of the nervous system.AIM: To investigate the functions and mechanisms of NG2 glial cells in the CNS.METHODS: 1)Use new born SD rat's cortex to purify and culture NG2 glial cells in vitro by shaking method,add PDGF-AA and b FGF in serum free medium to support these cells.2)Treat the cultured NG2 glial cells with 50 ng/ml LPS,collect cells after 6,12 and 24 h of LPS exposure,respectively,then detect the changes of proinflammatory factors IL-6,IL-1b,TNF-a by real-time PCR.3)Treat the adult C57 mice with intraperitoneal injection of 5 mg/kg LPS,use immunohistochemical staining to observe NG2 glial cells and microglial cells after 4,8,24 h after LPS challenge,respectively.4)Detect CX3CL1 expression levels in neurons after NG2 glial cells conditional medium treatment by using western blot and real-time PCR.RESULTS: 1)The shaking-off culture method we established vield NG2 glial cells with more than 90% purity.The cells cultures we cultured contained 90% of NG2 glial cells,about 3% of astrocytes and 2% of microglia cells,while oligodendrocytes was almost absent.2)NG2 glial cells did not express classic inflammatory factors after LPS stimulation both in vivo and in vitro.The m RNA levels of inflammatory cytokines such as IL-6,IL-1b and TNF-a of BV2 cells were significantly increased after LPS stimulation for 6h,12 h,24h,while m RNA levels of the three factors in NG2 glial cells were not significantly changed.NG2 glial cells did not express classic inflammatory factors after LPS stimulation in vivo.3)The way in which NG2 glial cells respond to inflammatory stimuli was different from microglial cells in vivo.Activation level of NG2 glial cells and microglial cells rised first quickly and then activation of microglial declined eight hours following LPS stimulation,but the microglia would reactivated after the decline.4)Treatment of cultured neuronal cells with conditional medium of NG2 glial cells increased the expression levels of CX3CL1 in neurons.Treatment of cultured neuronal cells with the conditional medium of NG2 glial cells significantly increased protein and m RNA levels of CX3CL1 in neurons in a dose-dependent fashion.The conditional medium of astrocytes also had the effect.While the conditional medium of BV2 glial cells was not effective in raising the expression level of CX3CL1 in neurons.5)NG2 glial cells regulated the expression of neuronal CX3CL1 through molecules whose molecular weight were less than 30 k Da.The <30 k Da fraction of NG2 glial cells' conditional medium still retained the ability to upregulate the expression levels of CX3CL1 in neurons,while the >30 k Da fraction did not.Classical pro-inflammatory molecules such as TGFb1,TGFb2,IL1 ra,IL-4,IL-10 or IL-13 could not raise the CX3CL1 expression in neurons.CONCLUSION: 1.NG2 glial cells respond to inflammatory stimuli in a different way compared with microglias.2.NG2 glial cells secrete active ingredients to modulate the expression of CX3CL1 in neurons,thus play a role in suppressing neuroinflammation.