The Effects And Mechanism Of Magnetic Stimulation On Astrocytes Migration By PEA-15

Background and Object Phosphoprotein enriched in astrocytes of 15KDa(PEA-15) is a small, lies in the Iq21-22,death effector domain(DED)–containing protein,a regulator of apoptosis and existes widely in the cytoplas. Extracellular signal-regulated kinases(ERK1/2) control diverse cellular functions, such as proliferation, survival, and migration. Magnetic stimulation can activate the ERK pathway, promoting the migration of astrocytes, playing an neural protection role in the inflammation of the central nervous system after injury. The phosphoprotein PEA-15 is reported to regulate the proliferative ERK/MAPK pathway by binding ERK and preventing its nuclear accumulation.PEA-15 may be related to astrocytes migration with the magnetic stimulation. In this study, we check whether PEA-15 affect the migration of astrocytes by regulate ERK with magnetic stimulation in vitro experiments and discussion on its mechanism.Materials and Methods 1.Astrocytes’ extracting, culturing, identification and cell fluorescence labeling: Anatomy of new 1-3 day Sprague-Dawley(SD) rats gets the cerebral cortex tissue and using isolated differential velocity adherent technique culturing to get the astrocytes, observation of the morphology and growth. Immunofluorescence was applied to identification of expression of GFAP. 2.Experimental groups:there were three groups in vitro Wound scratch assay according to different intensity of Magnetic stimulation.(1)control group(Do not receive magnetic stimulation, but with other cells to stimulate the group into the same environment);(2)A group(receive 30% maximum stimulus intensity);(3)B group(receive 60% maximum stimulus intensity). 3. Experimental groups: there were four groups in vitro Wound scratch assay according to either receive Magnetic stimulation or interference PEA-15 expression.(1) A group(neither receive Magnetic stimulation nor interference PEA-15 expression);(2)B group(interferenced PEA-15 expression, but did not receive Magnetic stimulation);(3)C group(exposed to receive stimulation, but did not interference PEA-15 expression);(4)D group(exposed to receive stimulation and interference PEA-15 expression). Observe the scratches healing, and Western Blot was applied to research the expression of PEA-15 and protein phosphorylation. Result 1.Astrocytes were isolated from the cerebral cortex tissue of neonatal SD rats and applying isolated differential velocity adherent technique culturing astrocytes. GFAP was identified by Immunofluorescence, DAPI stained nucleus presented that astrocytes with high purity. 2.Wound scratch assay in vitro, we found that with the increasing intensity of magnetic stimulation, the scratches area healed faster, 30%-60% maximum intensity of magnetic stimulation, the migration speed and intensity of the magnetic stimulation were positively correlated, and has significant difference compared with the control group(P<0.05). 3. Cells transfected with fluorescent confirmed that the transfection reagent with high transfection efficiency, the transfected cells with PEA-15 si RNA, Western Blot results indicated si RNA270 has the best interference effect. In the Wound scratch assay, transfection and magnetic stimulation groups were more obvious than normal group in the migration. The P-PEA-15 of Magnetic stimulation group was increased obviously.Conclusion 1. Applying isolated differential velocity adherent technique and shaking table to culturing astrocytes, we can get astrocytes with high purity. 2. Wound scratch assay in vitro, in the 30%-60% maximum intensity of magnetic stimulation can promote astrocytes migration. 3. Interference expression of PEA-15 can significantly promote astrocytes migration, and magnetic stimulation may promote astrocytes migration by phosphorylation of PEA-15.