| Objective1. To investigate the in vivo methods of isolation, cultivation and purification of marrow mesenchymal stem cells derived from bone marrow of male Wistar rats and to identify its phenotype.2. To study the protective effects of marrow mesenchymal stem cells (BM MSCs) allograft on the recovery of ischemia-reperfusion injury in rats and its possible mechanisms.Methods1. BM MSCs were isolated, cultivated and purified from femur of male Wistar rats by the density gradient centrifugation combined with adherent method. The morphology and growth condition of BM MSCs were observed in primary and passage cultures under inverted microscope and the expression of surface marker was detected by flow cytometry.2. Intestinal ischemia-reperfusion injury models were established in male Wistar rats (6-8weeks old with body weight180-200gram). All animals were fasted12hours with free access to water before operation. Standard sterile technique was used during operative procedures. We identified the principal branches of superior mesenteric artery of Wistar rats and occluded it with an atraumatic micro vascular clamp for30minutes to induce intestinal ischemia-reperfusion injury model.3. The third generation cultured cells were prepared as suspension (5×106/ml) in normal saline (NS). Intestinal ischemia-reperfusion injury models were established in72male Wistar rats, which were randomly divided into the experimental group (lml BM MSCs suspension was injected through the intestinal submucosa) and the control group (lml NS was inject through the intestinal submucosa), each with36rats. The rats were sacrificed on the postoperative2,6,24,72and120hours, respectively, with their serum and intestinal tissue samples collected for analysis. Luciferase tracing was used to detect colonization and survival of the transplanted cells. Serum diamine oxidase (DAO), D-lactate and TNF-α were tested by ELISA. Pathological changes in small intestinal tissue were observed under light microscope, and the changes of intestinal tight junction were observed by transmission electron microscope. And the tight junction protein ZO-1was tested by immunohistochemistry and Western blot.Results1. BM MSCs were isolated and cultured successfully with high purity and homogeneity. Most of the3rd generation of cultured adherent cells showed positive in CD29, CD90and RT1A, but negative in CD34, CD45and RT1B.2. Intestinal ischemia-reperfusion injury models were established successfully. Luciferase tracing showed submucosal injected BM MSCs were colonized in the intestine and survived long-term.3. DAO, D-lactate and TNF-a were tested by ELISA. DAO was11.36±1.89IU/ml and5.04±1.04IU/ml at6hour and24hour after injection in the experimental group, in contrast to14.27±2.09IU/ml and7.35±1.46IU/ml respectively in the control group. D-lactate was0.157±0.025mmol/L and0.109±0.013mmol/L at6hour and24hour after injection in the experimental group, in contrast to0.193±0.019mmol/L and0.141±0.007mmol/L respectively in the control group. TNF-a was266.09±8.84pg/ml and190.39±4.24pg/ml at6hour and24hour after injection in the experimental group, in contrast to286.81±11.54pg/ml and218.49±11.54pg/ml respectively in the control group. The DAO, D-lactate and TNF-a in the experimental group at6hour and24hour are lower than those of the control group (P<0.05).4. Pathological changes of the intestinal tissue:HE staining showed that edema of intestinal villi, necrosis of epithelial cells, congestion of interstitial tissue and infiltration of inflammatory cells were all relative slight in the experimental group compared with the control group. Under transmission electron microscope, the experimental group showed less damage in small intestinal villi, and also the intestinal tight junction and normal morphology of organelle were restored completely.5. Immunohistochemistry and Western blot results showed that ZO-1protein expression in the experimental group was significantly higher than that in the control group, suggesting that BM MSCs could promote the production of ZO-1protein.Conclusion1. Density gradient centrifugation combined with adherent method could purify BM MSCs from rats. The method was simple, low cost, and easy to use in which BM MSCs could proliferate in large volume in vivo.2. Intestinal submucosal injected BM MSCs could repair the intestinal epithelial structure, promote the expression of ZO-1, and thereby prevent the increase of small intestinal permeability that induced by intestinal ischemia-reperfusion injury. So we may say that the BM MSCs had protective effects on intestinal ischemia-reperfusion injury in rats. |
PDF Full Text Request