| The hypoxic-ischemic encephalopathy (HIE), a serious complication of neonatal asphyxia, is the major cause of severe neurodevelopment disability (cerebral palsy, epilepsy, mental retardation), even death in children. Despite advances in technology allowing better obstetric and neonatal care, the current clinical management of HIE has still been limited to supportive measures. Therefore, to find an effective prevention and treatment of HIE is extremely urgent.Hyperbaric oxygen (HBO) treatment is one potential therapy that has been used for the treatment of neonatal diseases including HIE for many years. Recent studies have shown that HBO is neuroprotective by promoting cell proliferation after brain injury. Our recent research also showed HBO therapy could promote the endogenous NSCs to proliferate, migrate and differentiate, thus restore the hypoxic ischemic brain damage, the proliferation of NSCs in neonatal hypoxic ischemic rats after HBO is correlated with the activation of Wnt/beta-catenin signaling. However this comes from in vivo experiment, the exact molecular mechanism of stimulating the NSCs proliferation and differentiation by HBO remain uncertain without in vitro experiment.The term "neural stem cell" is used loosely to describe cells that can generate neural tissue or are derived from the nervous system, have some capacity for self-renewal, and can give rise to cells other than themselves through asymmetric cell division. Neural stem cells which can be promoted proliferation after injury are primarily restricted to two brain regions, the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus, where neurogenesis persists into adulthood. But what promote the proliferation of endogenous NSCs is unclear. Our previous studies demonstrated that brain tissue extracts of HIBD rats can induce BMSCs differentiate into neural cells and HBO treatment can promote neurogenesis in neonatal HI rats. Role of HBO treatment on the proliferation and differentiation of NSCs treated with brain tissue extracts of HIBD rats is still unclear. Thus, in this study, we examined the effect of HBO treatment and brain tissue extracts of HIBD rats on neural progenitors differentiation into neurons in vitro.Neural stem cells can also be derived from more primitive cells that have the capacity to generate neural stem cells and stem cells of other tissues, such as bone marrow stromal cells (BMSCs), adipose tissue-derived stromal cells, amniotic epithelial cells, umbilical cord blood stem cell. Recent studies have demonstrated that BMSCs can be induced to generate not only the progenies of mesodermal lineages, such as adiposities, chondrocytes, and myogenic cells, but also cells from different germ layers, such as neuronal cells. These neurogenic capacities of BMSCs have attracted tremendous attention because of their great potential as donor cells for cell supplementary therapy of neuronal disorders, with the advantages of fewer ethical concerns and less immune rejection. But there were another studies show that only a few BMSCs can differentiate into neuron in brain after transplantation. To explore the effective way to improve the neuronal differentiation would expand the clinical application of BMSCs. We now investigate the effect of HBO on neuronal differentiate of BMSCs in cell-culture experiment to find more evidence for clinical therapeutic application.The BMP and Wnt signaling pathways have been previously shown to play a major role in the development of nervous system. Wnt signaling regulates patterning, proliferation and differentiation of NSCs during the early embryonic and later stages of development. Recent research also demonstrates the regulation effect on NSC differentiation by BMPs. So our study focuses on the molecule mechanism of HBO treatment on the differentiation of NSCs. Our investigation was divided into the following three parts:Part I Effect of hyperbaric oxygen on differentiation and Wnt3 expression of bone marrow stromal cellsObjective:To observe the effect of HBO on the differentiation and Wnt3 expression of BMSCs.Methods:Long bone of limbs was sterilely collected from rats, cut the metaphysis, with PBS wash carefully the bone marrow cavity, centrifuged and removal of the supernatant, the cells were re-suspended with DMEM medium containing 0.1 volume fraction of fetal bovine serum. The rat BMSCs of 3-5 passage were cultured in DMEM/F12 (1:1) medium with 10ng/ml bFGF,20ng/mlEGF and 2%B27 and induced by 24 hours. The induced BMSCs were randomly divided into two groups: Group control (CON) and Group HBO treatment (HBO), BMSCs were subjected to HBO (2 ATA,1 hr stabilizing pressure). The differentiated BMSCs were examined by neuron specific encloase (NSE); glial fibrillary acidic protein (GFAP) and 04 marked oligodendrocyte immunocytochemistry 24 hr later. The expression of Wnt3 protein in the differentiated BMSCs was detected by Western-blot.Results:BMSCs cultured in classic medium of neural stem cells could significantly induce the expression of nestin. The expressions of NSE or 04 of HBO group were greater than control group (P<0.01), but GFAP expression displayed no significant difference between these groups (P>0.05). Western-blot showed HBO could enhance the Wnt3 expression (P<0.05). Conclusions:HBO can induce BMSCs to differentiate into neural cells and oligodendrocyte, which is correlated with the activation of the Wnt3 protein.Part II Effect of hyperbaric oxygen and brain tissue extracts on differentiation of neural stem cells in vitroObjective:It was previously found that HBO therapy could promote the endogenous NSCs to proliferate, migrate and differentiate, thus restore the hypoxic ischemic brain damage. The purpose of this study is to investigate the role of HBO on the differentiation of NSCs treated with brain tissue extracts of HIBD rats in vitro.Methods:The cerebral cortices from newborn rats (0-3 days old) were sterilely, digested, and centrifuged. After removal of the supernatant, the cells were re-suspended with DMEM/F12 medium containing B27,bFGF and EGF. The NSCs of 2-3 passage were randomly divided into seven groups:a control (untreated) and 6 HBO treatment groups that NSCs were subjected to HBO treatment of different pressures (1,2 or 3 ATA) and different time (30 or 60 minutes). The NSCs were randomly divided into five groups:(1) Group control (CON); (2) Group HBO, NSCs were subjected to HBO (2 ATA,60 min stabilizing pressure); (3) Group normal brain tissue homogenate (NBH), NSCs were cultured in medium with normal brain tissue homogenate; (4) Group HIBH, NSCs were cultured in medium with tissue homogenate of HIBD brain; (5) Group HIBFI+HBO, NSCs were cultured in medium with tissue homogenate of HIBD brain followed HBO treatment. The differentiated NSCs were examined by neuron specific encloase (NSE), glial fibrillary acidic protein (GFAP) and O4 marked oligodendrocyte immunocy-tochemistrv 24 hrs later.Results:The percentage of NSH positive cells differentiated from NSCs was the highest in the 2ATA-60min group (P<0.01), followed by the 3ATA-60min,2ATA-30min,3ATA-30min, 1ATA-60min, 1 ATA-30min, and control group. In addition to 1ATA-30 group,the other HBO treatment groups had increased significantly percentage of NSE positive cells compared with the control group(P<0.01).Under the same pressure, the 60 min treated groups had increased significantly percentage of NSE positive cell compared with the 30 min treatment group(P<0.01). Compared with the CON, the NSE and 04 positive cells were increased in HBO, NBH, HIBH, HIBH+HBO (P<0.01). The NSE and O4 positive cells in HIBH, HIBH+HBO were higher than those in HBO, NBH (P <0.01). Compared with the HIBH, the NSE and 04 positive cells were increased in HIBH+HBO (P<0.01).The GFAP positive cells were higher in CON and HBO than those in NBH, HIBH, and HIBH+HBO (P<0.01).Conclusions:HBO treatment (2 ATA,60 min) produces a best effect in differentiation of NSCs into neurons. HBO, NBH or HIBH can promote NSCs differentiated into neurons and oligodendrocytes. Co-induce of HIBH and HBO plays strongest role in NSCs differentiate into neurons and oligodendrocytes. Brain tissue homogenate can reduce NSCs differentiated into astrocytes.PartⅢHyperbaric oxygen promoting the differentiation of neural stem cells via Wnt and BMP signaling pathwayObjective:HBO treatment promotes the differentiation of NSCs. The Wnt and BMP signaling pathway was associated with neurogenesis. Thereby this study examined whether HBO treatment could stimulate the differentiation of NSCs via the Wnt and BMP signaling pathway.Methods:The NSCs of 2-3 passage were randomly divided into five groups:CON,HBO,NBH,HIBH,HIBH+HBO. The expression of Wnt3 protein,β-catenin protein, or BMP2 protein in the differentiated NSCs was detected by Western-blot. The NSCs were randomly divided into two groups:NSCs were subjected to HBO (2 ATA,60 min stabilizing pressure) in the presence of Wnt inhibitor or not, secreted Frizzled-related protein 2 and 3 (sFRP2/3). The expression of Wnt3 protein,β-catenin protein, or BMP2 protein in the differentiated NSCs was detected by Western-blot.Results:The expression of Wnt3 protein and BMP2 protein of CON,HBO,NBH,HIBH,HIBH+HBO were increased gradually. The total expression of (3-catenin was nondistinctive, however the volume of (3-catenin in cytoplasm decreased gradually and increased in nucleus. Compared with the control group, NSCs were subjected to HBO in the presence of sFRP2/3, the expression ofβ-catenin nucleoprotein and BMP2 protein were down-regulated.Conclusion:The capability of HBO treatment and microenvironment of brain to stimulating the differentiation of NSCs is correlated with the activation of Wnt signaling. And differentiation of NSCs is regulated by combinatorial Wnt and BMP signaling pathways.The main contents and conclusions of the research were summarized as follows:1. HBO can induce BMSCs to differentiate into neural cells and oligodendrocyte, which is correlated with the activation of the Wnt3 protein.2. HBO treatment (2 ATA,60 min) produces a best effect in differentiation of NSCs into neurons.3. HBO, NBH or HIBH can promote NSCs differentiated into neurons and oligodendrocytes. Co-induce of HIBH and HBO plays strongest role in NSCs differentiate into neurons and oligodendrocytes. 4. The capability of HBO treatment and microenvironment of brain to stimulating the differentiation of NSCs is correlated with the activation of Wnt signaling. And differentiation of NSCs is regulated by combinatorial Wnt and BMP signaling pathways. |
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