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Objective To construct and identify the recombinant retroviral vector containing five copies of hypoxia responsive elements (5HRE)and neurotrophin-3 (NT-3 ).Methods Using PCR,enzyme digestion and DNA ligase,5HRE and human derived NT-3 were cloned into the retroviral vector plasmid (pLNCX)to construct the recombinant retroviral vector plasmid pLNCX-5HRE-SV40-NT3-IRES-EGFP.The retrovirus RV-5HRE-NT3 was packaged in the PT67 cells,and then it was purified and concentrated by high-speed centrifugation.After infected for 48 h with the concentrated retrovirus,the number of the EGFP positive cells in the NIH 3T3 cells was counted by fluorescence activated cells and sorted to calculate the retrovirus titer.Results The retroviral vector plasmid,pLNCX-5HRE-SV40-NT3-IRES-EGFP,was successfully constructed,and the retrovirus was packaged and defined as RV-5HRE-NT3.After purification and concentration,the retrovirus titer reached 9.1 × 10 6 cfu/mL. Conclusion The recombinant retroviral vector which carried out hypoxia-regulated expression of NT-3 was successfully constructed.It may provide basis for studies on hypoxia-regulated expression of the exogenous genes.
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Objective To investigate the neuroprotective effects of neurotrophin-3 (NT-3) expression controlled by five copies of the hypoxia-responsive elements after focal cerebral ischemia .Methods Three groups of rats re-ceived RV-5H-NT3, RV-5H-EGFP or saline injection .Three days after gene transfer , the rats underwent 90 min of transient middle cerebral artery occlusion ( tMCAO) , followed by 1-28 days of reperfusion .Immunohistostaining and western blotting were performed to detect ischemia/hypoxia-regulated expression of NT-3 controlled by HRE . The volume of brain infarction and the apoptosis were analysised by TTC and TUNEL staining .The neurological scoring was determined by neurological behavior tests .Results Three days after tMCAO , brain NT-3 expression was significantly increased in the RV-5HNT3-transduced animals compared with the RV-5H-EGFP or saline group (P<0.05), and brain infarct volume was smaller in the RV-5H-NT3-transduced group than the RV-5H-EGFP or saline group ( P<0.05 ) .The percentage of TUNEL-positive cells was reduced in RV-5 H-NT3-transduced brains compared with the RV-5 HEGFP or saline group 3 and 7 days after tMCAO ( P<0.05 ) .Furthermore , the neurolog-ical status of RV-5H-NT3-transduced rats was better than that of RV-5H-EGFP-or saline-transduced animals from 1 day to 4 weeks after tMCAO ( P<0.05 ) .Conclusions HRE may modulate NT-3 expression in the ischemic brain tissue and that the up-regulated NT-3 may effectively improve neurological status following tMCAO due to de-creased initial damage .
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<p><b>OBJECTIVE</b>To explore the effects of salidroside (sal) on the expressions of Bcl-2, Bax and caspases-3 proteins in cultured rat subventricular zone (SVZ) neural stem cells (NSCs) exposed to hypoxia injury.</p><p><b>METHODS</b>Primarily cultured SVZ NSCs from adult SD rats were incubated with salidroside (120 and 240 µmol/L) for 24 h prior to exposure to hypoxia. The cell viability was assessed with MTT assay, and the cell apoptosis was analyzed using TUNEL staining and flow cytometry. Western blotting was performed to detect the expressions of Bcl-2, Bax and caspase-3 in the cells.</p><p><b>RESULTS</b>Salidroside pretreatment of the cells for 24 h resulted in an obvious resistance to hypoxia-induced cell apoptosis and decrement of cell viability (P<0.05). Salidroside also antagonized the effect of hypoxia exposure in lowering Bcl-2/Bax ratio apoptosis of rat neural stem cells and decreased the expression of caspases-3 protein (P<0.05).</p><p><b>CONCLUSION</b>Salidroside can significantly resist hypoxia-induced. The neuroprotective effect of salidroside may be related to the modulation of expressions of apoptosis-related proteins.</p>
Subject(s)
Animals , Rats , Caspase 3 , Metabolism , Cell Hypoxia , Cells, Cultured , Flow Cytometry , Glucosides , Pharmacology , Neural Stem Cells , Phenols , Pharmacology , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Rats, Sprague-Dawley , bcl-2-Associated X Protein , MetabolismABSTRACT
Objective To explore the effect of Ligustrazine on neurogenesis in cortex after focal cerebral ischemia in rats. Methods Focal cerebral ischemia was induced by left middle cerebral arteryocclusion with asuture. Two hours later, injection of Ligustrazine (80 mg/kg, 1 time/d) was performed peritoneally. Four hours after the ischemia,5-bromodeoxyuridine (BrdU) (50 mg/kg, 1 time/d) was injected peritoneally. At 7 d, 14 d and 21 d after ischemia,BrdU positive cells in the cortex were observed by immunohistochemical staining. Results In ischemic model group, at 7 day, sparsely-distributed BrdU positive cells were observed in the Ⅱ - Ⅵ layers of the ipsilateral cortex, with a band-like distribution in ischemic penumbra. With the prolongation of ischemia, the number of BrdU positive cells increased.In Ligustrazine group, BrdU positive cells were also observed in the Ⅱ - Ⅵ layers of the cortex, with an intense distribution in ischemic penumbra. The numbers of BrdU positive cells at 7 d, 14 d and 21 d were more than those in ischemic model group respectively. Conclusion Ligustrazine increases the proliferated cells in cortex after focal cerebral ischemia in rats. The results suggest that it may be useful for promoting self-repair after ischemia.
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0.05).In SVZ,nNOS expression in ischemic model group was reduced on days 1-14,but increased on day 21;after Ligustrazine administration,nNOS expression was obviously decreased on days 3-14 in all Ligustrazine dose groups,but began to increase on day 21.In CC,nNOS expression in ischemic model group was reduced on days 3-14,and began to increase on day 21;in the different-dose Ligustrazine groups,nNOS expression was significantly decreased on days 3-14,especially in medium-and high-dose groups,but increased on day 21.In striatum and cortex peri-infarction,nNOS expression in ischemic model group was obviously decreased on days 3 and 7,but enhanced on days 14 and 21;in various-dose Ligustrazine groups,nNOS expression was decreased on days 3-21,especially in medium-and high-dose groups,but increased slightly on day 21.In DG and CA1 areas,nNOS expression in ischemic model group was reduced on days 3 and 7,but began to increase on day 14;nNOS expression in all Ligustrazine groups were decreased during 3-21d.There were significant differences between ischemic model group and different-dose Ligustrazine groups at different time points(P