| Hypoxia is a common pathological process.Although the hypoxic reactions vary from cell to cell,as hypoxia injury aggravates and exposure time prolongs,metabolic block and cellular malfunction happen,even cell death occurs.To fight against these hypoxic injuries,oxygen sensing and a set of downstream cellular signal transduction pathway will be activated,which lead to evoke of a serials of endogenous protection responses.Multiple studies have come to the conclusion that hypoxia inducible factor-1(HIF-1) may be a center part of the endogenous protection.However,pertinent mechanisms remain to bo elucidated.HIF-1 was identified as a nuclear factor which bounds to the cis-acting hypoxia response element(HRE) located in the 3'-flanking region of a number of hypoxia related genes.Over the last decade,it emerged as a global regulator of oxygen homeostasis which mediates hypoxic signal transduction and activates transcription of hypoxia related genes that will increase glycolysis efficiency(glycolytic enzymes),facilitate angiogenesis(VEGF), enhance transportation of glucose(glucose transproter-1),and so on.To this day,genes keep on being identified as HIF-1 regulated genes,studies on the function and mechanisms of HIF-1 are the front and hotspot in the field of hypoxic research.To confirm protective effect of HIF-1 on hypoxic injury and explore its mechanism, we applied the gene homologous recombinant principle and constructed a replication defective recombinant adenoviral vector,Ad-HIF-1α.Over expression of cellular of HIF-1αwas induced in HepG2 cell via adenoviral transfection,protective effects of replication defective recombinant adenoviral vector Ad-HIF-1αon hypoxic cell were investigated, and mechanisms were studied with molecular biological methods and gene microarray technique.Material and Methods:1.Replication defective recombinant adenoviral vectors(Ad-HIF-1α) and the contral(Ad-GFP) were constructed,amplified,and titrated with PCR and GFP expression. 2.Ad-HIF-1αwas transfected into the human HepG2 cells in vitro.The expression of HIF-1αwas detected with RT-PCR and Western Blot.3.24 hours after seeding,the HepG2 cells were divided into four groups:①Normoxic with Ad-GFP infection group(CC):After serum free medium was substituted and Ad-GFP added(MOI=75),cells were cultured normoxically for 48h.②Normoxic with Ad-HIF-1αinfection group(CH):After serum free medium was substituted and Ad-HIF-1αadded(MOI=75),cells were cultured normoxically for 48h.③Hypoxic with Ad-GFP infection group(HC):After serum free medium was substituted and Ad-GFP added(MOI=75),cells were cultured normoxically for 24h,then exposed to hypoxia(2%O2,5%CO2) for another 24h.④Hypoxic with Ad- HIF-1αinfection group(HH):After serum free medium was substituted and Ad- HIF-1αadded(MOI-75),cells were cultured normoxically for 24h,then exposed to hypoxia(2%O2,5%CO2)for another 24h.4.LDH leaking rate,cell viability,contents of NO and ROS,iNOS activity were evaluated.5.Expression of genes correlated to energy metabolism and mitochondrion were detected gene microarray.Results:1.The recombinant replication-defective adenovirus vector containing the complete human HIF-1αgene was constructed successfully.The combinant adenovirus titer was 1.15×1010 pfu/ml after amplification.2.High level of HIF-1αmRNA and protein were detected in Ad-HIF-1αtransfected HepG2 cells.3.LDH leaking rate was significantly higher and cell viability significantly lower in group HC than in group CC;cell viability was significantly higher in group HH than in group HC;no marked difference of LDH leaking rate was found between group HC and group CH.4.ROS was significantly higher in group HC than in group CC,while no marked difference was found either between group CH and group CC or between group CH and group HC.5.NO and iNOS activity was significantly higher in group CH and group HC than in group CC,while no marked difference was found either between group HH and group HC or between group HH and group CH.6.Compared with group CC,3 genes in group CH and 1 gene in group HH were down-regulted,10 genes in group HC and 6 genes in group HH were up-regulated.Conclusion:We have successfully constructed the replication defective recombinant adenoviral vectors Ad-HIF-1α,with which transfection,HIF-1αgene is over expressed in HepG2 cells. Ad-HIF-1αtransfection and HIF-1αover expression lead to protective effects against hpoxic injury in HepG2 cells,the mechanisms of which may be correlated with higher enery utilization efficiency and lower energy source consumption.
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