Effect Of Transfection Of Human Heat Shock Protein 70 Gene On Ischemic Reperfusion Injury Of Pulmonary Allograft In Rats

Objective1. To establish foundation in further gene therapy by heat shock protein 70(HSP70), study the construction of HSP70 gene recombination 5-type adenovirus vector .2. In order to provide a more stable experiment foundation by study lung transplantation reperfusion injury. Using the way of"Two-cuff-one-stent Technique"to establish an animal model about left lung orthotopic transplantation.3. Using the endogenous cytoprotection's bionomics of HSP70 to resist inflammation, lessen peroxidation and reject apoptosis and so on, which have the allogeneil graft left lung orthotopic transplantation animal model. Using objective gene of hHSP70 to induce ex vivo high express HSP70 and via perfuse the pulmonary vein and replication defective 5-type adenovirus vector to investigate the effect rat lung orthotopic transplantation on ischemia-reperfusion injury (IRI).Methods1. Construction of genetic carrier of hHSP70, preparation and packaging of adenovirus by technique of gene recombination.2. Using way of"Two-cuff-one-stent Technique"which have improvement in the manufacture of double-pipe and the technique of the pulmonary harvesting and perfusion. Anastomosis of the pulmonary artery, veins and bronchus can be completed under naked eye euthyphoria.3. Using Male SD rats as donator and receptor, establishment of allogeneil graft left lung orthotopic transplantation animal model by improved way of"Two-cuff-one-stent Technique". The experiment include three groups which have the control group of LPDG preservative fluid, the group of empty vector and the group of HSP70 gene transfected. Each group were in turn to perfused with 10℃Liquemine liquid of LPD-glucose liquid, pure adenovirus vector liquid and hHSP70 gene recombinant adenovirus vector liquid. All allografts were preserved for 3 hours at 10℃C before transplantation and assessed 4 hours after reperfusion . Several indexes were detected below: blood gas analysis,wet-to-dry(W/D);Grafted lung's malondialdehyde(MDA) , superoxide dismutase(SOD) and myeloperoxidase(MPO);Histopathologic examination; Heat shock protein70, tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ),interleukin-6(IL-6),NF-κB,Bax,Bcl-2 and apoptosis were also analyzed.Results1.The hHSP70 recombination 5-type adenovirus vector have been constructed successfully.2. Operation of rat left lung orthotopic transplantation has been completed in 20 consecutive cases. The achievement ratio of operation was 85 percent. The total time of surgical procedure was 65±10min. On 4 h after reperfusion, pathologic study found the typical pathologic change of lung orthotopic transplantation on IRI. 3. The group of gene transfection express specific gene product of hHSP70 via RT-PCR and immunohistochemistry 4h after reperfusion. Compare with control group, Partial pressure of oxygen (PaO2) levels in gene transfection group were higher, but W/D ratios was lower than other groups (p<0.05). MDA and MPO were lower ,but SOD and Bcl-2 were higer than the other groups (p<0.05). TNF-α,IFN-γ,IL-6,NF-κand Bax were reduced (p<0.05). It can be observed under light microscope that the group of transfection have fewer air sac interstitial edema, inflammatory cell and apoptosis index were decreesed.Conclusion1. Infectant reorganizational Adenovirus can be done up in 293 cells after the reorganizational Adenovirus carrier which carried hHSP70 gene was successfully made up, it was the base for its study and exercises.2. By using the improved way of"Two-cuff-one-stent Technique", orthotopic left pulmonary allograft transplantation animal model was successfully established. It can be completed under naked eye euthyphoria without the help of microsurgery. It is suit for application to study lung IRI for it can imitate clinical process of lung transplantation.3. Ex vivo lung via the pulmonary vein into extrinsic source objective gene is feasible and effective.4. Gene therapy by HSP70 can reject IRI effectively by restrain inflam cytokine and lessen peroxidization .5. Transgenic therapy in HSP70 can reduce the ratio of bax to bcl-2, also it can reduce apoptosis.