| Intestinal barrier and intestinal homeostasis play an important role in intestinal health and development.The proliferation and differentiation of intestinal stem cells can regenerate damaged tissues and replenish dying cells,it also is the source of epithelial cells' rapid self-renewal.Copper can regulate intestinal structure integrity and barrier function,which has great significance on intestinal growth and development.However,the reports about the effects of copper on intestinal stem cells(ISCs)are very limited.The model of intestinal organoids have many advantages,such as self-proliferation and differentiation,three-dimensional structure and contained various intestinal epithelial cell types,which can more truly simulate the intestinal environment.Therefore,intestinal organoids was used in this study to explored the influence of copper ions on ISCs and get the following results.The crypt tissues were isolated from the middle jejunum of C57 mice,wrapped in Matrigel and added the ENR(contained EGF,Noggin and R-spondin)medium.After 5 days,the organoids developed a "petal-like" three-dimensional structure.The morphology of intestinal organoids were stable and consistent in different passages.Ki67 immunofluorescence results showed that most of the cells were in the proliferative stage.Therefore,it proved that the model of intestinal organoids have been established successfully and can be treated experimentally.Mouse intestinal organoids were treated with 0,0.01,0.1,1 and 10 mg/L copper sulfate in ENR and EN(without R-Spondin1)medium for 72 h.The results showed that 0.1 and 1 MG/L of copper sulfate significantly increased the area and budding numbers of intestinal organoids in both ENR and EN medium compared with the control group.In addition,0.1 and 1 mg/L of copper sulfate enhanced Ed U fluorescence intensity and promoted intestinal organoids proliferation.Further studies showed that 0.1 and 1 mg/L of copper sulfate significantly increased the immunofluorescence intensity and protein expression level of the stem cell markers Lgr5 and Eph B3,as well as the immunofluorescence intensity and protein expression level of ?-catenin in the nucleus.In summary,0.1?1 mg/L copper sulfate promoted the growth of intestinal organoids and increased the number of intestinal stem cells.Which possibly through the Wnt/ ?-catenin pathway. |
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