Research On The Mechanism Of Recipient MSCs Inhibiting Acute Rejection Of Small Bowel Transplantation Of Rats

Part I The Establishment of Acute Rejection Model of Rat Heterotopic Segmental Small Bowel TransplantationObjective To establishof inbred BN-Lew rat eterotopic segmental small bowel transplantation animal models and observe histological of graft, determine the proper time for investigating acute rejection. Methods Using microsurgical techniques, constructed BN to Lew allogeneic and Lew to Lew heterotopic segmental small bowel transplantation animal models in rats. On day 1,3,5,7 after intestinal transplantation, Specimenfrom transplantated intestinal were harvested and HE Staining were performed. The severity of rejection were determined by histological analysis. Results Mild acute rejection on bowel allograft of BN-Lew were observed r 3 days after transplantation. Severe acute rejection appeared 5 to7days after transplantation. There is no acute rejection for Lew to Lew transplantation group.Conclusion Acute rejection did occur in BN strain to L ew strain rat heterotopic segmental small bowel transplantation model and the proper time for monitor acute rejection is day 7 after surgery.Part II The Seperation, Proliferation and Characterization of Rat Bone Marrow Mesenchymal Stem Cells and Determining their Immunesuppressive Activity in VitroObjective Isolate, culture and characterize the rat bone marrow mesenchymal stem cells and study the immune suppressive activity in vitro. Methods The whole bone marrow adherent method was used to isolate and proliferate MSCs. The phenotype of MSCs was determined by flow cytometry. The differential potential of MSCs were confirmed with osteogenic and adipogenic method. The mixed lymphocyte culture system were prepared using spleen cells as response cells, MSCs as stimulating cells, and ConA as T cell proliferation stimululating factor. Four 4 groups were compared:?Spleen cells alone;?spleen cells + MSCs;?spleen cells + ConA;?spleen cells + ConA + MSCs. After incubated 72 hours the expression of T cell subsets CD4+ and CD8+were analyzed by flow cytometry. Results?Most of primary MSCs were adherent in 48-72 hours. There wre 90% MSCs are converged three weeks after culcuration. The 3rd generation of MSCs were round and spindle.?Ninety-five percent of the 3rd generation MSCs were positive for surface markers CD29 and CD90, and less than five percent were positive for CD45.?The ratio of CD4+ cells: No difference between groups spleen cells + MSCs and spleen cells + ConA, significant differences between other groups.There is the most obvious difference between spleen cells + ConA and spleen cells + ConA + MSCs; CD8 expression: No difference between Spleen cells and spleen cells + MSCs?spleen cells + ConA?spleen cells + ConA + MSCs, but there are significant difference between spleen cells + MSCs?spleen cells + ConA and spleen cells + ConA + MSCs.CD4/CD8:In addition to spleen cells + ConA and spleen cells + ConA+MSCs, there were significant differences in the other groups. Conclusion MSCs can be prepared through bone marrow adherent culture with higher purity and strong proliferation ability. MSCs can be passaged for many times, with osteogenic and adipogenic differentiation potential. MSCs in vitro can inhibite T lymphocyte proliferation, mainly inhibiting CD4+, without significant inhibition for CD8+cells.Part?Research on the Mechanism of Recipient MSCs Inhibiting Acute Rejection of Small Bowel Transplantation of RatsObjective Study the mechanism of inhibiting intestinal allograft acute rejection by administration of recipient-derived bone marrow mesenchymal stem cells.Methods The experimental animals were randomly divided into 4 groups:?Lew to Lew syngeneic group;?BN to Lew allogeneic acute rejection group;?Acute rejection + FK506 treatment control group;?Acute rejection + MSCs treatment group.On day 7 after transplantation, transplantated intestinal specimens were harvested, PCR Array were performed to detect the transcription of immunological cytokines. Results Compared to syngeneic control, IDO and HGF transcription in allogeneic acute rejection group were significantly increased and Tnfsf15 transcription was significantly downregulated; Compared to acute rejection group, IDO and HGF expression was significantly inhibited and Tnfsf15 expression was upregulated in FK506 treatment group.; As compared to acute rejection group, Tnfsf15 and Igf2 dtranscription was increased significantly in MSCs treatment group, with Illa, VEGF-A, Igfbp6 upregulation significantly, lgf2,iNOs, Igfbp1, Tgfbl downregulation significantly; Compared to FK506 treatment group, IDO and Tgfbl transcription is significantly less in MSCs group, but VEGF-A?115and Ccr3 transcription were significantly increased; Compared to syngeneic group, VEGF-A and 1115 was significantly upregulated in MSCs group, with downregulation of IDO, Igf2, Ifng, Lta, Tgfb1, Ccl4 and 1118. Compared to syngeneic group, Ccl4?Ccr3 Igf2 andTgfb1 transcription were significantly decreased in FK506 treatment group. Conclusion?IDO, HGF and Tnfsfl5 mediated pathway played important role in acute rejection of small bowel transplantation in rats.?FK506 was able to fully suppress these three cytokines, which can effectively inhibit acute rejection of the rat small bowel transplantation.?MSCs treatment resulted in upregulation of Tnfsfl5 and partially inhibit the acute rejection of rat small bowel transplantation. The inhibitory effect of MSCs was weaker than that of FK506, but MSCs induced expression of factors related to immune tolerance, indicating that MSCs can not only inhibit acute rejection of small bowel transplantation, but may also induce immunetolerance.