Research On The Mechanism Of Miconazole Promoting The Development Of Oligodendrocyte Precursor Cell Differentiation And Myelination

Background:White matter damage(WMD),for which no effective therapy has been found yet,is one of the main causes for children with cerebral palsy or mental retardation.Oligodendrocyte progenitor cells(OPC)are the precursor cells of myelin-forming cells in the central nervous system,and OPC transplantation brings hope for WMD.However,the poor differentiation and myelination of transplanted human OPC in WMD rats inhibit the application of OPC.Many animal experimental studies have confirmed that though transplanted OPC can migrate to damage areas and live forever,most of cells can’t differentiate into mature cells,just remaining the stage of precursor cells.Therefore,it is necessary to establish promoting conditions for OPC differentiation and myelin regeneration.Objectives:1.Study of the protective effects of miconazole on corpus callosum of premature infants with cerebral WMD;2.To explore the influence of miconazole on neurobehavioral functions of premature infants with cerebral WMD;3.To investigate the mechanism of miconazole promoting OPC differentiation and myelin formation;4.To observe the effects of miconazole on OPC cultured in vitro.Methods:1.3 day old SD rats were subjected to the ligation of right carotid artery and hypoxia for 80min.Premature infants with cerebral WMD were received miconazole by intraperitoneal injection.HE and Myelin Basic Protein(MBP)immunohistochemical staining were used to identified the animal model.MBP immunohistochemical staining and Western blot detect the expression of cerebral white matter specific myelin basic protein(MBP)and to observe the change of myelin structure under an electron microscope.2.We test 28 day old rats’neurobehavioral functions with suspension experiment,slope and field tests.3.ERK and p-ERK expression level were detected by Western blot method in vivo and in vitro.4.NSCs are successfully isolated and cultured from human embryonic tissue,then induced and differentiated into OPC.Then morphological features of OPC were observed under inverted microscope,the cultured cells were identified by immunocytochemistry staining.Miconazole and phosphorylated inhibitor intervene the cultured OPC respectively,and then through the CCK 8 method to test whether miconazole has the function of promoting the growth and proliferation of OPC.To observe whether the OPC cultured in vitro can be induced to differentiate into mature OL by MBP/CC1 immunocytochemistry staining.Results:1.HE staining and MBP immunohistochemical staining confirm that we successfully established the model of premature infants with cerebral WMD;After treatment with miconazole,the quantity of MBP expression in corpus callosum increased significantly(P<0.05),and demyelination and thickness decrease of myelin sheath caused by ischemia and hypoxia have been obviously improved.2.Suspension experiment,slope and field test in miconazole intervention group were obviously better than the control group,and the difference was statistically significant(P<0.05).3.Western Blot confirmed miconazole significantly increased phosphorylated ERK expression(p-ERK),whereas phosphorylation inhibitors reduced its expression level.4.we successfully differentiated neural stem cells into OPC.The shape of OPC was round or oval,with typical bipolar protrusions,rare in three pole;The expressing of Sox 10,PDGF α,O4,GAIC were positive;Miconazole not only promotes the growth of OPC proliferation,and improves cell purity of OPC;However,MBP/CC1 immunohistochemical were negative.Conclusion:Miconazole protects premature infants with cerebral WMD induced by ischemia hypoxia by promoting the formation of the myelin sheath.