Study Of Manganese Superoxide Dismutase Gene Transfection Of Rat Bone Marrow Mesenchymal Stem Cells Protects Them From Radiation Injury

Premise MnSOD is a kind of antioxidase that can eliminate superoxideanion in somatic cell of organism. MnSOD's activity is different indistinguished type of constitutions, MnSOD blanket tumor's growth throughnoncytotoxic mechanism to tumor's organization , low level express ofMnSOD of tumor's cell can increase sensitivity of chems and radiotherapythat treated on tumor, and it can eliminate superoxide anion due to ionizationradiations' derivation, so MnSOD is considered antiradiation andanti-oncogene that have prospect to utilize.BMSCS have characteristic of stem cell multi-directional differentiationand of adherence growth..them are easy to collection out of body and isolatedculture ,their reproductive activity are so strong that it difficult to lost stem cellcharacters.They are also homing to medulla ossium ,they can locate in medullaossium and survive for long-term through self-renewal after transfusion backof body , they may express many kinds of objective genes these are remedialand exogenous but don't effect characteristic of stem cell , their imbeddingreaction are weak. In that, marrow stroma cells are thought a sort of idealtarget cells of gene therapy. We can draw a conclusion that MnSOD gene transfection havingradiation resistance may modify marrow stoma cells according to them arecharacteristic of homing to marrow .so we make more methods onhaematogenesis and immune functional reconstruction , marrow stroma cells'abilitivity resisting radiation is increased through reinforcing express ofMnSOD.Purpose We construct MnSODcDNA eukaryotic expression vector,transfecte marrow stroma cell and approach MnSODcDNA modifications'protection of ultimate death of marrow stroma cell.And we can c a mode ofgene-radiation therapy and establish a new way in order to elevate curativeeffect of clinical tumor radiation therapy and diminish damage of normaltissue and recover haematogenesis system.Method1. We can get out mRNA from human's HuH-7 of HCC and amplificatehuman's MnSOD cDNA by half nidus type PCR method, and insert thesite XhoⅠ/ XbaⅠof pMD18-T.Then we analysis pMD-MnSOD andsequencing target gene.We can build MnSOD eukaryotic expressionvector by XhoⅠ,XbaⅠdi-enzyme cutting pMD-MnSOD and pEGFP-C1by T4 ligase.2. We collect MSC of SD rat and separate and purificate it by densitygradient centrifugation and adherent culture.Then we make extraorganprimary culture and serial subcultivation and observe cell by invertedphase contrast microscope in order to make target gene transfection.3. We shift pEGFP-MnSOD into the first exponential phase of growthmarrow stroma cell by liposome.And we detect marrow stroma celltransfection masc clone by quantitate analysis RT-PCR and detectMnSOD activity and the number of viridis-fluor transfection cell.4. We deal with exponential phase of growth marrow stroma cell by differentX ray dose.Then we analysis the protection to radiation damage toMnSOD gene transfection. MSC of rat by cell survival rate and DNAspecial fragmentation of apoptosis cellResult1. We build eukaryotic expression vector of pEGFP-MnSOD after we detectenzyme cutting and sequencing and amplification cDNA of MnSOD.We get out stable passage MSC like stem cell through separate MSC fromcanna major and femur of SD rat by density gradient centrifugation andadherent culture.2. We are succeed to transfect pEGFP-MnSOD into the growth marrowstroma cell by liposome by RT-PCR,PCR –DNA and fluorescencemicroscope observation method.3. We analysis inhibition ratio to superoxide anion O2-and find the MnSODactivity of pEGFP-MnSOD of MSC which has been succeed to transfectby macroculture is super control group .4. The Msc of pEGFP-MnSOD transfection group,pEGFP-C1 transfectiongroup,and non-transfection by different X ray dose show anti-radiation ofMSC of pEGFP-MnSOD transfection group is super than the other two.conclusion MnSOD gene transfection can elevate expression of MnSOD ofMSC and enhance the protection to radiation to MSC.It's foundation to studyMnSOD gene transfection...