Intestinal Mucosal Barrier Protected After Small Intestine Transplantation In Rats By Implantation Of Bone Marrow Mesenchymal Stem Cells

Objective:1. To establish a stable acute rejection model of heterotopic small bowel transplantation in rats.2. To explore the expression pattern of tight junction associated proteins, ultrastructural and graft permeability after small intestine transplantation in rats.3. To explore the mechanism of intestinal mucosal barrier protected after small intestine hetetotopic transplantation in rats by implantation of bone marrow mesenchymal stem cells(BM MSCs). Methods:1. The rat hetetotopic small intestine transplantation models were divided into non-rejection (Lewis-Lewis, n=33) and rejection (BN-Lewis, n=43) groups. In auxiliary heterotopic small bowel transplantation, veins and arteries anastomosis under microscope. The proximal end of the donor s small bowel was anastomosed to stoma on the abdominal wall. Record the time of operation, compare two groups of survival, and weight. And the graft was observed the morphological and dynamic changes. The degree of histopathological changes was graded semiquantitatively using the histological injury scale previously HE staining in postoperative1,3,5,7,10day for intestinal transplantation.2. The rat hetetotopic small intestine transplantation models were divided into sham operation (n=30)、non-rejection (Lewis-Lewis, n=33) and rejection (BN-Lewis, n=43) groups. The injury of intestinal barrier was estimated through expression levels of diamine oxidase and D-lactic acid. Ultrastructural change of tight junction was observed to by transmission electron microscopy. Furthermore, the expression levels of Occludin and ZO-1affected by tumor necrosis factor-alpha, Interferon-gama and interleukin-10were measured using real-time PCR, Western blot.3. The rat hetetotopic small intestine transplantation models were divided into a non-rejection group (n=33), saline-treated experimental rejection group (via penile vein, n=43), and BM MSC-treated therapy group (via penile vein, n=33). The injury of intestinal mucosal barrier was estimated through expression levels of diamine oxidase (DAO) and D-lactic acid (D-LA). Tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), interleukin-10(IL-10), transforming growth factor-β (TGF-β) are detected by enzyme-linked immunosorbent assay (ELISA). Ultrastructural change of tight junctions (TJs) was observed to by transmission electron microscopy. Furthermore, the expression levels of Occludin and Zona occludens1(ZO-1) affected by the inflammatory factors were measured using real time PCR and Western blot. To explore the mechanism of intestinal mucosal barrier protected after small intestine hetetotopic transplantation in rats by implantation of bone marrow mesenchymal stem cells(BM MSCs).Results:1. The survival rate10d postoperative were90.91%,69.77%, respectively(P <0.05). Until7day, pathological symptoms of severe rejection obvious, weight loss average of5.23g during the daily. But, non-rejection is no significant rejection performance, the weight reduce average daily6.11g, started to get better to6d, average daily increase6g.2. Damage of intestinal mucosa barrier of small bowel transplantation rejection injury is no rejection group have significant differences,[diamine oxidase (U/mL):35.84±5.6vs.12.75±0.4,3d,46.50±0.8vs.16.27±0.6,5d;54.01±1.7vs.19.40±0.8,7d, P<0.05; D-lactic acid (g/L):8.01±0.78vs.6.20±0.15,3d,9.11±0.45vs.8.23±0.26,5d;12.60±1.0vs.10.37±0.6,7d, P<0.05). Intestinal microvilli and TJs were disrupted, and organelles were swollen with reduced electron density. Simultaneously, the expression levels of the two tight junction associated proteins Occludin and ZO-1decreased at both the mRNA and protein levels. It showed that TNF-a, IFN-y, IL-10and expression of tight junction proteins Occludin and ZO-1present correlation, reduced statistically significant in the no rejection group (bP<0.05);3. Density gradient centrifugation combined withadherent method could purify BM MSCs from Lewis rats. Purity of phenotypic characterization of stem cells more than95%flow cytometry. BM MSCs treatment group, found that the general situation improves each time point, the pathological scores were mitigate (rejection vs stem cell treatment group:69.5±2.43vs.41.83±2.93,P<0.05,5d), intestinal mucosal barrier permeability decrease [diamine oxidase (U/mL):46.50±0.77vs. 39.15±3.98,5d, P<0.05; D-lactic acid (g/L):9.11±0.45vs.7.031±0.18,5d, P <0.05]. BM MSCs therapy group can reduce DAO and D-LA, in graft, and inhibit production of TNF-a, INF-y, produce a large number of IL-10, TGF-β, increase expression of TJs proteins Occludin and ZO-1and mRNA, and is statistically significant with the rejection group (P<0.05).Conclusion:1.To establish a stable acute rejection model of heterotopic small bowel transplantation in rats.2. In the rat hetetotopic small intestine transplantation models, intestinal mucosal barrier and tight junction is impaired by the rejection reaction, and the expression of the tight junction associated proteins Occludin and ZO-1decreased along with the progression of rejection reaction in the intestinal tissues, closely related to the increase of TNF-a,IFN-γ.3. BM MSCs can improve intestinal barrier permeability and repair TJs, increased proteins of Occludin and ZO-1expression, protecting the intestinal mucosal after transplantation, and along with change of cytokines.