| Background and Objective:Spinal cord injury (spinal cord injury, SCI) is a serious central nervous system trauma, with high morbidity and caused motor nerve, sensory nerve missing,which has been a huge impact to the patient’s life. Spinal cord repair and treatment of injury always be difficult in world’s research. The discovery of stem cells is the study of human biology and disease development in a major breakthrough. Stem cells have the potential of proliferation and differentiation with the ability of self-renewal copied. It can produce highly differentiated cell function, and form of mammalian tissues, which makes pluripotent stem cells has great potential in biomedical engineering.Mesenchymal stem cells (BMSCs) have high self-replication and differentiation potential under certain induction conditions across mesoderm to differentiate into nerve cells, replacing defect nerve function; promoting remyelination; secreting neurotrophic factor; regulation of inflammation and reducing secondary injury toxic to neurons in situ. Many studies have shown that bone marrow mesenchymal stem cells can be directed to migrate to the damaged tissue and colonization (homing), which plays its power role of nerve repair. However, the transplant of bone marrow-derived mesenchymal stem cells in ischemic lesions just only 2% survival, which is caused by spinal cord injury secondary injury, including glutamate toxicity substance, overload oxygen free radicals, inflammatory cells, various cytokines and so on. How to improve the survival of bone marrow mesenchymal stem cells in the lesions become new research targets.Hyperbaric oxygen administrated in ischemic diseases of the nervous system has been widely recognized. The main mechanisms include:①nerve cells to oxygen dependence and sensitivity specially, as hyperbaric oxygen can significantly improve patients’ arterial oxygen partial pressure and tissue oxygen reserves, increase the oxygen diffusion distance to improve spinal hypoxia. ② HBO can increase red blood cell deformability, reducing capillary permeability. ③ hyperbaric oxygen can expand arteries and promote blood flow velocity, reduce platelet aggregation, reduce blood viscosity, thus increasing the blood supply to the spinal cord. ④hyperbaric oxygen correction of acidosis, spinal cord injury site to improve microcirculation and blood flow, maintain the energy metabolism of the nerve cells and restore neuronal function reversible damage. Studies have shown that hyperbaric oxygen could regulate apoptosis family Bax, Bcl 2-expression in spinal cord injury, maintain the stability of mitochondrial membrane potential, release of cytochrome C reduction, and blocking its downstream Caspase-3 activation to exert anti- apoptosis. In addition, hyperbaric oxygen can promote the proliferation of endogenous neural stem cells induced to differentiate into neurons, which may activit of Wnt/β-catenin pathway. It has been reported combination of hyperbaric oxygen and stem cell transplantation can significantly improve the neurological deficit symptoms, but among the mechanisms are not yet clear.Wnt pathway has play an important role in cell proliferation, differentiation, death, migration and polarization. β-catenin is the canonical Wnt pathway core regulatory factor, which determines the level of expression of an open pathway or not. Using an in vitro model of hypoxia and oxygenation (H/R) to mimic the ischemia reperfusion in spinal cord injury, we investigated the hypothesis that hyperbaric oxygencould protect rat MSCs against H/R-induced apoptosis and improve their proliferation,as well as the potential underlying mechanisms,which to provide experimental evidence for the development of cell therapeutics.METHODS:1, Rat bone marrow mesenchymal stem cells (BMSCs) collection, culture and flow cytometry to identify cell surface markers:MSCs were generated from the bone marrow of adult male SD rats aged 3-4 weeks and grown in 25cm2 flasks. Cells were determined by fluorescence activating cell sorting (FACS, Beckman Coulter, USA) analysis before the experiments, using directly conjugated antibodies against anti-rat CD44, anti-CD45 and anti-CD90.2, Groups:P3-P5 were randomly divided into four groups:normal control group, hypoxia/reoxygenation group, hypoxia/reoxygenation+hyperbaric oxygen group, hypoxia/reoxygenation+HBO+group YC-1. Hypoxia/reoxygenation group cells were washed with PBS and placed in serum free DMEM with 95% N2,5% CO2,98% absolute humidity for 9h, replaced with 5% complete medium 2h of reoxygenation. Normal control group replaced with complete medium and place the culture normoxic environment. HBO group was administrated after 2h of reoxygenation.3, HBO treatment:The cells were placed in a hyperbaric chamber in a mixed gas containing 98% O2 washing of 10 minutes, closed the door, with a progressive increase in pressure for 15 minutes, followed by 60 minutes of continuous exposure to 100% oxygen at 2.5 ATA. After 60 minutes of exposure, the pressure in the chamber was slowly reduced over a 15-minute period for every 12h with three times. A constant oxygen flow was given to maintain the oxygen concentration in the chamber at 98% or greater. The cells without HBO treatment were simultaneously placed outside the chamber for 90 minutes.4, Hoechst33258 staining:About 3×104/ml were grown in 6-well plates,the cells were fixed with 4% glutaraldehyde for 10min. After washing twice with PBS, 500ul/well of Hoechst 33258 was added for 10min. All samples were observed using a fluorescence microscope.5, CCK-8:About 3×104/ml the cells were grown in 96-well plates,10 ul CCK-8 solution were added to each well and the plates incubated for 1-2 h. The absorbance at 450 nm was measured using a microplate reader.The mean optical density (OD) of 4 wells in each group was used to calculate the percentage of cell viability.6, Flow cytometry analysis:About 5×104/ml cells were grown in 60mm dishes, cells were harvested and washed in ice-cold PBS, resuspended in 500 il binding buffer and incubated with 5 il Annexin V-FITC solution,then 5 il propidium iodide(PI) was added and incubated for 15 minutes at room temperature in the dark. The samples were immediately analyzed by bivariate flow cytometry on the FACSC-LSR equipped with Cell Quest software.7, Hoechst33258/PI staining:About 5×104/ml cells were grown in 60mm dishes,collecting the cells and concentration was adjusted to 106/ml by PBS.Taking 100ul cell suspension join a mix of Hoechst33258/PI (300ul:6ul) dye and incubated in the dark at room temperature 20min. Take lOul drops on a slide, add coverslip. All samples were observed using a fluorescence microscope.8, Western blot detection of protein expression levels:the ice after lysis protein in each experimental group were collected in lml EP tubes, centrifuged (12000 rpm, 4℃,20 min), the detection kit with protein concentration determination of total protein concentration. Turn after electrophoresis, transferred to a membrane, a primary and secondary antibody incubation, development and imaging. Gel imaging system scan and analyze HIF-1α, β-catenin, LEF-1, Cl.aspase-3, Bcl-2 protein expression.RESULTS:1. Cell growth, morphology, identification:Cultures of BMSCs are round, unequal in size, suspended, which mixed with a large number of hematopoietic stem cell.The cells began to paste the wall after static culture after 48 h, cells were round in irregular polygon. Adherent cells continued to increase with the prolongation of time, the fusiform cells have small protrusions,there is a small protrusion, part of colony-forming cells.The primary cell culture for 7 days can achieve a 80% to 90% fusion, which characterized by a spiral or radially arranged and can be passaged at the first time. After passage, cell proliferation is fast and cell purification is high.Flow cytometry showed high expression of CD29 and CD90, were 96.23+2.02% and 97.28±1.3%, while the low expression of the CD45 were 1.68±0.73%, which is in line with the general expression of mesenchymal stem cell surface markers.2.Hoechst 33258 staining:The cells with the typical morphological characteristics of cellular shrinkage and karyopyknosis were confirmed as exhibiting apoptosis. With the time of hypoxia/ reoxygenation, the number of apoptosis increasing number of blue light nuclei is gradually increasing compared with normal control group.3.CCK-8:Compared with normal group, the rate of survival in hypoxia/reoxygenation group was significantly decreased(P<0.05); after administration of hyperbaric oxygen treatment, cell viability was significantly increased(P<0.05); YC-1 was added 1h prior to treatment with hyperbaric oxygen,cell survival was significantly decreased (P<0.05).Compared with normal group, proliferation of bone marrow mesenchymal stem cells with hyperbaric oxygen treatments had no significant (P> 0.05).4. flow cytometry analysis:Compared with the normal group, hypoxia/reoxygenation group with prolonged hypoxia time, the percentage of apoptotic cells gradually increased,chose the hypoxia 9h/reoxygenation 2h to imitate the ischemia environment; compared with hypoxia/ reoxygenation group, the rate of apoptosis in hyperbaric oxygen treatment significantly decreased (P<0.05); administrated of YC-1 increased the rate of apoptosis (P<0.05).5. Hoechst33258/PI staining:Compared with hypoxia/reoxygenation group, PI-positive cells was decreased in hyperbaric oxygen treatment groups (P<0.05); YC-1 reduced cell apoptosis,which show that PI-positive cells was significantly increased (P<0.05).6. Western blot analysis:Western blot showed that HIF-1α expression in hypoxia/reoxygenation group was not significantly upregulated (P> 0.05); hyperbaric oxygen treatment can induce the expression of HIF-1α, which was significantly reduced by YC-1 (P< 0.05); Compared with hypoxia/reoxygenation group, high pressure oxygen treatment group increase the expression of P-catenin, LEF-1,β-catenin expression in nucleus also upregulated, this effect may be reversed by YC-1(P<0.05), it indicated that hyperbaric oxygen may play an anti-apoptotic ability by HIF-la. Compared with the normal group, hypoxia/reoxygenation upregulated Cl.aspase-3 expression, while decreased expression of Bcl-2(P<0.05);administrated of hyperbaric oxygen decreased expression of Cl.aspase-3, with Bcl-2 expression increased (P<0.05).CONCLUSION:Hyperbaric oxygen have the effect of anti-apoptotic to make bone marrow mesenchymal stem cells against hypoxia/reoxygenation injury. The mechanism may related to increase HIF-1α, while activating the Wnt/β-catenin pathway, promoting β-catenin into the nucleus and upregulating LEF-1 gene expression and this could be reversed by YC-l;in addition, Cl.caspase-3 can also be down-regulated in hyperbaric oxygen group,with upregulated the expression of Bcl-2; continuous use of hyperbaric oxygen does not promote bone marrow mesenchymal stem cell proliferation. |
PDF Full Text Request