| Neurodegenerative disease is a chronic and progressive neurological disease.This kind of disease has a high incidence of disease,a long period of illness,the lack of effective means of treatment and measures to the individual,family,society has brought heavy economic burden and social burden.Although the location and etiology of these diseases are different,but the destruction of myelin or demyelinating lesions is their common.Such as multiple sclerosis,is a common degenerative disease of the central nervous system.Although there are many studies on multiple sclerosis,the mechanism of oligodendrocyte reduction is still unclear.Oligodendrocytes(oligodendrocytes,OLs)are the only myelin forming cells of the central nervous system.The precursor cell of oligodendrocyte precursor cells(oligodendrocyte progenitor cell,OPC)has a good proliferation ability in vivo proliferation,migration,and differentiation into mature oligodendrocytes and myelin formation play an important role.How to obtain a large number of OPC is the key to the treatment of demyelinating diseases,whether endogenous or exogenous.We found that AST(astrocytes)and OPC co culture promote proliferation,but the specific mechanism is not clear.In this study,we investigated the effect of astrocytes on the proliferation of OPC.It provides a new experimental basis and theoretical basis for the treatment of demyelinating diseases.This study is divided into the following three parts:Part 1: astrocyte promotes the proliferation of oligodendrocyte precursor cells via Cx47In this part,the cell cycle and proliferation were detected by flow cytometry and EdU under different culture methods of astrocytes and OPC.All the differentially expressed genes under different conditions were analyzed by the two generation sequencing.The effective gene Cx47 was screened.The effective Cx47 siRNA,WB and PCR were used to detect the changes of Cx47 protein gene,the cell cycle was detected by flow cytometry,and the cell proliferation was detected by EdU.The main results are as follows:1.OPCs and ASTs direct contact co culture,light microscopy,flow cytometry and EdU assay showed that ASTs promoted OPCs proliferation compared with single culture group and the supernatant was more obvious,the cell cycle in S phase increased(p<0.05),the number of new cells increased(p<0.001).2.OPCs and ASTs direct contact co culture,Cx47 promoted the proliferation of OPCs.3 after interference with Cx47,the expression of Cx47 protein and gene decreased significantly(p<0.001),cell cycle arrest(p<0.01),the proliferation ability was reduced(p<0.001).These results suggest that ASTs may promote the proliferation of oligodendrocyte precursor cells by direct contact with Cx47.In the second part,astrocytes can promote the proliferation of oligodendrocyte precursor cells by up regulating S1PR3 expression by Cx47Confirmed in the co culture can promote the proliferation of OPCs precursors,we further compared with the single culture change signal downstream film under co culture,co culture group of differentially expressed genes in GO cell component analysis showed mainly in extracellular matrix,extracellular,cell membrane by contact.G-protein receptor activity cluster analysis showed that the hot spots in the S1 PRs family,the expression of three subtypes,S1PR1,S1PR3,S1PR5,especially the difference of S1PR3 was significantly higher than that of the single culture group.This part contact in ASTs and OPCs were cultured and cultured separately under the condition of further examined the mechanism of Cx47 on oligodendrocyte precursor cell proliferation,flow cytometry,confocal detection after Cx47 interference on the expression of Ca2+,WB,PCR detection of downstream S1PR3 expression,flow cytometry detected the cell cycle.Cell proliferation was detected by EdU.WB was used to detect the changes of S1PR3 protein,and the cell proliferation was detected by EdU.Flow cytometry detected the cell cycle.The main results are as follows:1.After Cx47 interference,the concentration of Ca2+ decreased(p<0.05)by flow cytometry,which was consistent with the confocal(p<0.001).2.the change of the downstream receptor signal was the most significant in the S1 PRs family in co culture and culture,especially in S1PR3.3.S1PR3 protein and gene expression decreased after Cx47 interference(p<0.001).4.After S1PR3 antagonised,cell cycle arrest(p<0.05)was detected by flow cytometry and cell proliferation was decreased by EdU(p<0.001).These results indicated that Cx47 stimulated Ca2+ influx in the co culture of OPCs and ASTs,and up-regulated the expression of S1PR3 to regulate the proliferation of OPCs.In the third part,astrocytes promote the proliferation of oligodendrocyte precursor cells by S1PR3 and activate the ERK1/2 pathwayIn this part,we studied the mechanism of Cx47 regulating the expression of S1PR3 to promote the proliferation of oligodendrocyte precursor cells in the condition of co culture of ASTs and OPCs.Cx47 interference,ERK1/2 blocked,WB detection of ERK1/2,p-ERK1/2 protein changes,PCR detection of changes in Id4.Cell proliferation was detected by EdU.After S1PR3 antagonism,the protein of ERK1/2 and p21 was detected by WB.The main results are as follows:1.After S1PR3 treatment,p-ERK1/2 decreased(p<0.001),p21 expression increased(p<0.05),cell proliferation ability decreased(p<0.001).2.After ERK1/2 blockade,the expression of Id4 was decreased(p<0.05),the cell cycle arrest(p<0.05)and the ability of cell proliferation were decreased(p<0.001).3.S1PR3 can increase the phosphorylation of ERK1/2,reduce the proportion of G1 phase of OPCs,promote OPCs into the S phase,thus regulating its proliferation.These results suggest that S1PR3 promotes the proliferation of oligodendrocyte precursor cells by activating ERK1/2 pathway... |
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