| Myelin is an insulating protective membrane that surrounds the axons of nerve cells and plays a vital role in the transmission of axons.It can transmit nerve impulses quickly and accurately downwards.As the society ages,the incidence of age-related neurodegenerative diseases is increasing year by year.More and more research supports Alzheimer’s disease,Parkinson’s disease,Huntington’s disease,multiple sclerosis,and multiple systems.Different degrees of myelin damage occur in neurodegenerative diseases such as atrophy,and myelin damage is also an important factor in its pathogenesis.How to promote regeneration and repair of myelin is the key to the treatment of such neurodegenerative diseases when myelin damage occurs in the body.However,oligodendrocytes(OLs),the only myelin-forming cells in the central nervous system,do not possess cell division and proliferation,and their proliferation depends on oligodendrocyte precursors(Oligodendrocyte precursors).The proliferation and differentiation of cells,OPCs).In our previous experimental studies,it was found that astrocytes(ASTs)can promote the proliferation of OPCs through direct contact with OPCs,but the mechanism is still unclear.In this experiment,three models of ASTs and OPCs direct contact co-culture,OPCs and ASTs supernatant culture and OPCs were cultured separately,and a series of interventions were carried out on OPCs.It was found that ASTs established information transmission channels through Cx47 and OPCs.ASTs transmitted proliferative information to OPCs,which stimulated the expression of Chi3l1 gene in OPCs.Chi3l1 acts on autologous and paracrine forms on Myh9 receptors on the surface of autologous or adjacent OPCs,up-regulating the expression of cyclin D1 in OPCs.Cyclin as a protein that regulates the cell cycle,cyclin D1 can promote the transition of cells from G1 to S phase,thereby regulating the cell cycle of OPCs and promoting the proliferation of OPCs.This study can provide new therapeutic targets and theoretical basis for neurodegenerative diseases.The study is divided into three parts:Part Ⅰ: ASTs establish information transmission channels through Cx47 and OPCs to promote the proliferation of OPCsIn this part,by constructing different in vitro culture models of OPCs,the cell proliferation ability was detected by inverted phase contrast microscopy,flow cytometry and EdU;the differentially expressed genes were analyzed by second generation sequencing of OPCs collected by the above culture methods,and the GO function enrichment analysis was used.The differential gene Cx47 was screened out by sequencing.After interference with Cx47 under direct contact co-culture conditions,the proliferation ability of OPCs was detected by flow cytometry and EdU.The main results are as follows:1.For OPCs culture alone,OPCs and ASTs supernatant culture,OPCs and ASTs under direct contact co-culture conditions,OPCs proliferative capacity detection,inverted phase contrast microscopy,flow cytometry and EdU results show that direct contact culture The proportion of OPCs in S phase was significantly higher than that in supernatant co-culture and culture conditions alone(p<0.01),and the proportion of new cells was also significantly increased(p<0.01).2.The OPCs under the above three culture conditions were collected for second-generation sequencing analysis of differentially expressed genes,and the differentially expressed genes with GO function annotation to channel activity were analyzed for heat,and Cx47 was screened and identified.3,OPCs and ASTs direct contact culture conditions for Cx47 siRNA interference,Western blot and immunofluorescence detected Cx47 expression decreased significantly(p <0.01),flow cytometry and EdU test results show that OPCs cell proliferation ability It decreases as the expression level of Cx47 decreases.The above results demonstrate that ASTs promote the proliferation of OPCs by inducing Cx47 expression of OPCs by direct contact with OPCs.Part Ⅱ: ASTs stimulates Chi3l1 secretion to promote OPCs proliferation through Cx47 channel established by direct contact with OPCsIn this part,the differentially expressed genes of the second-generation sequencing results in the first part of the experiment were further analyzed by GO function,and the differentially expressed genes enriched in extracellular secretion were analyzed by volcano mapping,and the differentially expressed gene Chi3l1 was screened;under contact co-culture conditions After interference with Cx47,the expression of Chi3l1 in OPCs was detected by Western blot and immunofluorescence.The proliferation of OPCs was detected by flow cytometry and EdU after adding exogenous Chi3l1 in OPCs alone.Then western blot analysis of cyclin D1 expression.The main results are as follows:1.GO analysis and volcano mapping showed that the expression of Chi3l1 was significantly different in OPCs under different culture conditions.2.After siRNA interference with Cx47,the results of Western blot showed that the gray value of Chi3l1 was significantly decreased(p<0.01).The immunofluorescence results showed that the fluorescence intensity of Chi3l1 was significantly attenuated after siRNA specific interference with Cx47(p<0.01).3.Exogenous Chi3l1 was added under the condition of OPCs culture alone.Flow cytometry showed that the proportion of OPCs entering S phase increased(p<0.05),and EdU showed an increase in the proportion of new cells(p<0.01).4.Chi3l1 was added under the condition of OPCs culture alone.The expression of cyclin D1 was increased by Western blot(p<0.01).The above results indicate that ASTs can stimulate the secretion of Chi3l1 to promote the proliferation of OPCs by up-regulating the expression of Cx47 in OPCs.Part Ⅲ: Chi3l1 stimulates the expression of cyclin D1 by binding to Myh9 of OPCs.In this part,the Chi3l1 antibody was mixed with the agarose beads and the protein homogenate of OPCs,and then the Pull-down experiment was performed.The pull-down protein was subjected to gel electrophoresis and Coomassie blue staining,and the stained bands were subjected to mass spectrometry to obtain Myh9 protein.Afterwards,Myh9 was verified by Co-IP experiment using Myh9 antibody and agarose beads and OPCs protein homogenate.The presence or absence of Chi3l1 was detected by Western blotting.The siRNAs under the condition of exogenous Chi3l1 were used to interfere with Myh9,the expression of Myh9 and cyclin D1 was detected by Western blot,the fluorescence intensity of Myh9 was detected by immunofluorescence,and the proliferation ability of OPCs was detected by flow cytometry and EdU.The main results are as follows:1.Mass spectrometry analysis showed that the Myh9 protein was contained in the pull-down protein band.2.In the Co-IP experiment,the protein band of Chi3l1 was significantly deeper than that of the goat IgG group in the Mye9 antibody group at the same protein concentration,confirming that Myh9 can bind to Chi3l1.The expression of Myh9 was decreased by Western blot and immunofluorescence after siRNA interference with Myh9(p<0.01).The results of flow cytometry showed that the proportion of OPCs entering S phase decreased with the interference of Myh9(p<0.01).The results of EdU assay for cell proliferation were consistent(p<0.01).Meanwhile,after siRNA interfered with Myh9,the expression of cyclin D1 was decreased by Western blot(p<0.05).These results indicate that Chi3l1 can bind to Myh9 on the surface of OPCs to activate the expression of cyclin D1 and promote the proliferation of OPCs. |
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