Study On The Protective Effects Of HO-1Transfected BM MSCs On The Damaged Intestinal Epithelial Barrier And Its Related Mechanism

Objective:1.To investigate the immunomodulatory effect of Bone Marrow Mesenchymal Stem Cells(BM MSCs) on T cell activation in vitro and vivo.2.To investigate the immunomodulatory mechanisms of BM MSCs and HO-1/MSCs in the monolayer enterocyte environment in vitro.3.To investigate the protection mechanisms of Heme oxygenase-1overexpression in BM MSCs(HO-1/MSCs) at the time of intestine Ischemiarepeffusion injury.Methods:1. BM MSCs were isolated from femur of4-5-week-old healthy male Wistar rats by the density gradient centrifugation in conjunction with adherent method. The expression of surface marker of the third generation of BM MSCs was detected by flow cytometry.2. Co-culture model establishment in vitro:CaCo-2cells were used to establish the model of single-layer intestinal epithelial environment in the6well culture plate. The third generation of BM MSCs or HO-1/MSCs (2×105/mL) and T lymphocytes of the rat’s spleen (1x106/mL) were co-cultured in each well. ConA (10μg/mL) was added to stimulate the proliferation of T lymphocytes in each well except in the blank group. Oh,24h and48h were selected as3different time points. Co-cultured cells were divided into7different groups: lymphocyte+HO-1/MSCs+CaCo-2+ConA (experimental group), lymphocyte+BM MSCs+CaCo-2+ConA (control group A), lymphocyte+CaCo-2+ConA (control group B), lymphocyte+HO-1/MSCs+ConA (control group C),lymphocyte+BM MSCs+ConA (control group D), lymphocyte+ConA (Proliferation group), lymphocyte (blank group). The activity of lymphocyte was detected by MTT. The cytokine TNF-a, IL-10and TGF-1β were detected by ELISA. The Foxp3+regulatory T cells were detected by flow cytometry.3.Imitate the ischemia-repefusion injury time of small intestine transplantation (SIT) to establish the intestinal model of ischemia-reperfusion injury(IRI):Select the well conditioned6-8-weeks-old wistar rats keeping fasting for12hours before operation. Isolate the superior mesenteric artery and block the artery for40minutes sterilely, then loose the artery forceps, inject the cell suspension or physiological saline with the same volume. Close the abdominal cavity to establish the intestine IRI model.4.Form the rats in4 groups.:experimental group、BM MSCs control group、NaCl control group、dummy treatment group.5.Test the cytokine by ELISA. Detect the pathology morpha by HE and immunohistochemistry.Results:1. The third generaion of BM MSCs expressed CD29, CD90and RT1A more than90%. The transfection efficiency of the third generaion of BM MSCs exceeded80%.2. Establish the co-cultured model in vitro successfully:①The lymphocyte eactivity in the experiment groups were lower than the control groups. The differences were statistically significant (P＜0.05).②The TNF-α, TGF-β1and IL-10level in the control groups were similar to the experimental groups at0h (69.865±18.953,181.933±6.19and275.92±9.714, respectively). The control groups of TNF-α at24h、48h were95.737±5.981、96.471±7.863, the experimental groups were57.444±18.41、63±8.718. The experimental groups of IL-10at24h、48h were586.154±15.783、638.718±41.154, the control groups were404.949±10.181、366.016±5.18.The experimental groups of TGF-β1at24h、48h were522.285±25.686、501.123±11.609, the control groups were441.27±12.716433.231±29.224. The differences were statistically significant (P＜0.05).③Detect the marker of T regulatory cells by flow cytometry:The expression rate of experiment groups were higher than the control groups.3.The results of the model of intestine IRI:①The TNF-a level in control groups of BM MSCs and the experiment groups at24h were51.877±2.2and31.827±4.681, respectively. The TGF-β1level were748.79±87.943and1097.566±198.582, respectively. The IL-10level were995.956±57.057and1357.963±115.003, respectively. The differences were were statistically significant (P<0.05).②The intestinal villi in the experimental groups were more severe in edema, putrescence, hyperaemia and lammatory infiltration than the control groups by HE stain.Conclusion:1. The density gradient centrifugation in conjunction with adherent method could isolate BM MSCs purely, and the method was simple and inexpensive.2. In the vitro co-cultured environment, BM MSCs could inhibit the expression of TNF-α by promoting the generation of immunity regulatory factor including TGF-β1, IL-10and T regulatory cells. And the effects could be enhanced by HO-1.3. In the model of small intestine IRI, BM MSCs could obviously repair the structure of small intestine and reduce its permeability. And the protection could be enhanced by HO-1.