Expression Of HTRX-PR39 Following Adeno-associated Viral-encoding Fusion Gene Transfer Induces Angiogenesis In Hypoxia Chick Embryo

BackgroundAt present,morbidity and mortality of cerebrovascular disease continue to rise,becoming a threat to human health and a hot issues of medical research.Clinical applications of new drug treatments and physical therapy methods,continuing to improve the success rate of resuscitation in the emergency room and to extend the life of the patient,still can't fundamentally reverse the pathophysiological changes of the patient,and it is difficult to restore the impaired brain function. According to cytology and molecular biology research progress in tissue repair after cerebral vascular disease in recent years,this topic design the rAAV with "Angiogenesis master switch"-PR39 and hTRX fusion expression,conduct it through the intervention approach to brain tissue and blood vessels with cerebral circulation insufficiency or/and with the tendency of infarct cerebrovascular disease,in order to start the expression and secretion of PR39,through its specific inhibition of the degradation of HIF-1αby Ubiquitin - proteasome,to increase the sustained high expression of angiogenie cytokine and receptor(VEGF,KDR,FLT-1 and FGF-1) in a long time,ultimately to achieve the neuronal repair and vascular re-build in the early stage after the occurrence of cerebral hypoxia and cerebral infarction;and at the same time to protect the injured nerve cells to survive and their function,through the anti-ischemic hypoxia,anti-inflammatory response function of PR39,through the anti-oxidation,anti-apoptosis,protection of ischemia-reperfusion injury function and the neurotrophic factor-like effects of hTRX.It will provide new theories and methods for the prevention and treatment of ischemic cerebrovascular disease.ObjectiveExogenous delivery of PR39 may provide a useful approach to the treatment of cerebral ischemia.The purpose of this study is to assess the value of AAV-mediated hTRX-PR39 fusion gene expression on anti-apoptosis in hypoxia vascular endothelial cell and on angiogenesis in chicken embryos,so as to discuss the protection of PR39 for cerebral ischemia.Methods1.The forward and reverse primer of PR39 were designed and synthesized.By means of asymmetrical primer/template,the fragment encoding PR39 was gained,which included EcoR721 and BamH I restriction enzyme sites.Then the synthesized fragment was cloned into vector pGEM-T easy.The positive clone was identified by restriction enzymes,and then the cloned amplified fragment was sequenced by dideoxy-mediated chain-termination method.2.Using hTRX as a template,the new hTRX cDNA which included EcoR721 and EcoRI restriction enzyme sites was gained.Then the synthesized fragment was cloned into vector pGEM-T easy.The positive clone was identified by restriction enzymes,and then the cloned amplified fragment was sequenced by dideoxy-mediated chain-termination method.3.After pGEM-T-hTRX and pGEM-T-PR39 were both digested by BamH I and EcoRI,the PR39/BamH I,EcoRI was cloned into recombinant vector pGEM-T-hTRX /BamH I,EcoRI.The recombinant vector pGEM-T-hTRX-PR39 was gained.4.When the recombinant vector DGEM-T-hTRX-PR39 was digested,the fusion gene hTRX-PR39 we got was inserted into the EcoRI-BamHI site of vector plasmid pSSCMV and the vecctor of hTRX-PR39 recombinant AAV was constructed.5.The recombinant AAV viral sock was packaged.Renal embryo 293 cells were co-transfected with the rAAV vector of plasmid pSSCMV/ hTRX-PR39,packaging plasmid pAAV/Ad and helper adenovirus plasmid pFG140.The recombinant adeno-associated virus was produced by homologous recombination of above 3 plasmids in renal embryo 293 cells and its titer was measured by quantitative dot blot hybridization.6.Then ECV304 were respectively transducted stably with AAV-NT4-TAT-His-PR39 and empty vector(EV),the fusion protein expression were confirmed by immunocytochemistry。7.ECV304 were divided into the AAV-hTRX-PR39 group and PBS group,at 1%, 5%O2 conditions,culture,and count living cells with the trypan blue staining method,observing the effect of hTRX-PR39 on hypoxia-cell protection.8.analysis the apoptosis of ECV304 cells under hypoxia conditions By flow cytometry(FCM).9.We detect the expression level of VEGF-A,VEGFR-1,VEGFR-2, FGFR-1,Syndecan-4 and PR39 Through quantitative PCR.10.120 embryos were randomly divided into AAV-hTRX-PR39 group and PBS group,and all the cells of each group will randomly set at hypoxia(1% O2,5%O2) and normoxia(20%O2) conditions for cultivation.The vessel density of the chicken embryos chorioallantoic membrane(CAM) were measured by Image Pro Plus(IPP) software.Results1 By DNA sequencing,we found that we obtained the sequence of hTRX which included EcoR721 and EcoRI restriction enzyme sites and the sequence of PR39 which included EcoR721 and BamH I restriction enzyme sites by pcr.Their sequences were consistent with those of gene bank.2.After analyzing the ORF of the cloned hTRX-PR39 cDNA by the DNASIS,,we found that amino acids encoded by the cloned hTRX-PR39 cDNA were also identical to the published results.3.The vecctor of rAAV/hTRX-PR39 was succssfully constructed.4.High titer of recombinant adeno-associated virus was obtained by homologous recombination in renal embryo 293 cells(3.46×1012~3.46×1013PFU/ml).5.It can be detected by immunocytochemistry that AAV vectors with recombinant hTRX-PR39 can be expressed in ECV304.6.Through quantitative PCR we found that the recombinant AAV-hTRX-PR39 can increase VEGF-A,VEGFR-1,VEGFR-2,FGFR-1,Syndecan-4 and PR39 expression level of hypoxia ECV304,which is significantly higher than PBS group(P＜0.001)7.By flow cytometry analysis of ECV304 cell cycle diagram in low-oxygen environment,the apoptosis of the experimental group was significantly smaller than PBS group.8.Vascular density of chick chorioallantoic in the experimental group was significantly greater than the PBS group under hypoxic environment.There is no significant difference between the vessel density of the two group in normoxic conditions.Conclusions1.The sequence of hTRX which included EcoR721 and EcoRI restriction enzyme sites was obtained by pcr.2.The sequence of PR39 which included EcoR721 and BamH I restriction enzyme sites was obtained by pcr.3.The recombinant vector pGEM-T-hTRX-PR39 was successfully constructed.4.The recombinant adeno-associated virus vector encoding gene hTRX-PR39 has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques,laying a foundation for further research of gene therapy of cerebral ischemic disease.5.Expression of Adeno-associated virus harboring hTRX-PR39 in ECV3046.The influence of Adeno-associated virus harboring hTRX-PR39 on the VEGF-A,VEGFR-1,VEGFR-2,FGFR-1,Syndecan-4 expression of Hypoxic vascular endothelial cells and promotion effect on angiogenesis of hypoxic chick embryo.