| Part I:The design and application of a self-regulated anoxia box using chemical reaction.[Background] In recent years, the incidence of ischemic stroke was clearly on the rise, so the effective prevention and treatment for ischemic stroke is very important. The cellular and molecular mechanism is the key in the study of ischemic brain injury. Oxygen glucose deprivation for cells cultured in vitro is the most commonly model to mimic the ischemia and anoxia in vivo. Constructing anoxia condition is the prerequisite for oxygen glucose deprivation experiment. At present, the common anoxia cell culture equipment is the anaerobic incubator; its main principle is to replace the air in the incubator by hypoxic mixed gas. However, because of the large incubator, the efficiency is low, and it needs continually to inject oxygen-free gas in order to maintain the state of anoxia. A simple and self-contained platform is urgently needed. The pyrogallic acid solution is relatively stable at ambient condition, and the alkaline solution can rapidly react with oxygen. This characteristic is obviously different from other oxygen scavenger, therefore, alkaline solution composed of pyrogallic acid and sodium hydroxide as oxygen scavenger, is an ideal choice.Buffer is able to maintain the pH of its solution in a stable contion.Na2HO4/NaH2PO4and NaHCO3/H2CO3are the common buffer pairs. Cell culture needs a stable pH condition; NaHCO3and carbon dioxide can keep the pH of the solution in a stable condition.Using these physical and chemical characteristics, we design a simple chemical self-contained anoxia box. The box should not only meet the anoxia but also satisfy the concentration of carbon dioxide.[Objective] To design a simple and practical anoxia cells culture box that can be regulated by chemistry reaction and investigate its practicability and methods.[Methods] The anoxia box was made from a1.5L sealed box.11.5g sodium hydroxide and2.6g sodium carbonate were dissolved in55ml water,18g pyrogallic acid dissolved in100ml water, and then injected into a sealed vacuum bag, to blend into pyrogallic acid alkaline solution. The mixed solution was injected into the anoxia.30mins later,110ml water solution with47.8g two aqueous sodium dihydrogen phosphate was injected into the box. NaH2PO4can react with NaOH and Na2CO3to form Na2HPO4, NaHCO3and H2CO3. Na2HPO4NaH2PO4was the basis buffer that can keep the pH in a stable condition, owing to its quantity larger than NaHCO3/H2CO3, the pH was lightly influenced by the release of CO2. In the box, the oxygen concentration was measured by oxygen analyzer, the carbon dioxide concentration measured by carbon dioxide measurement instrument, the pH of the chemical reaction solution measured by pH analyzer, the pH of the cell culture medium measured with blood gas analyzer. The anoxia box was tested with U87, U251and bone marrow mesenchymal stem cells and compared with95%N2+5%CO2as commonly used. Each group of experimental index was repeated5times.[Results]18g pyrogallic acid,11.5g sodium hydroxide and2.6g sodium carbonate were dissolved in water and injected into the anoxia box. The injection must be finished in 2mins, timing from the begin of the injection. The oxygen volume concentration was measured at5min,10min,15min,30min and24h, the results are0.592%±0.0798%,0.238%±0.0278%,0.200%±0.02236%,0.194%±0.01342%and0.194%±0.0321%. After10mins, the oxygen concentration tended to be stable, the results of10min,15min,30min and24h were compared, there were no significant differences (P>0.05).After30mins, sodium dihydrogen phosphate solution was injected into the anoxia box. At24h, the pH value of the chemical reaction solution was7.93±0.026, and the carbon dioxide concentration was5.03%±0.19%.The pH of cell culture medium was measured at15min,30min,1h and24h. The results respectively were7.341±0.0354,7.308±0.0365and7.311±0.0304. The cell culture medium pH had no obvious change. The anoxia box was tested with U87, U251and bone marrow mesenchymal stem cells and compared with95%N2+5%CO2injection, as commonly used. There were no significant differences (P>0.05).[Conclusion] In this experiment, we skillfully used the special chemical properties of pyrogallic acid and buffer to design the anoxia box that, with good effect, stable carbon dioxide concentration in the box, can meet the demand of hypoxia experiment.Part II:Resveratrol preconditioning protects neural stem cells from oxygen and glucose deprivation injury.[Background] The repair ability of nervous system is limited, neural stem cells and neural precursor cells may be able to provide repair for nervous system to replace the injured neuron. However, the survival and growth of the stem cells are seriously affected by the great changes of the intracellular environment after ischemic stroke and reperfusion. Resveratrol is a polyphenolic drug and widely exists in the nature plants. Studies found that Res can activate SIRT1, improve the tolerance ability of neural cells on anoxia, and protect neural cells through the SIRT1pathway. The research on the mechanism of Res preventing nerve system aginst injury, starting from the root causes of ischemic injury, will provide a more practical evidence and new ideas for nerve injury and repair. [Objective] To investigate resveratrol preconditioning possible protection on the neural stem cells against oxygen and glucose deprivation damage.[Methods] Neural stem cells were isolated from the brain of fetal rats from a15d pregnant SD rat and identified by immunohistochemistry, nestin expression, the character of neural stem cells. Neural stem cells were pretreated by culture medium with resveratrol10μ mol/L,20μ mol/L,40μ mol/L and60μ mol/L for lh, at the same time control group medium containing0.1%DMSO. The high sugar medium was replaced by low sugar medium, then the cells cultured in the anoxia box for oxygen and glucose deprivation for6hours. The cells activity was detected using methyl thiazolyl tetrazolium. Supernatant of culture medium was collected and the activity of lactate dehydrogenase in the supernatant of was detected by Lactate dehydrogenase Kit.[Results]5days later, the primary cells formed visible neurosphere, the immunohistochemistry results of cell climbing pieces and neurospheres showed nestin positive that is the identification neural stem cell. After6hours OGD, the MTT value for10μ mol/L and20μ mol/L RPC group were statistical significance to the control group (n=7, p<0.01), showing that10μ mol/L and20μ mol/L Res lhour pretreatment can protcet neural stem cells against oxygen and glucose deprivation injury. The LDH of20μ mol/L RPC group was lower than the control group (n=5, p<0.01).[Conclusion] Resveratrol preconditioning for lhour can prevent neural stem cells from oxygen and glucose deprivation injury, improve neural stem cell activity, reduce LDH exudation because of cells injury. |
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