Therapeutic Effect Of Human Umbilical Cord Mesenchymal Stem Cells On Inlfammatory Bowel Disease Model In Mice And Exploration Of Cell Transplantation Related Issues

Objective:To establish a model of inflammatory bowel disease induced by TNBS, this study aims to explore the effects of human umbilical cord mesenchymal stem cells (hUC-MSCs) on TNBS-induced inflammatory bowel disease model and the possible mechanism of its effects, as well as transplantation-related factors on the efficacy, such as cell infusion route, cell number, this study will provide the pre-clinical research data of the treatment.Methods:Ⅰ. Establishment of inflammatory bowel disease model:fifty SPF male BALB/c mice were randomly divided into7groups, including control group, single enema groups with different doses of TNBS (100,150,175,200,225mg/kg) and the group of skin sensitization combined with enema. The mice were sacrificed on day5, and the colons were observed under a stereomicroscope to evaluate the common morphological damage, and then embedded in paraffin to perform histological analysis.Ⅱ. To investigate the effects of human umbilical cord mesenchymal stem cells on TNBS-induced inflammatory bowel disease model:using skin sensitization combined with enema to establish inflammatory bowel disease model was to investigate the treatment effect of hUC-MSCs on intestinal damage. According to survival rate of animal, weight change, gross score and pathological grading score, curative effect was evaluated; the levels of IL-10,1L-6, TNF-α, IFN-γ in serum was detected; the application of CFSE and antinuclear antibody was used to track the implantation and survival of hUC-MSCs by immunofluorescence. It aims to explore the possible mechanism of hUC-MSCs in inflammatory bowel disease model. III. The discussion of transplantation related issues:1. Different infusion routes:hUC-MSCs implanted in vivo via tail vein and intraperitoneal, the differences in curative effect were observed by survival rate, gross score, weight change and pathological grading.2. Cell number: Enema mice were randomly divided into five groups, including control group, different doses hUC-MSCs group (2x104,6x104,2x105,6x105cells/mice), survival rate and body weight changes were observed in each group.Results:I. In the groups with low doses of TNBS (100,150mg/kg), there was no obvious ulcer formation in the colons, but in the groups with higher doses (175-225mg/kg), ulcers with different severity could be observed. The rate of ulcer formation was in direct proportion to the dosage of TNBS. In the dose of225mg/kg, the ulcer information was100%. However, the lesions of the colon were severe and disparity on great extent. Occasionally some animals had a side effect of mouse blind. By contrast, in the skin sensitization combine enema group, the ulcer information was also100%, but the lesions were moderate. There was no side effect was observed.II.hUC-MSCs could home to the gut via the tail vein, reduce the gross score and pathological score, also improved the survival rate. hUC-MSCs reduced inflammatory cytokines including IL-6, IFN-γ,TNF-α and up-regulate of anti-inflammation factors IL-10in serum. III. The curative efficacy was different after hUC-MSCs transplantation through different routes and cell numbers. Both of the routes could reduce gross score and pathological score, the intraperitoneal way was superior to vain tail way on improving the survival rate; the tail-vein graft was better than the intraperitoneal transplantation on repairing ulcer. The dosage of hUC-MSCs only within appropriate range could play therapeutic effect by improving the survival rate changes and changes in body weight effectively. Conclusion:The mouse IBD models could be established by using a single enema of TNBS in a dosage ranged from175mg/kg to225mg/kg, the group of skin sensitization combined with TNBS (150mg/kg) enema occours moderate lesions with100%ulcer formation rate and side effects. hUC-MSCs have therapeutic effects on TNBS-induced inflammatory bowel disease model,hUC-MSCs home to the gut after transplantation;the intraperitoneal way is superior to tail-vein way on improving the survival rate; the tail vein graft is better than the intraperitoneal transplantation on repairing ulcer. Cell number of hUC-MSCs plays a critical role on therapeutic effect.Thus the optimal dosage of hUC-MSCs needs to be confirmed through expansion of experimental objects.