| Objective The myelination of neurons axons directly affects the function of the nervous system.It is of great significance to study the formation of myelin in clinical treatment of neurological diseases.Krox-20(Egr2),a regulator of myelination,directly activates the transcription of Eph tyrosine kinase receptor 4(EphA4),but whether EphA4 affects Schwann cell(SC)myelination is unknown.This article will establish Schwann cell and dorsal root ganglion neuron co-culture model,Schwann cell differentiation model,in vitro,using a variety of molecular biology and cell biology techniques to study the biological behavior of EphA4 during myelination in Schwann cells.The effect of establishing a rat sciatic nerve crush injury model and injecting siRNA in situ to study the effect of EphA4 on remyelination of axons during nerve regeneration.Method1.Immunohistochemistry or cytochemistry staining detect the expression of EphA4 in localization of dorsal root ganglia,sciatic nerve,Schwann cells,and dorsal root neurons.RT-PCR detect the expression of Eph family and its ligands in Schwann cells and dorsal root neurons.2.Establishment of SD rat sciatic nerve crush model,Western-blot detect at different time points(1d,3d,5d,7d,14 d,21d)the expression of Mag,Krox-20,P0 and EphA4,the normal sciatic nerve as a control.Immunohistochemistry staining detect the expression of EphA4 and Mag in rat sciatic nerve crush model at different time.3.Establish a myelination model in vitro to detect the expression of Mag,Krox-20,P0 and EphA4 during myelination.4.Design 3 siRNA of EphA4,RT-PCR and Western-blot screening efficiency of interference best siRNA,and based on the best siRNA design cholesterol methylated siRNA.5.Establish the Schwann cells differenciation model in vitro,and Western-blot detect the expression of Mag.6.Transfect the modified siRNA into Schwann cells and examine the effect of EphA4 on its proliferation,migration and differentiation.7.The modified siRNA was injected into the rat sciatic nerve crush in situ.After 14 days,the effect of EphA4 on myelination was detected by immunohistochemistry and Western-blot.Result1.Immune-histochemical and immune-cytochemical staining results showed that EphA4 cells expression of cytoplasm and membrane,and mainly expressed in the cell bodies of dorsal root neurons,and the axon portion is hardly expressed.RT-PCR results showed that EphA4 is expressed in Schwann cells and dorsal root neurons,and compared to other Eph family,EphA4 with higher receptor expression in a degree.2.The expression of Mag,Krox-20,P0 and EphA4 at different time points after sciatic nerve injury detected by Western-blot showed that EphA4 and the early myelin regulating factor Krox-20 showed a common expression.Immunohistochemical staining detection of MAG and EphA4 at different time points.Results showed that EphA4 and Mag have co-location in 7 days,14 days,21 days.It was speculated that EphA4 may be involved in the process of nerve regeneration in the early correlation of axon remyelination.3.After the myelination of Schwann cells and dorsal root neurons,there were obvious phase strips,and the expression of P0 and Mag increased significantly,indicating the successful establishment of in vitro myelinating model.4.RT-PCR detection of the process of myelination in vitro.The results showed that the expression level of EphA4 increased in the early stage of co-culture and decreased in the later stage,indicating that EphA4 may be involved in the early development of myelination.5.RNA interference and cytological experiments showed that EphA4 play a crucial role in early myelination.The learning of Schwann cell behavior(proliferation,migration and differentiation)of EphA4,which is to promote proliferation,depress differentiation,and show less of an effect on migration.6.Injecting modified siRNA into the pinch in situ interferes with the expression of EphA4.After 14 days the results of immunohistochemistry and Western-blot showed that compared to the control group,the number of myelin sheaths in the EphA4 interference group was relatively high,and it was speculated that EphA4 had a negative regulation on myelination.Conclusion1.EphA4 expressed in Schwann cells and cell bodies of dorsal root ganglion neurons.2.The myelinating process in vitro,EphA4 negative regulate myelination by promoting Schwann cell proliferation,and inhibiting differentiation.3.EphA4 inhibit remyelination of axons in nerve regeneration. |
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