Heme Oxygenase-1 Gene Modified Bone Marrow Mesenchymal Stem Cells In Reducing Acute Rejection During Rat Small Bowel Transplantation

Objective: To establish a stable and repeatable acute rejection model of rat heterotopic small bowel transplantation(SBTx) and determine whether heme oxygenase-1(HO-1) gene modified bone marrow mesenchymal stem cell(BMMSCs)could reduce acute rejection and improve outcomes for SBTx in rat.Methods:Firstly, BMMSCs were isolated from the femur and tibia of Lewis rats and cultured in a cell culture incubator by gradient centrifuge and adhesive culture methods. The third-passage BMMSCs were transfected with empty adenovirus vector or adenovirus carrying HO-1 gene to form Ad/BMMSCs or Ad/HO-1/BMMSCs,respectively. Then, acute rejection model of heterotopic SBTx was performed allogeneically from BN rats to Lewis rats. Lewis to Lewis SBTx served as control.Living condition and histopathology change of the recipients were assessed to determine whether the acute rejection model was estabished successfully. On the basic of successful modeling, recipient rats in the Ad/HO-1/BMMSCs treatment group were treated with 1 × 107 Ad/HO-1/BMMSCs through the superficial dorsal veins of the penis immediately after surgery. Control rats were treated with 1 × 107Ad/BMMSCs or 1 × 107 BMMSCs. Isopycnic saline infusions served as the basic control. The animals were sacrificed after 1, 5, 7 or 10 days for specimen collection.Small bowel histology and apoptosis were detected, HO-1 protein and m RNA were analysed by immunohistochemical staining, Western Blot and PCR. In addition,serum samples were collected to measure cytokine concentrations in serum,lymphocytes were isolated from the recipient spleens to assesse NK cell activity and numbers of T regulatory(Treg) cells at each time point.Results: Our result showed that BMMSCs isolated from rat femur and tibia have the characteristics of easy cultivation, large quantity and high purity. BMMSCs transfected with recombinant adenovirus did not changed in terms of biological property. We finally estabilished the acute rejection model of SBTx by repetitious practice, and we found there was no obvious acute rejection within 3 days post operation. While, mild or moderate acute rejection was observed on day 5 and moderate or severe acute rejection was observed on day 7. All recipient suffered severe acute rejection on day 10. The intestine graft presented heavier cellular infiltration into the muscle layer and mucosa, more apoptotic cells and severer architectural distortion and mucosal ulceration, with development of the acute rejection. All recipient died within 2 weeks and the median survival time was 10 days.BMMSCs treatment significantly improved animal living condition and changes of histopathology. Acute rejection grading scores, apoptosis and NK cell activity significantly reduced. Serum IL-10 and TGF-β concentrations, which related to anti-inflammatory or Tregs differentiation, significantly increased. While, Serum IL-2,IL-6, IL-17, IL-23, TNF-α and IFN-γ concentrations, which related to proinflammatory or Th17 differentiation, significantly decreased. Besides, BMMSCs induced the production of Tregs. However, these effects usually maintained within 7days and decreased on day 10. HO-1 gene modification significantly improved the survival rate of BMMSCs in vivo and inhanced the protective effect in SBTx. The recipient survival rate, changes of histopathology and ultrastructure and apoptosis further improved, NK cell activity significantly reduced while Tregs level significantly increased, Serum IL-10 and TGF-β concentrations significantly increased while serum IL-2, IL-6, IL-17, IL-23, TNF-α and IFN-γ concentrations significantly decreased, compare to that in the BMMSCs treated group.Conclusion: Rat femur and tibia are the ideal source of BMMSCs as the characteristics of easy cultivation, large quantity and high purity. It’s feasible to modify BMMSCs by adenovirus transfection. BMMSCs treatment reduces acute rejection for heterotopic SBTx in rat, but the effect may be limited by the low survival rate in vivo. HO-1 gene modification inhances the effect of BMMSCs on reducing acute rejection and improving the outcomes of SBTx in rat.