Biological Effects Of ECSOD Gene In Vitro Transfection On Rhesus Bone Marrow Mesenchymal Stem Cells

With the rapid development of science and technology, application on nuclearenergy and nuclear technology has become increasingly widespread in industry,agriculture, medicine and military field. More and more countries have own or aredeveloping nuclear weapons. The exploitation and utilization of nuclear powertechnology is extraordinarily rapid. Although people have pay attention to the radiationprotection when using the nuclear energy technology, nuclear emergencies still happenfrequently, resulting in acute radiation sickness (ARS) among high dose ionizingradiation exposed population. Based on exposure dose and disease course, ARS can bedivided into bone marrow form, intestinal form, and brain form, and bone marrow formARS can be subdivided into mild, moderate, severe and extremely severe degrees. Atpresent, the main treatment measures to ARS patients are anti-radiation drugs,hematopoietic stem cell transplantation and comprehensive symptomatic therapy.Severe degree or less bone marrow form ARS can be successfully cured by thesemeasures, but therapeutic effect of extremely severe degree bone marrow form ARS ispoor, with existing measures can only prolong survival time of patients. The treatmentof extremely severe degree bone marrow form ARS is very difficult and bone marrowhematopoietic function failure, immune function loss and multi-organ failure are themain reasons that cause those patients of severe degree bone marrow form ARS death.It is well known that body injuries cased by rays consist of direct injuries andindirect injuries. Direct injuries effect time is short but damage quickly.. They aremanifested mainly as internal molecular structure changes and loss of biological activity,with direct shielding being only protection measure. Indirect damage is a complicatedprocess, the body contains a lot of water (accounting for80%of the organisms dryweight). Rays can cause activation of water molecules and free radical generation. Freeradicals can attack the bases, ribose, and phosphodiester bonds in DNA molecules, resulting in oxidation of base and ribose, bond breakage, and protein cross linking. Atthe same time, free radicals can damage the bio-membrane system through a series ofreactions, resulting injuries of material transport, energy conversion, as well as signaltransduction and recognition. All above is referred to as indirect injuries. These aresame naturally occurring free radical scavenging enzymes inside the body. For example,catalase and peroxidase can decompose H2O2, superoxide dismutase can catalyseO2-dismutation reaction, and glutathione transferase can clear the hydroxyl peroxide.However, these enzymes can mainly clear endogenous free radicals, which are lesseffective to large amount of exogenous free radicals generated by rays. Althoughinternal superoxide dismutase can resist radiation through free radical scavenging, theyhave no ideal protection effects on tissues and organs directly acted by rays, as well asearly stage injuries caused by free radicals. There is an unmet medical need to develop anew therapeutic approach which can effectively clear early stage free radicals and caneffectively repair multi-organ failure.Bone marrow mesenchymal stem cells(MSC) are a class of non-hematopoieticstem cells derived from bone marrow, which can be amplified in vitro and havemultilineage differentiation potential. In specific induction conditions, MSC candifferentiate into osteoblasts, chondrocytes, adipocytes, tendon cells, myotubes, nervecells and matrix supporting hematopoietic stem cells. Several studies have confirmedMSC homing into damage tissues and differentiation into appropriate tissue cells afterimplantation. MSC can be combined with a variety of viral vectors and can undergo avariety of gene modified, which has the potential to be a new cell drug for the treatmentof ARS.Based on above background, this study aimed to use adenovirus expression vectorbearing extracellular superoxide dismutase (ECSOD) and reporter gene EGFP totransfect rhesus bone marrow mesenchymal stem cells, to investigate biological effectsof ECSOD gene in vitro transfection on rhesus bone marrow mesenchymal stem cells(R-BMSC),and to provide experimental basis for clinical management of extremelysevere degree bone marrow form ARS.First, using density gradient centrifugation and adherent culture way to isolate,culture and purify R-BMSC to culture cells’ fibrous adherent growth. To prove thatnon-hematopoietic cells are in line with the reported mesenchymal stem cell surfacemarkers, training the third generation of cells surface antigens detected by flow cytometry. In order to prove R-BMSC of multipotent differentiation ability, inducingR-BMSC differentiation to the osteogenic and adipogenic cells.Second, structuring carrying gene of ECSOD and EGFP adenovirus expressionvector in use of gateway technology in vitro, receiving adenovirus with good vigorthrough packaging in293cells. infecting R-BMSC with different MOI adenovirus invitro, making application of fluorescence microscopy and flow cytometry detectedinfection efficiency can reach more than90%; cell culture supernatant ECSOD proteinexpression was detected by ELISA after infection.Finally, in order to determine the impact of the ECSOD gene transfection on thebiological properties of R-BMSC, detecting MSC-ECSOD cell surface antigens usingflow cytometry, the results show that MSC-ECSOD is similar to the phenotype of theR-BMSC. Further testing the multipotent differentiation ability of MSC-ECSOD, theadipogenic inducement14days, oil red O staining showed dyed red fat droplets; theosteogenic inducement17days, alizarin red staining showed dyed red bone nodules,which show that ECSOD transfection has no influence on the R-BMSC multipotentdifferentiation ability. There is no statistical difference in the proliferative capacity ofcells before and after infection by MTT assay.In summary, the R-BMSC harvested by density gradient centrifugation andadherent culture has similar biological characteristics to human bone marrowmesenchymal stem cells. The phenotype and multipotent differentiation ability of theR-BMSC were successfully identified. In vitro infection of R-BMSC with adenovirusvectors showed higher than90%infection efficiency. ECSOD has no influence onphenotype and biological properties of R-BMSC. This study provides experimentalbasis for management of extremely severe degree bone marrow form ARS with ECSODgene-modified R-BMSC.