Regulation And Mechanisms Of Neural Stem Cell Exosomes On Neural Stem Cell Differentiation And Vascular Endothelial Cell Function

Part One: Regulation and mechanisms of neural stem cellderived exosomes on neural stem cell differentiation Backgrounds:Neurodegenerative diseases are diseases in which the gradual loss of neuronal or glial cell structure and function or even death causes neurological dysfunction,and increasing the number of functional neurons and glial cells to promote neurogenesis and gliogenesis is an effective measure to treat neurodegenerative diseases.In our previous studies,we observed that induced neural stem cell-derived exosomes reprogrammed from primary cultured mouse embryonic neural stem cells and astrocytes have the potential to promote neural differentiation,but the exact role of neural stem cell-derived exosomes on the differentiation of neural stem cells into neurons and astrocytes and the specific mechanism remains largely unknown.Objective:The aim of this study was to investigate the regulatory role and potential mechanisms by which neural stem cell-derived exosomes promote the differentiation of neural stem cells into neurons and astrocytes.Methods:1.Mouse embryonic neural stem cells were cultured in primary,exosomes were extracted by gradient centrifugation and identified by transmission electron microscopy,protein immunoblotting(western blot,WB),nanoparticle tracking analysis and nanoflow cytometry,PKH67-labelled exosomes were co-cultured with neural stem cells and the uptake of exosomes by neural stem cells was observed.2.Mouse embryonic neural stem cells were co-cultured with their exosomes and the effect of exosomes on the differentiation of neural stem cells into neurons and astrocytes was observed by immunofluorescence and real-time quantitative PCR(RTq PCR).3.To detect the expression abundance of miRNAs in exosomes using small RNAs(micro RNAs,miRNAs)microarrays and RT-q PCR to identify target miR-9.4.Mouse embryonic neural stem cells were transfected with miR-9 agonist and antagonist agents,respectively,and the effects of miR-9 on the differentiation of neural stem cells into neurons and astrocytes were observed using immunofluorescence and RT-q PCR.5.Mouse embryonic neural stem cells were treated with miR-9 antagonist,exosomes were collected,and the expression of miR-9 in these exosomes was detected by RT-q PCR;exosomes with knockdown miR-9 were co-cultured with mouse embryonic neural stem cells,and the effect of knockdown miR-9 on the differentiation of neural stem cells into neurons and astrocytes was observed by immunofluorescence and RT-q PCR.6.RT-qPCR was used to screen miR-9 downstream target gene Hes1.WB was used to observe the effect of miR-9 on the expression of downstream target gene Hes1.Dual luciferase reporter system was used to determine the direct action of miR-9 in relation to downstream target gene Hes1.7.Hes1-siRNA was transfected into mouse embryonic neural stem cells and the effect on the differentiation of neural stem cells into neurons and astrocytes was observed by immunofluorescence and RT-q PCR.8.Mouse embryonic neural stem cells were treated with both Hes1-siRNA and knockdown miR-9 exosomes,and the effects on the differentiation of neural stem cells into neurons and astrocytes were observed by immunofluorescence and RT-q PCR.9.Hes1-siRNA transfection of mouse embryonic neural stem cells was followed by RT-q PCR to examine the expression of Notch signaling pathway genes Dll1,Notch1 and Notch2 in neural stem cells.Results:1.Exosomes of mouse embryonic neural stem cell origin promote the differentiation of neural stem cells into mature neurons and astrocytes.2.MiR-9 was expressed in highest abundance in mouse embryonic neural stem cell-derived exosomes.3.Overexpression of miR-9 promoted the differentiation of mouse embryonic neural stem cells and the maturation of neurons and astrocytes;knockdown of miR-9 inhibited the differentiation of mouse embryonic neural stem cells into mature neurons and astrocytes.4.Knockdown of miR-9 exosomes inhibits the differentiation of mouse embryonic neural stem cells into mature neurons and astrocytes.5.Hes1 is a target gene of miR-9 and overexpression of miR-9 in mouse embryonic neural stem cells inhibits Hes1 expression.6.Knockdown of Hes1 in mouse embryonic neural stem cells promotes the differentiation of neural stem cells into mature neurons and astrocytes.7.Knockdown of Hes1 in mouse embryonic neural stem cells restores the potential of miR-9-deficient exosomes to inhibit neural stem cell differentiation.8.Knockdown of Hes1 in mouse embryonic neural stem cells promoted the expression of Notch signaling pathway genes Dll1,Notch1 and Notch2 in neural stem cells.Conclusion:The mouse embryonic neural stem cell-derived exosome miR-9 promotes neural stem cell differentiation into mature neurons and astrocytes by targeting Hes1 to mediate neurogenesis and gliogenesis,providing a new strategy for developing therapeutic options for neurodegenerative diseases.Part Two: Regulation and mechanisms of neural stem cell-derived exosomes on brain microvascular endothelial cell functionBackgrounds:Under normal physiological conditions,the nervous and vascular systems regulate each other,especially the signal exchange between neural stem cells and peripheral cerebral microvascular endothelial cells throughout the life course can regulate neurogenic angiogenic processes,including the ability of neural stem cells to promote cerebral microvascular endothelial cell differentiation and angiogenesis.Under pathological conditions,neural stem cells can promote the restoration of endothelial cell function.However,the regulatory role of neural stem cell-derived exosomes on cerebral microvascular endothelial cell function and the underlying mechanisms have not been fully elucidated.Objective:The aim of this study,together with the results of the previous part of the study,was to investigate the mechanisms by which mouse embryonic neural stem cell-derived exosomes regulate the proliferation,migration,survival and death of cerebrovascular endothelial cells via miR-9.Methods:1.Mouse embryonic neural stem cell-derived exosomes were co-cultured with mouse brain microvascular endothelial cells under normoxic and hypoxic conditions,and cell proliferation was detected by CCK8,cell migration by scratch assay and cell survival and death by Calcein-AM/PI staining.2.Mouse brain microvascular endothelial cells cultured under normoxic and hypoxic conditions were transfected with miR-9 antagonist,and cell proliferation was detected by CCK8,cell migration by scratch assay,and cell survival and death by Calcein-AM/PI staining.3.The exosomes with knockdown miR-9 were co-cultured with mouse brain microvascular endothelial cells under normoxic and hypoxic conditions,and cell proliferation was detected by CCK8,cell migration by scratch assay,and cell survival and death by Calcein-AM/PI staining.4.Hes1-siRNA transfected mouse cerebral microvascular endothelial cells cultured under normoxic and hypoxic conditions,cell proliferation was detected by CCK8,cell migration by scratch assay and cell survival and death by Calcein-AM/PI staining.5.Mouse brain microvascular endothelial cells cultured under normoxic and hypoxic conditions were cotransfected with miR-9 antagonist and Hes1-siRNA,and cell proliferation was detected by CCK8,cell migration by scratch assay and cell survival and death by Calcein-AM/PI staining.Results:1.Under normoxic conditions,mouse embryonic neural stem cell-derived exosomes promote proliferation and reduce cell mortality of mouse brain microvascular endothelial cells.Under hypoxic conditions,mouse embryonic neural stem cell-derived exosomes promoted the proliferation and migration of mouse brain microvascular endothelial cells and reduced cell mortality.2.Overexpression of miR-9 promoted the proliferation and migration of mouse brain microvascular endothelial cells and reduced cell mortality under normoxic and hypoxic conditions,while knockdown of miR-9 inhibited the proliferation and migration of mouse brain microvascular endothelial cells and increased cell mortality.3.Under normoxic and hypoxic conditions,knockdown of miR-9 exosomes promoted the proliferation and migration of mouse brain microvascular endothelial cells and reduced cell death.4.Under normoxic conditions,knockdown of Hes1 promoted proliferation and reduced cell mortality in mouse brain microvascular endothelial cells.Under hypoxic conditions,knockdown of Hes1 promoted the proliferation and migration of mouse brain microvascular endothelial cells and reduced cell mortality.5.The effects of knockdown of miR-9 on proliferation,migration,cell survival and death of mouse brain microvascular endothelial cells under normoxic and hypoxic conditions were partially reversed by knockdown of Hes1.Conclusion:Neural stem cell-derived exosomes target Hes1 via miR-9 to promote brain microvascular endothelial cell proliferation,migration and reduce cell mortality,which provides a new opportunity for neural stem cell-derived exosomes to regulate brain vascular endothelial cell function recovery to promote cerebral angiogenesis for neurodegenerative diseases.