| Objective: To construct a recombinant adenoviral vector carring HIF-1α gene for further application in cerebral ischemia gene therapy. Methods: The AdEasy System was used in the experiment. HIF-1α gene was amplified by PCR and linked to pAdTrack-CMV plasmid. Perform minipreps using the conventional alkaline lysis method. Linearize the shuttle plasmid with restriction enzymes(PmeⅠ).The PCR product of recombinant shuttle plasmid was confirmed by DNA sequencting analysis. Then homologous recombinant was performed in E.coli BJ 5183 between Linearize the shuttle plasmid pAdTrack-CMV-HIF-1α and adenovirus backbone plasmid pAdEasy-1 by electroporayted method.A recombinant adenoviral vector carring HIF-1α gene plasmid was identified by PCR amplification , pulsed-field gel electrophoresis, followed by PacI digestion. Results: we have successful cloned the HIF-1α gene to the plasmid pAdTrack-CMV. The restriction analysis and the sequencing analysis of PCR product confirmed that correct recombinant adenoviral plasmid was constructed. Conclusion: we have successful constructed the recombinant adenovirus plasmid pAdHIF-1α and control recombinant adenovirus plasmid pAd。Objective: To find methods for reproduction , purification and titre measurated of recombined adenoviral vector carring HIF-1α gene for further application in cerebral ischemia gene therapy. Methods: Before transfection, recombinant adenoviral plasmids were digested with PacⅠ. Then, the digested recombinant adenoviral plasmid was trasfected to 293 cells ( human embryonic kidney cells) and packaged out recombinant adenoviral AdHIF-1α . Transfections viral productions were monitored by GFP( green fluorescent protein) expression. To amplify recombinanted adenovirus in 293 cells for further experiment ,then to test the infective efficiency by virus titer in cells with GFP and limited dilution method, The recombinanted adenovirus was purified by gradient centrifugation with chloride cesium and thus titre was increased, then to assay the HIF-1α expression of recombinant adenovirus in Hela cells by Western blotting. Results: After transfection, the fluorescence was observed in 293 cells. The titre of amplified recombinant adenovirus AdHIF-1α and Ad were 6.6×108pfu/ml ,8.2×108pfu/ml respectively. After centrifugation ,the titre increased to 8.4×109pfu/ml ,9.6×109pfu/ml respectively; The infectiveefficiency is not alike to diffence target cells. The infective efficiency is lowest to normal cells. The expression of the HIF-1α gene was detected by western blotting. Conclusion: Culturing and reproduction of 293 cell can reproduce recombined adenovirus AdHIF-1α successfully . AdHIF-1α could be effectively infect to target cells , and could be expressed stably in cells. The volume of purified AdHIF-1α can meet the needs of in vivo gene transfection at laboratory study.Objective: To study the protective effect and mechanism of recombinant adenovirus vector containing HIF-1α gene transfer on cerebral ischemic reperfusion (CIR) in rats .It will establish a solid theory basis for cerbral ischemia gene therapy. Methods: (1) We established the Middle Cerebral Artery Occlusion 2h reperfusion 1,3,7,14,21d models in rats .We injected recombinant adenovirus (Ad) containing green fluorescent protein(GFP)into ischemic ventricle after MCAO model, the expression of GFP was observed .(2) We established the Middle Cerebral Artery Occlusion 2h reperfusion 72h models in rats. The rats were randomly divided into four groups: sham group , cerebral ischemic reperfusion group(CIR group),recombinant adenovivus group(Ad group), recombinant adenovirus containing HIF-1α gene group(AdHIF-1α group). In addition to sham group, the other three group were injected phosphate buffered saline (PBS),AdHIF-1α and Ad into ischemic ventricle after MCAO model, respectively. Its cerebral infarctvolume on cerebral ischemic reperfusion was evaluated by 2,3,5-Trihenyltetrazolium chloride(TTC) staining of the frontally sectioned brain removed at 72h. (3) We established the Middle...
|
PDF Full Text Request