| Background:Inflammatory Bowel Disease (IBD) is an unclear, intestine injured, degestive disease, above Ulcerative Colitis (UC) and Crohn’s Disease (CD) and Indeterminate Colitis. Bone marrow mesenchymal stem cells (BMMSCs) are a kind of non-hematopoietic stem cells in bone marrow. They have many biological advantages, such as low immunogenicity, multipotential differentiation, immune regulating function, and accessible. They can promote the progress of healing of injured IBD intestinal mucosa and rectify the effect of mucosal immune dysfunction. So, they are applied for the treatment of IBD.In previous studies, we have found that the number of intravenous transplanted BMMSCs migrated and planted on diseased colon was low, limiting its therapeutic effect. Stem cell homing behavior involves a variety of chemokines and their receptors. The Chemokine Receptor-4/Stromal cell-derived Factor-1 (CXCR-4/SDF-1) are confirmed on promoting stem cell homing. According to this theory, the CXCR-4 gene transfected BMMSCs could be able to improve the targeting property and high efficiency of the treatment.Objective:By comparing the plantation and therapeutic effect between the normal BMMSCs and CXCR4-BMSCs on the experimental IBD model to explore its mechanism, so as to provide the theory basis for confirming a proper way on targeting BMSCs to treat IBD.Contents:1. Incubating and identifying BMMSCs.2. Constructing and identifying CXCR4-BMSCs and assessing their migration rate in vitro.3. Observing the therapeutic effect and evaluating the migration rate on damaged intestinal mucosa in experiment IBD model.4. Exploring the mechanisams of targeted BMSCs homing and impairment on intestinal lesions.Methods:1. Whole bone marrow adherent culture method was used on incubating BMMSCs; The tracer (CM-Dil) was labeled on BMMSCs.2. Morphological characters of BMMSCs were observed; Flow cytometry was applied to identify BMMSCs; The ability of BMMSCs on osteogenic and adipogenic lineages was examined.3. CXCR4-Lentivirus were transfected to BMSCs to construct CXCR.4-BMMSCs.Real time PCR were applied to detect and compare the expression of CXCR-4 in two groups.4. Morphological characters of CXCR-BMMSCs were observed; The ability of CXCR-BMMSCs on osteogenic and adipogenic lineages was examined; Flow cytometry was used to determine the surface markers; the cell cycle experiment and Trypanblau dying were applied to determine cell viability of two groups.5. Transwell migration system was used to compare the ability of migration to SDF-lin two groups.6. Female BALB/C mice of 6-8weeks were randomly divided into 6 groups. 〤ontrol group:Normal saline (00μl/per mouse) was used to enema.0.1ml PBS was injected through tail vein on the next day after enema. ②TNBS group (labeled T group):TNBS 2.0mg/50% alcohol(100μl/per mouse) was used to enema.0. lml PBS was injected through tail vein on the next day after enema. ③ BMMSC transplant group (labeled MT group):TNBS 2.0mg/50% alcohol (100μl/per mouse) was used to enema.0.1ml PBS containing 1×106 BMMSCs was injected through tail vein on the next day after enema.④CXCR-BMMSC transplant group (labeled CMT group):TNBS 2.0mg/50% alcohol (100μl/per mouse) was used to enema.0.1ml PBS containing 1×106 CXCR-BMMSCs was injected through tail vein on the next day after enema.The day of transplantation was labeled D0,1-13 days after transplantation, respectively for D1-13.7. The degree of diseased IBD models and therapeutic effect of different treatments were evaluated by survival rate, general condition, weight changes, clinical symptom scores, gross colons and their changes of length, and histological scores.8. Three mice in each group were killed, and serum, intestinal tissue, mesenteric lymph nodes and spleen samples were collected on D0、D1、D3、D5、 D7、D9、D11、D13.9. Red fluorescence locations in colon were observed by fluorescence microscopy. The relative expressions of Sex-determining gene (SRY) were detected by Real time PCR.10. Immunohistochemistry was applied to detect the expression of Lgr-5, Occludin and VEGF.11.Western Blot was applied to detect the expression of PI3K.Results:1. BMMSCs incubated by whole bone marrow adherent culture method were growing as a long spindle and beam pattern. The surface markers of BMMSCs like as CD90.2+; CD105+; CD45-; CD11b-. There was obvious results on osteogenic and adipogenic differentiation. CM-Dil tracer has no influence on the biological character of BMMSCs.2. Compared with BMMSCs, high expression of CXCR-4 was determined after CXCR-4 gene transferred BMMSCs..3. CXCR-BMMSCs were growing as a long spindle and beam pattern. The results of surface markers didn’t change (CD90.2+; CD105+; CD45-; CD11b-). There was obvious results on osteogenic and adipogenic differentiation. Trypanblau dying showed about 90% cell viability. The above outcomes of BMSCs in two groups showed no difference (p>0.05). In the CXCR-BMMSC group, the copy period in cell cycle increased (p<0.05).4. The migration experiments in vitro showed that the number of different BMSCs had moved to SDF-1 were CXCR-BMMSC group> BMMSC group (p<0.05).5. TNBS induced experimental model, the survival rate, mental state, body weight and decreased significantly, clinical symptom and histological score obviously increased; the gross colons were significantly shortened accompanied hyperemia, hemorrhage and narrow, and histopathological found that ulceration, a large number of neutrophil infiltrated in the mucosa and submucosa (p<0.05). On the second day after transplantation, compared with T group, the MT and CMT groups conditions were inmroved (p<0.05). The therapeutic effect of CMT group was better (p<0.05).6. Red fluorescence were observed in colon of MT and CMT group; SRY gene were detected in the male, MT and CMT group; the engraftment rate was CMT group> MT group.7. The expressions of Lgr-5, Occludin and VEGFsignifantly increased in MT and CMT group.8. Western Blot results showed:compared with T group, The relative expression of PI3K in the MT and CMT group was increased, especially in the CMT groups (p<0.01).Conclusion:1. The transfection of CXCR-4 gene couldn’t change the biological characters of BMMSCs2. Migration experiments in vivo and in vitro showed that the over-expression of CXCR-4 could increase the mobility of BMSCs, and they could be coordinated to strengthen the migration characteristics of BMSCs.3. The rate of migration was positively related with curative effect in the treatment of experimental IBD by different BMSCs transplantation. BMSCs could accelerate the differentiation of intestinal stem cells, rebuild the tight junction, predominately inhibit excessive activation of PI3K signal pathway, as to promote regeneration and repairmen of intestinal mucosa, reconstruct the epithelial barrier and angiogenesis... |
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