| Background and IntroductionInflammatory bowel disease(IBD)is featured by chronic idiopathic inflammation of the gastrointestinal(GI)tract.The pathogensis of IBD remains unclear and is closely related with genetic predisposition,environmental factors,intestinal microbiota and immune system.Host-microbiome interactions and abnormal immune response to commensal microbiota are considered as the central part in the occurrence and progression of IBD.Impairment and dysfunction of epithelial barrier is also an essential part in the development of intestinal inflammation.Mesenchymal stem cells(MSCs)refer to a heterogeneous population of spindle cells with the capacity to differentiate into osteoblasts,chondrocytes and adipocytes in vitro.MSC’s self-regeneration and differentiation ability,immunomodulatory property and trophic functions make this kind of cells a promising candidate in the treatment of a variety of diseases.Human embryonic stem cell derived mesenchymal stem cells are used in the thesis.Previous research work has confirmed its therapeutic effect in the treatment of chemically induced colitis in mice.However,the underlying mechanism is still unclear.The thesis aims to explore the molecular basis and the key protein that can explain the therapeutic function of embryonic stem cell derived MSCs.Methods1.MSC treatment in DSS induced mice colitis and its in vivo tracking(1)Both acute and chronic DSS induced colitis models were established and mice were given intravenous MSC administration.The therapeutic effect was evaluated by body weight,colon length,colon pathological sections and serum inflammatory cytokines.(2)Mice were given Dil labeled MSCs intravenously and frozen sections of the colons,livers and lungs were observed to figure out the distribution of MSCs in different tissues.Immunofluorescence stains of CD31 and F4/80 were performed to explore the co-localization with other types of cells.2.MSC promote colon epithelium repair and regeneration by increasing circulating IGF-1 level(1)Micro-array test of mice serum samples was performed to screen potential therapeutic proteins.(2)Serum IGF-1 was detected in different stages of MSC treatment model to confirm the elevated level in treatment group.The origin of elevated IGF-1 was detected by qPCR and immunohistochemistry stain of liver tissues.(3)Inhibitors of IGF-1 receptor were used to block its secretion and the therapeutic effect was re-evaluated.IGF-1 levels of different tissues were detected by ELISA.Typical colon proliferation markers BrdU and Ki-67 were examined by immunohistochemistry stain.Downstream signal pathway of IGF-1 was observed by immunoblotting of colon total protein and colon epithelium protein.(4)RNA-sequencing of colon tissues was performed to figure out the differentially expressed genes.3.In vitro study of IGF-1 stimulation on the proliferation of colon cells and organoids(1)CCK-8 assay was performed in a normal colon epithelium cell line NCM460 to figure out the effect of in vitro IGF-1 stimulation on cell proliferation.BrdU incorporation assay was used to check IGF-1 stimulation on cell cycle.Annexin V stain and flow cytometry analysis was adopted to explore IGF-1 stimulation on TNF-α induced cell apoptosis.(2)Mice colon organoids were cultured and IGF-1 stimulation on the proliferation of organoid was evaluated.Results1.MSC alleviate mice colitis by increasing circulating IGF-1(1)Intravenous treatment of human embryonic stem cell derived MSCs showed significant therapeutic effect in both acute and chronic DSS colitis models by improving body weight,alleviating colon inflammation and decreasing serum inflammatory cytokine levels.Dil labeled MSC mostly distributed in the lungs and liver.MSCs trapped in the lungs were mostly within the vascular and MSCs in liver showed co-location with macrophages.(2)Intravenous treatment of MSC increase serum IGF-1 level.Colon protein and colon epithelium protein showed elevated expressions of downstream IGF1 R-AKT-PI3K pathway.MSC treatment increase the expression of BrdU and Ki-67 in colons.RNA-sequencing showed a significant difference in gene expression.Inhibitors of IGF-1 receptor abolished the therapeutic effect of MSC treatment.2.In vitro study showed MSC stimulation promote the proliferation of colon cells(1)IGF-1 stimulation promoted NCM460 proliferation,decreased TNF-α induced cell apoptosis and increased cell percentages in S stage of the cell cycle,(2)IGF-1 stimulation promoted the growth and expansion of mouse colon organoid.Conclusions1.Intravenous MSC treatment is effective in mouse DSS colitis.2.MSC promote colon epithelial regeneration by elevating circulating IGF-1... |
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