| Hypoxic ischemic encephalopathy (HIE) is the major recognized perinatal cause of neurological morbidity in full-term newborns and can result in mental impairment, seizures and permanent motor deficits, such as cerebral palsy. At present, there are no effective measures of treating HIBD (hypoxic-ischemic brain damage, HIBD)Ever since a long time ago, people considered that the central lesion could not be recovered. But now we have found that there are many endogenous neural stem cells(NSCs) at subventricular zone (SVZ) and hippocampus dentate gyrus(DG) lifetimes these cells can proliferate, differentiate by stimulation of pathologic impairments. It is found that NSCs were capable of being activated to proliferation and differentiation. However the capacity for recruiting endogenous NSCs is limited.Thus,other strategies used to modify endogenous neurogenesis after ischemic brain gamage have been described.Study showed there were proliferation of endogenous neural stem cells in hypoxic-ischemic neonate rats . Hyperbaric oxygen therapy has been used in HIE for decades, many studies have reported improved neurological outcomes with hyperbaric oxygen therapy. We also have found HBO therapy promoted the proliferation of endogenous neural stem cells in hypoxic-ischemic neonate rats. In a review, about hyperbaric oxygen therapy and cerebral ischemia, with an emphasis on the mechanisms of hyperbaric oxygen therapy-related neuroprotection, relevant literature was located in the National Library of Medicine and National Institutes of Health Database, and from bibliographies of articles reviewed. In the last 15 years,there have been no fewer than 65 animal and human studies of hyperbaric oxygen therapy in cerebral ischemic injury. Numerous studies have demonstrated a protective effect of hyperbaric oxygen therapy in experimental ischemic brain injury, and many physiological and molecular mechanisms of hyperbaric oxygen therapy-related neuroprotection have been identified. Hyperbaric oxygen therapy has been shown to ameliorate brain injury in a variety of animal models including focal cerebral ischemia, global cerebral ischemia, neonatal hypoxia-ischemia and subarachnoid hemorrhage.Human clinical studies have shown hyperbaric oxygen therapy may be beneficial in chronic cerebral vascular disease or in the setting of cardiopulmonary bypass to date.Recent studies have shown that HBO therapy may alleviate neuronal injury and promote the recovery of HIBD in rats an effect that is correlated with Wnt-3 protein. Several studies have shown that Wnt signaling is involved in cell fate decision during neurogenesis and embryonic development as well as in adult stem cells differentiation in the nervous system, the Wnt signaling plays a key role on modulating cell differentiation or proliferation states.β-Catenin is a critical downstream mediator of the canonical Wnt pathway, which functions in gene transcription and cell adhesion, and plays vital roles in the proliferation and differentiation of neural progenitor cells. Overexpression ofβ-catenin in cortical neural precursors leads to expansion of the precursor population and cortical overgrowth.On the contrary, conditional deletion ofβ-catenin from cortical neural precursors results in delamination of neuroepithelial cells, loss of adherens junctions, impaired radial migration of neurons, decreased cell proliferation. Studies have shown that in the presence of Wnt signals, (3-catenin is stabilized and translocates to the nucleus,where it interacts with transcription factors of the LEF/TCF(lymphoid enhancer-binding factor/T cell-specific tran-scription factor) family to induce changes in gene expression, theβ-catenin/TCF complex appears to directly regulate the promoter of neurogenin1(Ngn1), a gene implicated in cortical neuronal differentiation. Ngn1 promotes neural progenitor cell differentiation into neurons. Bone morphogenetic protein 4(BMP4) belongs tothe TGF-b (transforming growth factor-b) superfamily. Study has shown that BMP and Wnt signaling could antagonize each other for self-renewal or differentiation of stem cells at specific tissues and times, and BMP4 has a potential to repress neurogenesis and induce astrocyto-genesis of neuroepithelial cells .Therefore, transfectingβ-catenin siRNA into NSCs and then treatting with HBO after cultured with HIBD brain tissue extract conditioned cultures and normal brain tissue extract conditioned cultures respectively as previously described in vitro, we tested the hypothesis thatβ-catenin regulated the proliferation of rats NSCs after HBO therapy.Our investigation was divided into the follwing three parts. PartⅠConstruction and screening of eukaryotic expression plasmids containing short hairpin RNA targeting at the ratβ-catenin geneObjective To construct eukaryotic expression plasmids containing short hairpin RNA (shRNA) that target at the neural stem cells (NSCs)β-catenin gene, and to select the plasmids that silenceβ-catenin gene most efficiently.Methods Three pairs of shRNAs that target atβ-catenin gene were designed. The eukaryotic expression plasmids (named shRNA1-3) were constructed and identified sequencing analysis. The plasmids were then transfected into NSCs by electroporation. The transfection rate of recombinant plasmids was measured 48 h after transfection, andβ-catenin mRNA and protein expression was determined using reverse transcriptase-polymerase chain reaction and Western blotting.Results The expression plasmids were confirmed by sequencing analysis. The transfection rate of recombinant plasmids in NSCs was approximately 62.63%±15.9%. Forty-eight hours after transfection,theβ-catenin mRNA and protein levels of shRNA1-3 group were tested.Conclusions The shRNA eukaryotic expression plasmid targeting atβ-catenin gene is constructed and selected successfully. Theβ-catenin mRNA and protein expression was suppressed significantly in NSCs by this given plasmid.we can use it to study the role of the wnt/β-catenin signaling in the development of NSCs.PartⅡInfluence of HBO on NSCs differentiationObjective To investigate the influences of HBO and rat brain extracts on proliferation of rats neural stem cells in vitroMethods Immunocytochemical staining was performed simultaneously on the 9 groups of NSCs on precoated chamber slides. The NSCs were processed for immunofluorescent labeling of FITC and CY3. The nucleus were counterstained with Hoechst 33258. The percentages of FITC-positive and CY3-positive cells were determined. NSE-positive NSCs,O4-positive NSCs and GFAP-positive NSCs were counted. The data represent the mean±SD of six separate experiments.Results In vitro HBO alone promoted ncNSCs (NSCs infected with negative control siRNA) differentiate into neurons and oligodendrocytes but not depressed astrogliosis;HIBD or normal brain tissue extract cultures promoted ncNSCs differentiate into neurons and oligodendrocytes but depressed astrogliosis, the effectus of HIBD brain extract cultures was superior to the latter and could be further increased by HBO.β-catenin siRNA decreased the NSE-positive neurons and increased GFAP-positive astrocytes in the siNSCs (NSCs infected withβ-catenin siRNA) in vitro,the effectus can not be inversed by HBO, though can be alleviate.Conclusions (1) HBO could promote ncNSCs differentiate into neuronal or Oligodendrocyte, and inhibited ncNSCs differentiate into astrocytes (2)β-catenin siRNA depressed neurogenesis, and promoted astrogliosis. (3) HBO therapy promotes the proliferation of NSCs in vitro, an effect that is correlated withβ-catenin protein. PartⅢInfluence of HBO on the expression of Ngn1 and BMP4 in the NSCsObjective To investigate the influences of HBO and rat brain extracts on the expression of Ngn1 and BMP4 in the NSCsMethods Quantitative RT-PCR was used to detect the relative contents of Ngn1 mRNA and BMP4 mRNA in the 9 groups of NSCs. Western blot was used to detect the relative contents of Ngnl protein and BMP4 protein in the 9 groups NSCs.Results HBO alone increased the level of Ngn1 mRNA and decreased BMP4 mRNA of ncNSCs; HIBD and normal brain tissue extract cultures increased the level of Ngn1 mRNA and decreased BMP4 mRNA of ncNSCs, the effect of HIBD brain extract cultures is superior to the latter and treatment with HBO further increased it; transfection ofβ-catenin siRNA could down-regulate the expression of Ngn1 mRNA and up-regulate BMP4 mRNA of NSCs in vitro respectively. HBO alone increased the level of Ngn1 protein and decreased BMP4 protein of ncNSCs;HIBD and normal brain extract cultures increased the level of Ngn1 protein and decreased BMP4 protein of ncNSCs,and the effectus of HIBD brain extract cultures is superior to the latter; it is more significant for HIBD accompany HBO to increased the level of Ngnl protein; transfection ofβ-catenin siRNA could down-regulate the expression of Ngnl protein and up-regulate BMP4 protein of NSCs in vitro respectively. Conclusions (1) HBO alone increased the level of Ngn1 gene and decreased BMP4 gene of ncNSCs, (2) transfection ofβ-catenin siRNA could down-regulate the expression of Ngnl and up-regulate the expression of BMP4 of NSCs in vitro (3) there is potential cooperative actions of BMP4 and Ngn1 on differentiating rat neural stem cell in cerebral ischemic brain. The ability of Ngnl to promote neurogenesis may allow Ngn1 to act as a potent neuronal commitment factor.The main contents and conclusions of the research are summarized as following:1. The shRNA eukaryotic expression plasmid targeting atβ-catenin gene is constructed and selected successfully.2. HBO could promote NSCs cultured with HIBD brain extract cultures differentiate into neuronal or Oligodendrocyte, and inhibited them differentiate into astrocytes.3. HBO therapy promotes the proliferation of NSCs in vitro, an effect that is correlated withβ-catenin protein.4. HBO therapy could promote neurogenesis byβ-catenin-induced activated Ngn1, could repress astrocytogenesis byβ-catenin-induced down regulated BMP4. |
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