Experiment Study Of HO-1Modified Adipose-derive Stem Cells Transplantation For Treatment Of Asthma In Mice Models

Part â… : Isolation, cultivation and identification of mice adipose-derive mesenchymal stem cells in vitroObjective: To isolate, culture murine adipose-derived mesenchymal stem cells（ADSCs）, to identify ADSCs by flow cytometry and stem cell pluripotent differentiationtechnology，to master stem cells culture techniques in vitro and understand the biologicalcharacteristics of ADSCs,Material and methods: To take inguinal and abdomen adipose tissue of BALB/cmice aged6-8weeks by sterile technique. Adipose tissue was digested with type Icollagenase and got ADSCs, the cells were cultured in Dulbeco’s modified eaglemedium（DMEM） containing10%fetal bovine serum（FBS）, ADSCs were isolated byadherent, ADSCs were subcultured and purified in vitro. Cell growth was observed usingan inverted microscope every day. Cell adhesion rate was calculated and growth curve ofADSCs was draw. Stem cell surface markers CD90, CD44, CD11b and CD45wereidentified using flow cytometry. ADSCs were induced into adipocyte, osteocyte,chondrocyte using different induction medium. ADSCs were identified using oil red Ostaining, modified Gomori calcium cobalt method, Von Kossa staining, Alcian bluestaining method.Results: The cells sparsely attached to the surface of the flask as fusiform, a fewtriangular, polygon on the second day after inoculation. When the medium was changed onthe third day, most cells were adherent. On the fifth day, cells were colony-like growth, theshape of cells were the fibroblast-like spindle. On the ninth-tenth day, cell colonies were about80%confluence, cells were uniform spindle-shaped and spirally arrange. About twoweeks later, the cells could cover the bottom. Passaged cells grow in a uniformlydistributed manner, cells were almost completely confluent on the fifth-seventh day, aweek later cells could be subcultured one time. About2hours after cell inoculation, somecells were adherent, adherent rate was29.4%±1.3%, it was97.3%±1.8%24hours later.Cells began to grow on the first day after inoculation, but cell growth is relatively slowduring the first3days, cell growth was accelerated during the fourth-eighth day, cells werealmost confluent on the eighth day. Cell morphology showed uniform long spindle orfibrous until three generations. The determination of CD90, CD44-positive cells reached96.6%and85.3%, CD11b, CD45positive cells were reached0.5%, they could differentiateinto adipocytes, osteoblasts, and chondrocytes in different medium. It indicated that thecells were ADSCsConclusion: we could get lots of adipose-derived mesenchymal stem cells by type Icollagenase and adherent in vitro, cell surface markers CD90, CD44were positive andCD11b, CD45were negative, the cells were with multipotent differentiation in vitro.Part2: Establishment of lentiviral vector with HO-1gene andtransfection for adipose-derived mesenchymal stem cellsObjective: To prepare the lentiviral vector with HO-1gene and the HO-1genesequence was tested; lentiviruses were packed in293T cells, the high titer lentivirus withHO-1gene were obtained, ADSCs were transfected by lentivirus with HO-1gene.Material and methods: Total RNA was obtained from fresh spleen tissues of mice,total RNA was reversely transcribed to cDNA. According to mouse HO-1gene （GeneBanknumber: NM₀10442.2）, the primer of HO-1was designed and synthesized, the cDNAfragment of HO-1gene was obtained using RT-PCR technology and connected with thepMD18T vector. The pMD18T-HO-1was obtained. And then it was connected with thepCDH-MCS-EF1-GFP vector linearized by digestion, the product transformed thecompetent cells XL10-gold. The clone was identified; the positive clones were selected to be sequenced. Lentiviruses were packaged in293T cells, lentiviruses were collected andconcentrated, the titer of virus was determined. Finally ADSCs were transfected bylentiviruses with HO-1gene. The expression of HO-1on ADSCs was determined inRT-qPCR and Western-Blot way.Results: Agarose gel electrophoresis and sequencing confirmed that the recombinantVector pCDH-CMV-HO-1was successfully constructed; HO-1gene wassuccessfully introduced into the pCDH-CMV-MCS-EF1-GFP-Puro vector. Lentiviruseswith HO-1gene were successfully packaged in293T cells, the virus titer was3×108TU/ml. Lentiviruses with HO-1gene was successfully transfected into ADSCs, HO-1wasabundantly expressed in ADSCs.Conclusion: Lentiviruses with HO-1gene was successfully constructed, and ADSCswere successfully transfected with lentiviruses with HO-1gene.The high qualified ADSCswith HO-1gene were provided for the animal experiment.Part3: Experimental study about transplantation ofADSCs with HO-1gene in the treatment of asthmaObjective: Mice were as models of asthma, the effect was researched about ADSCswith HO-1gene in the treatment of bronchial asthma, and the impact on cytokinesinvolved in the pathogenesis of asthma was discussed. The effect was compared betweenADSCs and ADSCs with HO-1gene in asthma. The feasibility of gene therapy in asthmawas evaluated.Material and methods:32female BALB/c mice were chosen, weight is18-22g,and animal model of asthma was established via OVA sensitization and challenging. Micewere divided into four groups: control group, asthma group, HO-1/ADSCs group andADSCs group. Mice were sensitized with200μl OVA on d0,7,14and challenged viainhaling3%OVA solution from d21to d27,30minutes every time, once a day. On thetwentieth day, the asthma group was injected with0.1ml natural saline through tail vein.The procedure of ADSCs group and HO-1/ADSCs group were same as that in the asthma group, but on the twentieth day, they were injected with0.1ml2×106ADSCs or ADSCswith HO-1gene. Control group received saline instead of OVA, the procedure is same asthat in the asthma group. On the twenty-eighth day, mice were sacrificed after spirometrytest to observe the changes of lung tissue, inflammatory cells, cytokines in BALF andexpression of HO-1in lung tissue.Results: Mice model of asthma was successfully established. Compared with thecontrol group, in asthma group airway resistance was increased, lung compliance wasdecreased, the total number of cells and eosinophils were significantly increased in BALF,cytokines like IL-4, IL-5, IL-13,TNF-were significantly increased and the cytokine likeIL-10, IFN-γ was significantly decreased. HE staining of lung tissue shows inflammatorycells infiltration around the airway, PAS staining showed mucus cells were increased inairway mucosa, expression of HO-1was increased through immune-histochemistrystaining. In treatment groups airway resistance could be reduced, lung compliance could beincreased, the total number of cells and eosinophils in BALF could be reduced, cytokineslike IL-4、IL-5、IL-13、TNF-could be reduced, cytokines like IFN-γ、IL-10could beincreased, infiltration of inflammatory cells around the airways and mucus cells in airwaymucosa could be reduced, HO-1expression in lung tissue could be increased. The effect inHO-1/ADSCs group was better than that in ADSCs group; the difference between thegroups was statistically significant.Conclusion: ADSCs with HO-1gene and ADSCs had therapeutic effect on asthma,but the effect in ADSCs with HO-1gene was better than that in ADSCs, the gene therapywas feasible in the treatment of asthma.