Experimental Study On The Therapeutic Effect Of Hepatocyte Growth Growth Factor Gene Modified Mesenchymal Stem Cells On Bronchiolitis Obliterans Syndrome

Allogeneic transplantation has become a widely accepted treatment for hematologic malignancies. Despite of new and better treatments for infectious complications, and better immunosuppressive drug treatments, pulmonary complications are still the main cause of long-term morbidity and mortality following hematopoietic stem cell transplantation(HSCT). Bronchiolitis obliterans syndrome(BOS) is a serious, steadily, noninfectious, and fatal pulmonary complication. The underlying pathogenic mechanisms are poorly understood and chronic graft-versus-host disease(c GVHD) has been suggested to be involved in the development of BOS. At present, the treatment and prevention of BOS remain still a difficult issue. So developing new treatment strategies is of great importance.Hepatocyte growth factor(HGF) is a multifunctional factor with the target cells from a variety of tissues. In animal models of bleomycin-induced lung fibrosis, HGF could modulate the epithelial-mesenchymal transition and reduce the fibrin deposition at least partially by inhibition of TGF-β1 secretion, exerting its anti-fibrotic and anti-inflammation effect. However, the short half-life of HGF protein in vivo requires multiple dosing and, intravenous drug delivery can’t guarantee the deposition of HGF in the injured tissues. Mesenchymal stem cells(MSCs) are pluripotent progenitor cells with the properties of regulating immune reaction and homing to the sites of inflammation and injury. Our previous study suggested that recombinant adenovirus hepatocyte growth factor(Ad-HGF) modification could increase the expression of HGF by human umbilical cord mesenchymal stem cells( UCMSCs) and prolonged the survival time of UCMSC in the lungs of mice. HGF-modified MSCs reduce pulmonary inflammation and fibrosis in the radiation-induced lung injury and fibrosis. Gene modified stem cells, posseses the dual advantage of stem cell therapy and gene therapy, have been recognized and concerned widely in the field of gene therapy. In the present study, the therapeutic effect of Ad-HGF modified UCMSCs on transplant-related BOS was observed and the mechanisms were investigated. To evaluate the curative effect of HGF gene modified UCMSCs on the prevention and treatment of bronchiolitis obliterans syndrome, pathological morphology of transplanted trachea were observed and percentages of airway obstruction were calculated. To clarify the mechnisms, the experssion levels of inflammatory cytokines in the transplanted trachea and serum were detected by Q-PCR and ELISA respectively. Spleen lymphocyte phenotype changes were detected via FACS.Tracheal transplantation was performed as described by Hertz et al. To evaluate the MSCs distribution in mice of BOS, bone marrow MSCs from EGFP-transgenic mice were used to observe the distribution of MSCs, with EGFP as the tracing marker. Prior to the transplantation, MSCs were infected with 150 MOI Ad-Null or Ad-HGF for 36 h. MSCs-Null or MSCs-HGF at a dose of 1×106 suspended in 0.2 m L saline were administered intravenously via the lateral tail vein of each recipient mice. The peripheral blood, transplanted trachea, lung, liver, spleen and kidney of the recipients were collected at the indicated time points. Genomic DNA was then extracted according to the DNA Kit operation manuals. EGFP was quantified using the 7500 Fast Real-Time PCR System.After tracheal transplantation, the recipient mice were injected 1×106 human umbilical cord mesenchymal stem cells(h UCMSCs) infected with Ad-Null or Ad-HGF of 150 multiplicity of infection(MOI) or saline via the tail vein. At day 10 or 21 post transplantation, the serum was collected for determination of the concentrations of cytokines. The spleens were collected for lymphocyte phenotype analysis. Implanted trachea was removed for RNA isolation and histopathological analysis.The results showed that the administrated MSCs mainly distributed in the transplanted trachea, blood, liver and lung of the recipients; HGF could enhance the engraftment of MSCs. The histopathological recovery of allografts tracheas was improved significantly after MSCs-Null and MSCs-HGF treatment and the beneficial effects were particularly observed in MSCs-HGF-treated mice. Furthermore, the allo-transplantation induced immunophenotype disorders of the spleen, including Treg, Th1, Th2 and Th17, were attenuated in both cell treated groups. MSCs-HGF treatment reduced expression and secretion of inflammation cytokines interferon-gamma(IFN-g), and increased expression of anti-inflammatory cytokine IL-4 and IL-10. It also decreased the expression level of the profibrosis factor TGF-β1.In conclusion, we have shown that HGF-modified MSCs can prevent the formation of the airway fibro-proliferative lesion associated with tracheal graft rejection by means of modulating the inflammation and fibrosis process. The present study appeals further clinical trials to evaluate the role of this approach in the prevention of BOS.