Regulatory Mechanism About Myelination Of CXCL12/CXCR4 On Rat Oligodendrocyte Precursor Cells

BackgroundAxonal demyelination of spinal cord injury is an important pathological changes,seriously affecting the retention function of axons.Transplantation of oligodendrocyte precursor cells(OPCS)can promote axonal remyelination,however remyelination exact mechanism is not very clear,thin and short myelin regeneration,and the original form and function of myelin have certain distinction.Therefore,to clarify the mechanism of axonal remyelination has important implications for the treatment of spinal cord injury.There are experiments showed increased expression of CXCL12 after spinal cord injury,and the migration and differentiation of OPCs closely.RAF-MEK-ERK and PI3K/Akt pathways involved in remyelination of axons and is CXCL12/CXCR4 common downstream pathways.This topic speculate: CXCL12 by interacting with its receptor CXCR4,not only to mediate transplant directional migration and proliferation of OPCs in vivo,and can be directly involved in remyelination of axons regenerated through ERK and PI3 K pathways affect axon myelination.Studies using a variety of techniques intended to clarify the role of CXCL12/CXCR4 molecular signals in myelination of axons in for the treatment of spinal cord injury provides a new idea.Method1.Sprague-Dawley(SD)rats in newborn 1-2 days,modified oscillatory separation and differential adherence methods for primary culture of OPCs,identified by the immunocytochemical technique.2.Detection of concentration-effect correlation,The expression of myelin basic protein(MBP)and proteolipid protein(PLP)were detected by Western blot under different concentrations of CXCL12(0 ng/mL,5 ng/mL,10 ng/mL,20 ng/mL).The expression of MBP and PLP was showed by immunofluorescence assay.3.Detection of time-effect correlation,the expression of MBP and PLP were detected by Western blot at different time(0h,12 h,24h,48 h,72h)under the condition of CXCL12(20 ng/mL).The expression of MBP and PLP was showed by immunofluorescence assay.4.The expression of CXCR4 was inhibited by CXCR4 siRNA.The expression of MBP and PLP was detected by Western blot under the condition of CXCL12(20 ng/mL).The expression of MBP and PLP was showed by immunofluorescence assay.5.The activity of PI3K/AKT pathway was inhibited by LY294002,and the activity of ERK pathway was inhibited by U0126.The expression of MBP and PLP was detected by Western blot under the condition of CXCL12(20 ng/mL).Immunofluorescence assay was used to detect the expression of MBP and PLP.Result1.A large number of OPCs with high purity can be obtained by modified oscillatory separation and differential adherence method.The cell body is approximately circula with symmetrical bipolar or tripolar cell processes.The cell body is small with diameter of 10?m and well refractivity,the expression of PDGFR-? is positive.The mid-term OPCs are round,the expression of O4 is positive.A large number of reticular processes appeared in the late cell body,the expression of MBP is positive.2.In a certain concentration range,increase the concentration of extracellular CXCL12,MBP and PLP expression increased gradually.Compared with 0 ng/mL,the expression of MBP and PLP was significantly increased(P<0.01,P <0.01).when the concentration of CXCL12 was 20 ng / ml.Immunofluorescence assay showed that both MBP and PLP were observed under the condition of CXCL12(10 ng/mL and 20 ng/mL).3.The expression of MBP and PLP increased gradually over a period of time under the condition of CXCL12(20 ng/mL).Compared with 0h,the expression of MBP and PLP was significantly increased at the time of 72h(P<0.01,P<0.01).Immunofluorescence assay showed that both MBP and PLP were observed under the condition of CXCL12(20 ng/mL)at the time of 48 h and 72 h.4.The expression of MBP and PLP was significantly decreased after CXCR4 inhibited by CXCR4 siRNA compared with the control group(P<0.01).Immunofluorescence assay showed that the expression of MBP and PLP was significantly decreased after CXCR4 siRNA interference compared with the control group.5.The activity of PI3K/AKT and ERK pathway was inhibited by LY294002 and U0126.The expression of p-ERK and p-AKT in CXCL12 group was higher than that in control group(P<0.01;P<0.05).The expression of p-ERK and p-AKT was significantly increased by U0126 inhibition by LY294002.Compared with CXCL12 group,the expression of p-AKT in CXCL12+LY294002 group was significantly decreased(P<0.01),while the expression of p-ERK in CXCL12+U0126 group was also significantly decreased(P<0.05).Western blot and immunofluorescence assay showed that the expression of MBP and PLP in both CXCL12+LY294002 and CXCL12+U0126 group was significantly lower than that in CXCL12 group.Conclusion1.In this experiment,a large number of OPCs with high purity can be obtained by modified oscillatory separation and differential adherence method.2.In a certain concentration range of extracellular CXCL12 over a period of time,the expression of MBP and PLP increased gradually.3.The expression of MBP and PLP was significantly reduced after inhibit CXCR4 expression,AKT and ERK pathway activity.