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
Objective To observe erythropoietin (EPO) and its mRNA expression changes in rats cortical neurons when suffering hypoxia and investigate the endogenous EPO protective effect of hypoxia neuronal cells.Methods Cultured cortical neurons were prepared from hypoxia rats and divided into control,hypoxia 12,24,48,72 h group.Using immunohistochemistry,reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot,we observed EPO and its mRNA expression in cells,and also observe the culture medium containing lactate dehydrogenase to evaluate the activity of neurons in the whole process.Results Immunohistochemistry,RT-PCR and Western blot analysis showed:the EPO (20.79 ± 2.98) and its mRNA (0.78 ± 0.05) at 12 h had a basic expression in hypoxia group,compared with the control group (EPO:17.12 ± 1.99; mRNA:0.39 ± 0.05),and the difference was statistically significant (t =2.51,P ＜ 0.05 ; t =13.51,P ＜ 0.01) ; the strongest expression was observed at 48 h (EPO:28.88 ± 3.41,mRNA:1.45 ± 0.07),the difference was statistically significant (t =7.29,P ＜ 0.01 ; t =33.24,P ＜ 0.01) ; and neuronal activity was strongest.Lactate dehydrogenase activity was significantly decreased after hypoxia 72 h,and also a statistically significant difference was found between the groups at each point.Conclusions The EPO and EPO mRNA expression are increased after hypoxia in neuron cells,and may enhance the activity of neurons.Our study suggests that EPO might be involved in the development process of neuronal hypoxia and play an important role in neuronal hypoxia process.
ABSTRACT
Objective To investigate the expression of hypoxia-inducible factor 1α (HIF-1α)gene in rat cerebral cortex neurons under hypoxia conditions,and provide more experimental basis for clinical treatment of hypoxic brain disease.Methods The rat cerebral cortical neurons in primary culture and hypoxia model were prepared and identified by immunocytochemistry analysis.The expression of HIF-1α in normal and hypoxic neurons was detected at 4 time points (12 h,24 h,48 h and 72 h) by immunohistochemical analysis.Results The expression of HIF-1α positive cells in normal control (NC) group was less at each time point,and it showed no statistically significant within groups.The weak expression of HIF-1α was found at 12 h in hypoxic group,and the expression augmented along with the time extended,it increased obviously at 24 h,reached the peak at 48 h(IOD＝0.27±0.02,F＝35.703,t＝11.795,P＜0.01),and declined until 72 h,it showed statistical significance between two groups at each time point.Conclusions The expression of HIF-1α is increased after hypoxia.HIF-1α has a protective effect on neurons after hypoxia.