Effects And Mechanism Of Bone Marrow Mesenchymal Stem Cells Genetically Modified By Extracellular Superoxide Dismutase On Cardiomyocytes Protection Against Hypoxia/Rperfusion Injury

Background and aimsAccording to the2012China health statistics yearbook, in2011, mortality ofcardiovascular disease in China cities is136.72/100,000, accounting for19.51%of thetotal mortality, and in China countrysites is128.13/100,000, accounting for17.45%.With establishment and application of thrombolytic, coronary artery angioplasty, arterybypass surgery and other methods, reperfusion of myocardial tissue after ischemia,mortality of early myocardial infarction significantly reduce. However, long-term heartfailure and mortality caused by Ischemia-reperfusion injury remained stubbornly high.Therefore, prevention of ischemia-reperfusion is noticed by more and more people. Oxidative stress reflects an imbalance between the systemic manifestation of reactiveoxygen species and a biological system’s ability to readily detoxify the reactiveintermediates or to repair the resulting damage. Disturbances in the normal redox stateof cells can cause toxic effects through the production of peroxides and free radicalsthat damage all components of the cell, including proteins, lipids, and DNA. Oxidativestress is thought to be involved in the development of cancer, Parkinson’s disease,Alzheimer’s disease, atherosclerosis, heart failure, myocardial ischemia/reperfusion,etc. It is closely associated with cardiovascular disease (CVD), including coronaryheart disease, hypertension, myocardial infarction and other cardiovascular diseasedevelopment. Therefore, reducing oxidative stress is one of key strategy to prevent andtreat myocardial ischemia-reperfusion and to reduce mortality.Extracellular superoxide dismutase is discovered and named by Marklund in1982.It is an enzyme and one of the superoxide dismutase protein family. SODs areantioxidant enzymes that catalyze the dismutation of two superoxide radicals intohydrogen peroxide and oxygen. The product of this enzyme is thought to protect thebrain, heart, and other tissues from oxidative stress injury.Gene therapy is a new method to treat disease, which use of genetic engineeringtechnology as tool to import the purpose gene, repair abnormal genes or makecorresponding protein expression. The basic principle is that using the vector (usuallyvirus) carry the target gene to defective cells in order to repair a defective gene or tosecrete the corresponding proteins. Gene therapy is first conceptualized in1972. In1990, the FDA approved gene therapy experiment to treat severe combinedimmunodeficiency(SCID) at first time. Since then, more than1700clinical trialsabout gene therapy have been conducted. Although there are many questions aboutgene therapy caused by failure of early clinical trials, a renewed interest in genetherapy have been lead by success in treatment of Leber’s congenital amaurosis, SCID and Parkinson’s disease, etc.Our study intends to explore the effects of BMSCs genetically modified byec-SOD on cardiomyocytes under hypoxia/reperfusion and its possible mechanisms.MethodsPart Ⅰ: Cardiomyocytes and bone marrow mesenchymal stem cells were isolated,cultured and identified in vitro. Cardiomyocytes (CMs) and bone marrowmesenchymal stem cells (BMSCs) were isolated by adhesion dissociation method asdescribed from neonate Sprague–Dawley rat hearts and C57mice bone marrow. Cellmedium is DMEMF-12containing10%FBS. And the medium were changed every3days. Morphological characteristics of cardiomyocytes and BMSCs were identified byinverted microscope. Cardiomyocytes were identified by cTnI staining assay. Surfacemarkers of BMSCs were identified by Flow cytometry assay.Part Ⅱ: Effects of BMSCs genetically modified by ec-SOD on CMs. CMs andBMSCs were isolated and allocated into4groups: control group; H/R control group;conditioned medium group; co-culture group. Secretion of ec-SOD was measured byElisa assay. CMs were refreshed by the supernatant medium from ecSOD-BMSCs at48hours post transfection, or co-cultured with transfected-BMSCs. All the cells weresubjected to hypoxia for6hours and re-oxygen2hours, CMs cell viability wereidentified by MTT assay. The apoptosis rate was detected by TUNEL assay.Part Ⅲ：Mechanisms of BMSCs genetically modified by ec-SOD on CMs’protection. CMs Cells were allocated into4groups: control group; H/R control group;conditioned medium group; co-culture group. They were under a state of hypoxia for6hours, after which oxygen was recovered2hours, the content of ROS was detected byDHE staining assay. Protein expression of p38MAPK and phosphorylated p38MAPKof CMs were measured by Western blot. Results:1. After cultured for3days, CMs extend pseudopodia interweaving net. Pulsationis synchronous and powerful. And frequency of pulsation is100~120by min. Theimmunostainning assay showed that CMs were positive for cTnI. BMSCs were showedtypical spindle, fibrous, and arrangement closely. The result of surface markersdetected by Flow cytometry assay is that CD44, CD90is positive and CD34, CD45isnegative.2. BMSCs genetically modified by ec-SOD can significantly reduce the mortalityof CMs. At the same time, it can also significantly reduce apoptosis of CMs.3. BMSCs genetically modified by ec-SOD can significantly reduce ROS levels.At the same time, it is possible to suppress the phosphorylation of p38MAPK.ConclusionBMSCs genetically modified by ec-SOD can reduce oxidative stress of CMsunder H/R injury, which may be associated with the reducing expression ofphosphorylated p38MAPK.