| Part 1 The effects of IFN-γ on the activity,proliferation,differentiation and oxidative stress expression level of NSCs in vitroObjective To investigate the effects of IFN-γ on the activity,proliferation,differentiation and oxidative stress level of NSC in vitro,and compare with other four cytokines such as BDNF,VEGF,TGF-β1 and IGF-1.Methods To extract primary NSCs from fetal rats,and identify cell-specific markers by immunofluorescence and observing cell morphology.NSCs were treated with different concentrations of IFN-γ,the cell morphology evaluation and CCK8 assay were used to check whether IFN-γ was toxic to NSCs,and compared with other four kind of cytokines such as BDNF,VEGF,TGF-β1 and IGF-1 to evaluate the proliferation and differentiation of NSCs.The effects of IFN-γ on cell activity,signaling pathway and oxidative stress level were detected by WB and qPCR.Results The primary NSCs were neurosphere-like in vitro.We confirmed that NSCssignificantly expressed the specific marker Nestin by immunofluorescence.And NSCs successfully differentiated into neurons(Tuj1),astrocytes(GFAP)and oligodendrocytes (MOG).CCK8 test showed that IFN-γ had no toxic effect to NSCs,and 20 ng/ml concentration of IFN-γ can promote cell proliferation,but showed no significant difference compared with other four kind of cytokines.Under differentiation condition,IFN-γ can promote NSCs to differentiate into more neuronswhich were Tuj1 positive compared with other four kind of cytokines.In addition,the results of WB and qPCR shown that IFN-γ activated NSCs,activated Jak1/Stat1 downstream pathwayand increased intracellular SOD2 expression level which was a marker of oxidative stress.Conclusions Primary NSCs were successfully extracted from the hippocampus of fetal rats.IFN-γ had no obvious toxic effect to NSCs.IFN-γ can promote NSCs to differentiate into neurons under differentiation condition.IFN-γ affected the status of NSCs in the early stage,increased cell ability to resist oxidative stress,and activated Jak1/Stat1 downstream signaling pathway.Part 2 The effect of combined IFN-γ withNSCs transplantation therapy for cerebral ischemic rats in vivoObjective To investigate the potential therapeutic role of IFN-γ combined withNSCs in the treatment of ischemic stroke rats.Methods Adult male SD rats were subjected to transient focal cerebral ischemia by MCAO method.Different concentrations of IFN-γ(50ng or 500ng)were injected alone or in combination withNSCs into rats,the neurological functions were evaluated by m NSS and Rotarod tests.TTC staining was used to calculate the volume of cerebral infarction after treatment for 28 days.The Nissle and TUNEL staining were used to evaluate nerve cell degeneration and necrosis.Double-labeled immunofluorescence staining(Brdu+DCX/GFAP)was used to detect the differentiation of transplanted NSCs in cerebral ischemic area of rats.The expressions of microglia(Iba1)and T cells(CD4+ and CD8+)around cerebral ischemic regions were evaluated.Furthermore,the expressions of four pro-inflammatory factors(IL1β,IL6,TNF-α and IFN-γ)and two anti-inflammatory factors(IL-10 and TGF-β1)were detected by ELISA which were from cerebrospinal fluid of rats.And the downstream pathway and oxidative stress level were also investigated.Results Low concentration of IFN-γ(50ng)synergistically increased the therapeutical effects of transplanted NSCs on MCAO rats.The improvement of neurological functions at 14 and 28 days after injection was better than that of NSCs alone,and the infarct volume was further reduced.IFN-γ synergistic therapy also promoted the nerve cell survival and increased the neuronal differentiation of transplanted NSCs.However,high concentration of IFN-γ(500 ng)increased the level of inflammatory response(such as microglia and T cell activity)in vivo,but did not affect the therapeutical efficiency of transplanted NSCs.After co-administration withNSCs,NSCs can neutralize the effects of high concentration of IFN-γ,including reduced level of inflammation.Finally,IFN-γ also activated the Stat1 pathway.Conclusions Transplantation of IFN-γ alone had no effective in rats with cerebral ischemia.Low concentration of IFN-γ(50 ng)combined withNSCs can exert synergistic therapeutical effects to further promote neurological recovery,reduce nerve cell apoptosis,and promote the neuronal differentiation of transplanted NSCs.High concentration of IFN-γ(500ng)increased the level of neuro-inflammation,but NSCs neutralized or attenuated the high level of neuroinflammatory.Part 3 The roles of exosomes derived from hNSCs and stimulated by IFN-γ in vitro and in vivoObjective Due to the successful acquisition of human NSCs(hNSCs),subsequent experiments were performed on h NCSs.To explore the potential functions of exosomes derived from normal and IFN-γ stimulated hNSCs,and to compare their potential functions in vitro and in vivo.Methods Primary hNSCs were extracted from fetal cerebral hippocampus,their characteristics were identified mainly by cell morphology and immunofluorescence assays etc.Theexosomes were extracted from cell culture supernatant by ultracentrifugation using exosomes extraction kits and their morphology and size were identified by TEM,NTA and WB.Cellular stress model was induced by hydrogen peroxide(H2O2)then exosomes were added to evaluate cell viability and apoptosis by CCK8,live-dead cell assay and immunofluorescence etc.PKH67 was used to label exosomes in order to observe their migration and distribution in vitro and in vivo.Furthermore,exosomes were stereotactically transplanted into MCAO rats in order to evaluate their therapeutical ability,including neurological functions,infarct volume,nerve cell activity and neuro-vascular remodeling.Results Primary hNSCs showed typical neurospheres in vitro,expressed specific markers Nestin,SOX2 and Musashi1,and successfully differentiated into three types of cells.Exosomes derived from hNSCs presented typical morphology and size characteristics of exosomes,expressed specific markers Hsp70,CD63 and Tsg101.Exosomes significantly increased cell viability and reduced cell apoptosis under H2O2 stress model in vitro experiments.Furthermore,exosomes can alter the differentiation direction of hNSCs.Exosomes effectively promoted the neurological recovery of MCAO rats,reduced infarct volume,promoted the survival of thenerve cell,increased the neurogenesis and angiogenesis.Exosomes exerted more efficiency in vivo experiments which were derived from hNSCs under IFN-γ stimulation.In addition,PKH67-labeled exosomes migrated into cells and to distant areas in vitro and in vivo.Conclusions Exosomes derived from hNSCs were successfully extracted.Exosomes can increase cell proliferation and decrease cell apoptosis under H2O2 stress.Transplantation of exosomes can effectively improve the neurological functions of cerebral ischemic rats,reduce infarct volume,and promot the neuro-vascular remodeling,especially with more efficiency in exosomes which were produced under IFN-γ stimulation.Part 4 Experimental study on miRNAs expression profile of exosomes derived from normal and IFN-γ stimulated hNSCs by NGSObjective High-throughput sequencing was used to detect the differential expression miRNAs in exosomes derived from normal and IFN-γ stimulated hNSCs,then to verify and study the potential functional miRNAs,and to explore their potential regulatory mechanism.Methods Exosomes derived from normal and IFN-γ stimulated hNSCs were obtained by ultracentrifugation.After extracted their RNA,high-throughput sequencing was performed.Differential expressed miRNAs were analyzed,such as their target genes,GO and KEGG pathway.qPCR was used to verify the differential expressed miRNAs.After transfected miRNA minics into hNSCs,the cell apoptosis was evaluated under H2O2 stress model.And the specific cel-miR39-3p was transfected with exosomes in order to evaluate exosomes’ transduction effects on hNSCs.Results RNA extracted from exosomes which were derived from normal and IFN-γ stimulated hNSCs was analyzed(including total Reads number and nc RNA annotation).A total of 47 differential expression miRNAs were found by database correction(24 up-regulation and 23 down-regulation).After adjusted differential fold and P values(|log2(Fold Change)|≥2,and P<0.01),7 differential significant miRNAs(hsa-miR-206,hsa-miR-133a-3p,hsa-miR-3151-5p,hsa-miR-205-5p,hsa-miR-3656,hsa-miR-34c-3p and hsa-miR-4677-5p)were verified by qPCR.And exosomes were successfully transfected with cel-miR-39-3p into hNSCs for expression.Under H2O2 stress model,hNSCs which were transfected with hsa-miR-133a-3p and hsa-miR-3656 significantly reduced cell apoptosis compared with control group.Conclusions NGS successfully analyzed the miRNAs expression profile of exosomes derived from normal and IFN-γ stimulated hNSCs.Differential expressed miRNAs were screened,and 7 miRNAs with significant difference were identified.Exosomes can play biological role by transmitting exosomal miRNAs into cells.And exosomes derived from hNSCs stimulated by IFN-γ reduced cell stress ability and cell apoptotic level,mainly by transmitting hsa-miR-133a-3p and hsa-miR-3656 into cells. |
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