The Mechanism Of Astrocyte Promotes Oligodendrocyte Precursor Cells Proliferation Via Cx47 Activating SphK1/s1PR3

Degenerative disease of central nervous system is a kind of chronic diseases which is characterized by neurological degenerative changes.Myelin degeneration and damage is a common characteristic,with the amount decreased and the function missed of oligodendrocyte.Oligodendrocyte precursor cells have stable structures,and will proliferation,migration and differentiation into oligodendrocyte to generate myelin.So the study on OPCs proliferation could provide new ideas for demyelinating diseases.This research was divided into the following three parts.The first part: The astrocyte direct contact cultured with oligodendrocyte precursor cells to promote its proliferation via Cx47This part established different OPCs cultivation system,using 5-Ethynyl-2’-deoxyuridine and light microscope to observe cell growth.The second generation sequencing results were applied to analyze differentially expressed genes under different cultivation.The second generation sequencing results were applied to analyze the differentially expressed genes under different culture modes,and RT-PCR was used to verify its accuracy.The main results as followed:1.According to the light microscope results,direct contact group had more new cells,and proliferation ability better over time.Newborn cells number proportion increased significantly in direct contact group tested by EdU(P<0.01).2.The gene ontology function analysis of enrichment found that main functions of differentially expressed genes belong to the biological process,cell components and molecular functions between direct contact group and individual culture group.3.The second generation sequencing results showed that the FPKM value in direct contact group was obviously higher than that of other two groups.The multiple of RT-PCR was the same as RNA sequencing,which could verify the accuracy of the second generation sequencing results.The experimental results showed that ASTs might promote OPCs proliferation by Cx47.The second part: Cx47 activated sphingosine kinase 1/sphingosine 1-phosphate to promote oligodendrocyte precursor cells proliferationOn the basis that direct contact culture could promote OPCs proliferation,we made a further investigation about the signaling pathways downstream under different culture conditions.Enriched with GO function analysis found differentially expressed genes mainly distributed in the cell membrane and extracellular region and extracellular matrix.The G protein coupled receptor clustering analysis revealed that OPCs mainly express three subtypes,S1PR1、S1PR3 and S1PR5 respectively,with the differences of S1PR3 was mostly pronounced under different culture conditions.S1 P not only can function as a second messenger in the cell,but also secrete to the outside of the cell and to its specific receptors of S1 PRs,then playing a biological role such as cell proliferation,migration and differentiation,which are regulated by the activity of SphKs.This partial experiment needed to observe the expression of SphKs under different culture conditions by clustering analysis heat maps.SphK mainly express the two subtypes,which respectively is SphK1 and SphK2.High performance liquid chromatography was used to detect the concentration of S1 P in the supernatants of OPCs after Sph K1 blocked.CCK-8、EdU、FCM and light microscope observed the effects of S1 P on OPCs.Heat map analysised S1 PRs expression difference under different culture conditions and growth situation after S1 PRs blocked.The main results as followed:1.Heat maps showed that SphK1 was clearly different in direct contact group,and FPKM values were higher than the other two groups,but SphK2 had no obvious change in three groups.2.High performance liquid chromatography found S1 P concentration was lower in SphK1 blocked group than the control group(P<0.01).3.Both light microscope and CCK-8 results indicated that S1 P could promote OPCs proliferation and the mechanism was associated with the change of cell cycle(P<0.01).4.Both heat maps and FPKM values revealed that the expression of S1PR3 was higher in direct contact group than individual culture group.5.The newborn cells reduced after S1PR3 blocked(P<0.01).The ability of proliferation of S1 P was reduced after adding S1 P into S1PR3 blokers.The experimental results showed that the mechanism of Cx47 promote OPCs proliferation might be related to the activation of SphK1/S1 P.The third part: The S1P/S1PR3 could activate PI3K/Akt pathway of oligodendrocyte precursor cells to promote its proliferationWe had confirmed that S1 P could act on its specific receptor S1PR3 to promote OPCs proliferation,but what about downstream signaling pathways.This part detected the signaling pathways downstream of S1P/S1PR3 mainly by WB、FCM and Ed U.The main results as followed:1.The expression of protein PI3 K decreased after S1PR3 blocked;The expression of Akt had no obvious change,but p-Akt was significantly reduced after PI3 K blocked.2.The number of OPCs reduced(P<0.01)and many cells were arrested in G1 phase(P<0.01);The experimental results showed that S1P/S1PR3 activate PI3K/Akt signaling pathways to promote OPCs proliferation.