Research On BMMSCs Regulate The Imbalance Of Immune Function Of Experimental IBD Mouse Through The TGF-?/Smad Signaling Pathway

Background:Inflammatory Bowel Disease(IBD),above Ulcerative Colitis(UC)and Crohn's Disease(CD),is a series of immune associated,intestine inflammatory injured disease.Even the several of causes of IBD have been found and been classified,the pathogenesis of this disease still not been completely understood.Bone marrow mesenchymal stem cells(BMMSCs)are a kind of stem cells origin from the bone marrow.BMMSCs have many powerful biological functions,such as proliferation and differentiation and special character and ability,such as low immunogenicity and immune regulation.Therefore,these cells have been tried to treat several diseases,like myocardial infarction,diabetes and IBD.Some research have found the BMMSCs could promote the intestinal mucosa heal.However,for the immune regulation of BMMSCs on IBD,there is not valuable research so far.The previous studies have revealed the relationship between the TGF-?/Smad signaling pathway and the immune regulation,which is associated with the immune cells(Thl7/Treg cells)cand the cytokines(IL-17a/IL-10)change.Therefore,it is important to do a research on BMMSCs regulate the imbalance of immune function of experimental IBD mouse through the TGF-?/Smad signaling pathwayObjective:By using the BMMSCs to treat the IBD,evaluating the curative effect,then do some researches on the mechanisms of this therapy.Based on these mechanisms,exploring the key gene that both associated the progress of BMMSCs treat IBD and the TGF-?/Smad signaling pathway,so as to further promote the clinical application of stem cells and expand the new idea of IBD therapy.Contents:1.Cultivating and identifying BMMSCs,using these cells to treat IBD mice model,and evaluating the curative effect.2.Studying on the mechanism of immune regulation on BMMSCs treat IBD,exploring the key gene on the TGF-?/Smad signaling pathway.3.Testing the function of key gene on the progress of BMMSCs treat IBD.Methods:1.BALB/c male mice(3 days)were selected to be the origin of BMMSCs,using the improved method to cultivate the BMMSCs,these cells be identified by several methods.2.BALB/c female mice(8-10 weeks old)were randomly divided into 3 groups:the control group(Group C),the model group(Group T)and the BMMSC transplantation group(Group M).Group T and Group M get the TNBS-ethanol enema(0.1ml a mice)once a week.After four weeks,Group M inject the BMMSCs(1*106 a mice)3.Evaluating the curative effect according to the mortality,general status,clinical manifestation and pathological score.Observe the location of BMMSCs on intestine.4.The biological specimens(including blood,spleen and intestines)of mice with different intervention groups were collected,and preparing the test samples according to the experimental and store requirements.5.Performing several associated experiments to study the specific mechanism of the BMMSCs treatment of IBD(1)Using immunohistochemistry to detect the expression of some functional proteins on intestine tissue;(2)Using cell flow cytometry to detect the percentage of Th17 cells and Treg cells;(3)Using QPCR and CBA to detect the expression of cytokines(IL-17a,and IL-10).6.Using Roche NimbleGen gene expression Chip(12 x 135K)microarray and global signal transduction network to explore the key gene.7.Using the lentiviral vector(LV)and si-RNA to build the carriers,and transfecting these carriers to BMSCs.8.Transplanting the different BMMSCs to IBD mice model and repeat the Method five.Results:1.BMMSCs cells showed long fusiform shape,like most fish like ordered,few in a spiral volute arrangement.The expression rates of cell surface markers CD90.2 and CD 105 were high,and CD 11b,CD45 were at a lower level.BMMSCs could differentiate to osteogenic cells adipogenic cells.2.The mice in the enema module were in accordance with the model standard of IBD in mortality,general status,body weight change,clinical manifestation and pathological score.After the BMMSCs transplantation,the general situation of the mice in the model group improved significantly,and all indicators could be mathematically quantified improved significantly.3.The mechanism of BMMSCs treatment on IBD mice model(1)BMMSCs transplantation can promote the repair of intestinal mucosa in IBD mice model.First,BMMSCs promote angiogenesis lesion.Second,the BMMSCs helps to restore the close connection between cells.(2)BMMSCs has a regulatory effect on immune cells.For Thl7 cells,the proportion of T mice is 3.20%,while the proportion of Th17 cells in M group is reduced to 1.87%,indicating that BMMSCs transplantation has an inhibitory effect on Th17 cells.For Treg cells,the percentage of that in T group was 3.13%,and the proportion of M group was 3.41%,which increased slightly.This indicates that BMMSCs transplantation can promote the proliferation of Treg cells in IBD mice.(3)BMMSCs plays a regulatory role in immune cell related cytokines.It is as follows:IL-17a:After BMMSCs transplantation,the expression level of the factor decreased significantly(**p<0.01)mindicating that BMMSCs has a inhibitory effect on the release of the factor.IL-10:the expression level of the factor was higher in the M group after BMMSCs transplantation(**p<0.01).These results suggest that IL-10 may have a negative regulation of inflammation,and BMMSCs has the effect of enhancing the release of IL-10.4.By transfection of lentivirus and si-RNA into target cells,we obtained TGF-BMMSCs 3 overexpression mouse BMMSCs(enhanced expression group)and TGF-beta 3 gene silencing mice BMMSCs(silent expression group).5.The immunoregulation effect of TGF-beta 3 gene in the treatment of IBD by BMMSCs(1)The TGF-beta 3 gene has a down-regulation effect on Th17 cells and has an up-regulation effect on Treg cells.For Th17 cells,in the normal cell group,the ratio of the cells was 4.53%:in the enhanced expression group,the proportion of Th17 cells was 2.75%,while the proportion of silent expression group was 7.12%.For Treg cells,the percentage of that in the three groups of normal cells,enhanced expression group and silent expression group was 0.79%,1.74%and 0.53%,respectively.(2)TGF-beta 3 gene has a negative correlation with IL-17a,but this gene could promote the release of IL-10.For IL-17a:the expression of IL-17a on mice intestinal tissues combined with TGF-beta3 silencing expression was higher than the ordinary cell group(*p<0.05),and the difference with the change of time increase.For IL-10:the expression level of IL-10 in the intestinal tissue of mice increased with the TGF-beta 3 gene over-expression(*p<0.05).Correspondingly,the expression level of IL-10 in the intestinal tissue was decreased due to the inhibition of TGF-beta 3 gene expression(**p<0.01).Conclusion:1.BMMSCs transplantation can treat IBD effectively by repairing the mucosal damage and relieving the clinical manifestation.2.The mechanism of BMMSCs treatment on IBD mainly involves promoting mucosa repair and the regulation of systemic and local immunity imbalance,as follows:BMMSCs promote angiogenesis and cell tight junction on local lesion;change the number of immune cells(Thl7/Treg cells)and regulate the cytokines(IL-17a,IL-10)release level.3.TGF-beta/Smad signaling pathway is an important pathway related to immune regulation in the process of BMMSCs treatment on IBD.The TGF-beta 3 gene in this pathway may play a key role in regulating a series of complex immune functions(regulating the immune cells and cytokines).