The Research Of Transplantation Of Bone Marrow-Derived Mesenchymal Stem Cells For Treatment Of Experimental Colitis And Synergistic Effect Of Compound Sophorae Decoction

Part Ⅰ:Isolation, culture and identification of the Bone marrow mesenchymal stem cells in vitroObjective:To isolate and culture of rat bone marrow mesenchymal stem cells in vitro, to identify them, to lay the foundation for subsequent experiments.Methods:4-week-old SD rats were used to isolate and culture bone marrow mesenchymal stem cells in vitro; The cells were serially splitted in order to be purified; The phenotype wereidentified by flow cytometry.Results:Bone marrow mesenchymal stem cells were fibroblast-like morphology, spindle or spindle-shaped, spiral or radial arrangement. Up to3passages of BMSCs, no obvious signs of aging was found. Flow cytometric analysis showed that the BMSCs were high expression of CD29and CD90, without expression on hematopoietic stem cell surface markers such as CD45and CD11b.Conclusion:BMSCs can be isolated and culture by the means of whole bone marrow adherent in vitro successfully. The cells form a uniform and state stability, with a high purity and morphology. Flow cytometry identified in line with the characteristics of mesenchymal stem cells. Part II:Transplantation of BMSCs to treat of experimental colitis and synergistic effect of Compound Sophorae decoctionObjective:To observe the changes of disease activity index (DAI) and pathology and histology of the colon after the BMSCs transplantation and the treatment of Compound Sophorae decoction.Methods:8-week-old SD rats were used to be constructed for the model of ulcerative colitis by using TNBS/ethanol complex method; lentivirus-mediated GFP were used to transfect BMSCs; the labeled BMSCs were transplanted after modeling and Compound Sophorae decoction intervened at the same time; the expression of GFP were detected by immunofluorescence staining and weston in colon tissue of rats in each group;the disease activity index (DAI) and the pathology and histology of the colon were assessed and scored.Results:Two week after intravenous administration of BMSCs and drug therapy, immunofluorescence staining and weston blot showed that the expression of GFP could be observed in both BMSCs group and the bivalent group in damaged area of the colon;Compared with the control group, the DAI scores and the histopathological scores increased significantly in PBS group;Compared with the PBS group, the scores in BMSCs group and the bivalent group declined.Conclusion:BMSCs transplantation and the treatment of Compound Sophorae decoctioncan effectively alleviate the symptoms of colitis in rats, while the damage of the colon tissue repaired. Part Ⅲ:Immune mechanisms for BMSCs treatment in experimental colitisObjective:To study the related immune mechanisms for BMSCs treatment of experimental colitis.Methods:Isolate and culture of BMSCs in vitro, then the cells were identified by flow cytometry; BMSCs were labeled by beening transfected with LV-GFP;The animal models of ulcerative colitis were induced by TNBS, and divided into four groups:including control group, PBS group, BMSCs group, BMSCs and Compound Sophorae decoction group;BMSCs were transplanted via the tail vein, two joint group were treated by Compound Sophorae decoction intervention;The expression of Treg in peripheral blood were detected by flow cytometry; The expression of TNF-α in serum were detected by ELISA;The cytoplasm and nucleus of NF-κ Bp65expression were detected by Western blot;The expression of Foxp3mRNA and TNF-α mRNA were detected by RT-PCR.Results:1, Compared to the control group, the level of Treg in PBS group decreased significantly; after the intervention, compared with the PBS group, the level of Treg in BMSCs and bivalent group increased.2, Compared to the control group, the expression of Foxp3mRNA in PBS group increased significantly;after the intervention, compared with the PBS group,the expression of Foxp3mRNA in BMSCs and bivalent group decreased.3, Compared to the control group, the expression of NF-κ Bp65in cytoplasm and nucleus in PBS group increased significantly; after the intervention, compared with the PBS group, the expression of NF-κ Bp65in cytoplasm and nucleus in BMSCs and bivalent group declined significantly.4, Compared to the control group, the expression of TNF-α in PBS group increased significantly; after the intervention, compared with the PBS group, the expression of TNF-α in BMSCs and bivalent group decreased significantly. Conclusion:The symptoms of experimental colitis can effectively be alleviated by the transplantation of BMSCs;The mechanisms may be related to the redeployment of the proportion of Treg between peripheral blood and intestinal mucosa, as well as the regulation of NF-κ B-mediated proinflammatory, and Compound Sophorae decoction take a synergistic effect.