The Role Of Bone Marrow Mesenchymal Stem Cells Modified By Chemokine Receptor CXCR3 And Heme Oxygenase-1 Gene In Protecting The Transplanted Small Intestine And Its Protection Mechanism

Objective: To investigate the effect of bone marrow mesenchymal stem cells(BMMSCs)co-modified with chemokine receptor CXCR3 gene and heme oxygenase-1(HO-1)gene on relieving the rejection after small intestinal transplantion and repairing the function of transplanted intestine and its mechanism.Methods: 1.Healthy male Lewis rats at the SPF level were used to extract their femoral and tibial bone marrow.BMMSCs were prepared by whole bone marrow adherent screening method,and were identificated by morphological,differentiation and surface markers.2.Adenovirus carrying different target gene transduction into BMMSCs to prepare Ad/BMMSCs,Ad-HO-1/BMMSCs,Ad-CXCR3/BMMSCs and Ad-(CXCR3+HO-1)/BMMSCs.3.The IEC-6 cells were treated with TNF-α to establish the injury model.The injured IEC-6 cells were co-cultured with different kinds of BMMSCs and normal lymphocytes.The chemotactic ability of different kinds of BMMSCs was detected by crystal violet staining and viable cell workstation.Western blot,RT-PCR and immunohistochemistry were used to detect the expression of p38-MAPK pathway-related molecules.The activity of T lymphocyte was detected by flow cytometry and ELISA.4.BN as donors,Lewis as recipients,establish ectopic small bowel transplantation rejection model.Pretreatment of recipients before transplantation and the experimental specimens were collected at 0 h,1 d,3 d,7 d,10 d and 14 d.The recipients’ general status and survival time were observed and the survival rate was analyzed.Intestinal rejection and injury were analyzed by HE staining.Western blot,RT-PCR and immunohistochemistry to detect the expression of HO-1,CXCR3 and the related molecules of the p38-MAPK pathway in small intestine.Flow cytometry to detect the level of Tregs.The activity of NK cells were detected by lactate dehydrogenase(LDH)method.The concentrations of DAO,proinflammatory cytokines and anti-inflammatory cytokines were detected by ELISA.Results: 1.BMMSCs were successfully obtained by whole bone marrow adherent screening method.Ad-(CXCR3+HO-1)/BMMSCs were successfully obtained and their biological characteristics were not changed compared with BMMSCs.2.The model of injured intestinal cell IEC-6 was successfully established in vitro.The acuterejection model of small intestine transplantation was established by BN rat-donated intestine transplanted to Lewis rat.3.Different genes screened by microarray were associated with the p38-MAPK pathway.4.Western blot,RT-PCR and immunohistochemistry showed that BMMSCs could inhibit the phosphorylation of p38-MAPK protein in damaged intestinal cells,leading to inhibition of the phosphorylation of downstream molecules ATF2,CHOP10 and MEF2 C,decreased apoptosis,and increased expression of PCNA and ZO-1.Among them,(Ad-(CXCR3+HO-1)/BMMSCs)showed the most significant repair effect,and CXCR3-modified BMMSCs decreased T lymphocyte activity significantly.5.In the rejection model of allogeneic intestinal transplantation,combined treatment of Ad-(CXCR3+HO-1)/BMMSCs could significantly prolong the survival time of recipients,reduce the proinflammatory cytokines IL-2,IL-6,IL-17,IL-23,IFN-γ and TNF-α,reduce intestinal epithelial cell apoptosis and NK cell activity,increase the anti-inflammatory cytokines IL-10 and TGF-β and the level of Tregs.Analysis of the transplanted intestine in different groups on 7 day after surgery found that,Ad-(CXCR3+HO-1)/BMMSCs could inhibit the phosphorylation of both p38-MAPK protein and downstream molecules of p38-MAPK pathway such as ATF2,CHOP10 and MEF2C,and increase the expression of PCNA and ZO-1 more significantly.Conclusion: Whole bone marrow adherent screening method can successfully produce BMMSCs that meet the experimental needs.It is feasible to transfect BMMSCs with adenovirus carring with different genes.BMMSCs modified with combined CXCR3 and HO-1 genes showed more significant effects than both BMMSCs and HO-1 gene modified BMMSCs in postponing the onset of acute rejection,repairing damaged structure and function of intestine,and prolonging the survival time of the recipient.At the same time,the p38-MAPK signaling pathway was involved in this protective effect.