Paracrine Protection Of Swine Bone Marrow Mesenchymal Stem Cells Transfected Ex Vivo By Human Hemeoxygenase-1(hHO-1) On Myocardium Under Hypoxia/Reoxygenation Condition In Vitro

Background and ObjectivesBone marrow Mesenchymal stem cells(BMSC) have been increasingly tested experimentally and clinically for cardiac repair,and cumulative data have proven that more than 90%of grafted cells would die in the ischemic heart.However,MSC remain attractive as a cellular vehicle for gene delivery,providing an alterative and applicable path by modification with well-designed exogenous gene(s).Hemeoxygenase-1(HO-1) participates in defense mechanisms against agents that may induce oxidative injury such as metals,endotoxin,heme/hemoglobin,and various cytokines.It is speculated that such protections lie in not only HEME/HO response system,but also interactive reaction of paracrine cytokines,cell signalling pathway,and so on.The main objects of this study are to identify Whether adenovirus-mediated HO-1 gene transfer can enhance the survival of transfected BMSC and better protect myocardium in vitro under hypoxia/reoxygenation condition which are principal components of myocardial ischemia and reperfusion and have distinctive effects on the tissue.Method:1.The recombinant adenoviruses expressing hHO-1 were established through Adeasy system and the titer of Ad-HO-1 was measured by TCID50 assay.2.The optimal MOI was chosen for both highest hHO-1 protein expression and enzyme activity of MSC transfected with Ad-HO-1.Whether hHO-1 gene transfer affect the properties of BMSC was observed.3.hHO-1 modified mini swine bone marrow MSC(BMSC-HO-1) were incubated in hypoxia chamber for indicated time in which LDH release and apoptosis index were measured.Rat myocardium were incubated in planned conditioned culture medium under hypoxia/reoxygenation condition.Cell viability and SAPK/JNK signal pathway were measured by trypan blue pigmentation and western blot analysis.Results:1.Ad-HO-1 was successfully generated and purified with a titer of 2×10¹⁰pfu/ml.2.The optimal MOI chosen by balancing transfection efficiency and cell viability was 100pfu/cell.Interestingly,HO-1 gene modification reduced LDH release and apoptosis index under hypoxia and serum starvation conditions.3.12 hours after hypoxia,myocardium in vitro incubated by GFP H-M,HO-1 N-M and HO-1 H-M conditioned culture medium showed statistically low apoptosis index compared with control group.While 2 hours after reoxygenation,only myocardium in vitro incubated by HO-1 H-M represented better cell viability. myocardium in vitro incubated by HO-1-transfected group showed statistically decrease on P-JNK after 1 hour of reoxygenation.Conclusions:1.Through successful virus vector construction,amplification and purification, high-titer,safe and high-efficient recombined adenovirus(Ad-HO-1) were obtained.2.Adenovirus-mediated gene transfer strategies provide an efficient and safe tool for ex vivo modification of MSC.3.hHO-1 modification strategy provide BMSC elevated tolerance to hypoxia and serum-deprived injury in vitro.The reduced hypoxia/reoxygenation cell injury of myocardium invitro incubated by BMSC-HO-1 hypoxia-conditioned culture medium are in part due to the change of SAPK/JNK Cell signal pathway.These reulsts indicate that hHO-1 modification strategy may play an important role on survival of grafted BMSC and paracrine cytokines "cocktail" of BMSC-HO-1 may provide a potential effect on cell protection under pathological circumstances.