| Objective:1. In vitro expriments:To explore the effects of the rat bone marrow mesenchymal stem cells (BM MSCs) on the proliferation of lymphocytes.2. In vivo expriments:Heme oxygenase-1gene carried in adenovirus was transfected into the3rd generation of wistar rat bone marrow mesenchymal stem cells. To explore the protective effects of BM MSCs transplantation combined with HO-1gene therapy on intestinal ischemia-reperfusion injury.Methods:1. In vitro expriments:The mesenchymal stem cells (BM MSCs) were identified and isolated from Wista rats, then added with lymphocyte and concanavalin to set up the co-culture system. When cultured for0hour,24hours and48hours, the expression of TNF-α, TGF-β1and IL-10were evaluated by ELISA, changes in the number of lymphocytes were measured by the MTT method, and percentage of regulatory T cells (Treg) were analyzed by flow cytometry.2. In vivo expriments:(1)BM MSCs were isolated, cultivated and-purified from femur of male Wistar rats by the desity gradient centrifugation combined with adherent method. The morphology and growth condition of BM MSCs were observed in primary and passage cultured under inverted microscope, and the expression of surface marker of the3rd generation of BM MSCs was detected by flow cytometry.(2)Luc cells were injected in the intestinal mucosa of healthy Wistar rats, and luciferase tracing was used to detect colonization and survival of the transplanted cells.(3)The intestinal ischemia-reperfusion models were made in healthy aged-matched male Wistar rats. All the rats were divided into exprimental group (1ml HO-1/MSCs or1ml BM MSCs were implanted in the intestinal mucosa), and control group (lml NaCl was implanted by the same way).(4)The serum samples were collected at different times after surgery (Oh,2h,6h,24h,72h and120h), and were detected by enzyme-linked immunosorbent assay (ELISA), light microscopy, transmission electron microscopy, western blot and immunohistochemical methods.Results:1. In vitro expriments:BM MSCs inhibited the secretion of TNF-α, and increased the secretion of TGF-β1and IL-10. The optical density (OD) was significantly lower in the BM SMCs group than that in the group without BM MSCs (P<0.05), and the amount of Treg was increased in the BM SMCs group.2. In vivo expriments:(1)BM MSCs were isolated and cultured successfully with high purity and homogeneity. Most of the3rd regenation of cultured adherent cells showed positive expression of CD29, CD90and RT1A (the rate were97.12%,97.07%and98.07%, respectively), but were negative in CD34, CD45and RT1B.(2)Luciferase tracing showed that the B16-F10-Luc-G5cells were colonized in the intestine2h after injection. After getting the intestine and washing it repeatedly, the cells were still colonized and survived long-term.(3)When clipping the superior mesenteric artery, the artery pulse disappeared, and the bowel became white, cramped, collapsed and lost the peristalsis. But after the arterial pulse recovering, the bowel turned red gradually.(4)Morphological changes:HE staining showed that intestine villi edema, epithelial cells necrosis, intestinal interstitial congestion and inflammatory were significantly reduced in the experimental group compared with the control group. And the effects in HO-1/MSCs group were more significant than those in the BM MSCs group. The damage of intestinal villi and the tight junction integrity under electron microscope was reduced in the experimental group, the organelle also restored to mormal morphology. These effects were more significant in HO-1/MSCs group.(5)Serum ELISA:the serum content of DAO, D-lacticacid and TNF-α of the experimental group were lower than the control group, and the HO-1/MSCs group decreased more significantly, with all the difference was statistically significant.(6)Immunohistochemistry and western blot showed Occludin protein expression of the experimental group was significantly higher than the control group, especially in HO-1/MSCs group, which suggested HO-1may help BM MSCs to promote the expression of Occludin.Conclusions:1. In vitro expriments:(1)Density gradient centrifugation combined with adherent method could purify BM MSCs from rats. The method was simple, low cost, and easy to carry out, which made BM MSCs proliferated in large volume in vivo to provid adequate cell source for follow-up experiments.(2)BM MSCs showed inhibitory effects on lymphocytes, and the effect was not time-dependent. TGF-β1, IL-10and Treg may be involved in this immunosuppressive process..2. In vivo expriments:(1)Clipping the superior mesenteric artery could make rats intestine ischemia-reperfusion injury models successfully.(2)Intestinal submucosal injected HO-1/MSCs and BM MSCs could repair the intestinal epithelial structure, promote the expression of Occludin, and thereby prevent the increase of small intestinal permeability induced by intestinal ischemia-reperfusion injury. The effects were more significant in HO-1/MSCs group. |
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