ABSTRACT
Objective:To investigate the transfection of recombinant adenovirus vectors containing the hypoxia inducible factor-1α gene (AdHIF-1α)in rat brain microvascular endothelial cells(BMECs) and to provide a theoretical basis for the treatment of hypoxic BMECs by AdHIF-1α.Methods:Rat BMECs were isolated, identified, and cultured in a maintenance medium containing 100 μmol/L cobalt dichloride (CoCl 2), establishing a hypoxia model of BMECs; then AdHIF-1α was transfected into hypoxic BMECs.The transfection of fluorescent protein was observed under a fluorescence microscope. Results:Transfection of AdHIF-1α into BMECs was monitored under a fluorescence microscope at 12 h, 24 h, 48 h and 72 h, respectively.Minor fluorescence began to appear at 12 h (0.13±0.01), and the fluorescence expression increased at 24 h (0.46±0.03, q=25.88, P<0.01), was most obvious at 48 h (0.97±0.05, q=40.00, P<0.01), and decreased at 72 h (0.38±0.02, q=46.28, P<0.01). Conclusions:Recombinant adenovirus vectors containing AdHIF-1α can be transfected into hypoxic BMECs in vitro.
ABSTRACT
Objectives To investigate the changes of erythropoietin(EPO)expression in rats after focal cerebral ischemia/reperfusion injury.Methods Male Sprague-Dawley rats were randomly divided into normal,sham,cerebral ischemic/reperfusion(CIR)groups.Middle cerebral artery occlusion(MACO)model was established by Longa's method,and reperfusion was followed 2 hours after occlusion in CIR group.The rats' brain neurological deficit scores were evaluated at 24 h,48 h,72 h and 96 h after reperfusion.The protein expression of EPO was determined by immunohistochemistry staining and Western blotting in each time points.Results The rats' brain neurological deficit scores at 48 h,72 h and 96 h were significantly increased(3.40±0.32,3.60±0.17,3.70±0.21,all P<0.05)compared with those at 24 h(3.00±0.22)after reperfusion in CIR group.The results of immunohistochemistry staining and Western blotting showed that the positive expression of EPO proteins in rats started at 24 h(0.36±0.05,140.20±0.30)after cerebral ischemic/reperfusion injury,increased significantly at 48 h(1.09±0.10,145.40±0.16),reached the peak at 72 h(1.29±0.09,156.23±0.12),began to decline at 96 h(0.98±0.04,141.56±0.36).Conclusions Cerebral ischemia and reperfusion injury can induce increased expression of EPO protein,which suggests that EPO may have protective effect on nerve cells under the condition of ischemia and reperfusion.
ABSTRACT
Objective To investigate the expression of Caspase-9 and heat-shock protein-90 (HSP 90) in rats after focal cerebral ischemia-reperfusion injury.Methods The male SD rats (200-250 g) were divided into three groups by the random number table: normal group, sham group and cerebral ischemia-reperfusion (CIR) group.Each group was sorted into four subgroups including group 6 h, group 24 h, group 48 h and group 72 h according to the reperfusion time.Suture-occluded method was adopted to prepare focal cerebral ischemia-reperfusion(CIR) injury in rat model.Enzymelinked immunosorbent assay (ELISA) method was used to detect the variations of Caspase-9 and HSP-90 expression in rats.Results The changes in Caspase-9 and HSP 90 expression in the brain cells were observed by ELISA method.The expression of Caspase-9 and HSP-90 was weakly expressed in sham group, and was at peak in CIR group within 24 h-48 h, then began to decline at 72 h after the reperfusion time.The differences in the expression of caspase-9 and HSP-70 between sham group and normal group were not statistically significant.Conclusions Apoptotic cells gradually increase along with reperfusion time and reach the peak at 48 h after cerebral ischemia-reperfusion.In ischemia half dark stripe, the expression of Caspase-9 and HSP 90 is increased in neuronal cells after cerebral ischemia-reperfusion, and the positive cells number is at peak at 48 h after cerebral ischemiareperfusion.Apoptosis of neuronal cells after cerebral ischemia and reperfusion is a dynamic evolutionary process.The expression of Caspase-9 and HSP 90 in nerve cells plays an important role in regulating cell apoptosis.