Effects of Tumor Necrosis Factor-α on Mouse Small Intestinal Organoid Growth,Barrier Function and Intestinal Functional Cells
[Objective]The effects of tumor necrosis factor-α(TNF-α)on small intestinal organoid growth,tight junction proteins,and various functional cell marker genes were studied to establish disease damage models for small intestinal organoids.[Method]The crypt cells were isolated from mouse small intestine with gentle cell dissociation reagent(GCDR)digestion solution and cultured in intestinal organoid medium.Small intestinal organoids were stimulated with 0(control group),50,250 and 500 ng/mL TNF-α for 48 h.The growth of organoids was observed under an optical microscope,and cell proliferation was tracked by Edu staining.The expression levels of cell proliferation,barrier function and intestinal functional cell marker genes mRNA were detected by Real-time fluorescent quantitative PCR.[Result]① Compared with control group,50 and 250 ng/mL TNF-α significantly reduced the germination rate of small intestinal organoids(P＜0.05),and TNF-α stimulation had no effect on organoid formation rate(P＞0.05).250 and 500 ng/mL TNF-α resulted in a significantly higher rate of small intestianl organoid necrosis(P＜0.05).②Compared with control group,250 ng/mL TNF-α significantly reduced the mRNA expression of the small intestinal organoid tight junction protein Occludin(P＜0.05).500 ng/mL TNF-α increased mRNA expression of Claudin-1(P＜0.05),a tight junction protein in small intestines.③Compared with control group,different concentrations of TNF-α led to a significant increase in the expression of mRNA of TNF-α(P＜0.05),but had no significant effect on the expression of IL-6(P＞0.05).250 and 500 ng/mL TNF-α resulted in a significant increase in mRNA expression of IL-1β(P＜0.05).④Compared with control group,250 ng/mL TNF-α led to a significant decrease in mRNA expression of the marker genes Ki67 and Pcna of proliferating cells(P＜0.05).⑤Compared with control group,50 ng/mL TNF-α stimulation significantly reduced the mRNA expression of Lgr5 gene(P＜0.05),250 and 500 ng/mL TNF-α stimulation significantly reduced mRNA expression in Muc2,Chga and Lyz genes.250 ng/mL TNF-α stimulation significantly reduced the mRNA expression of Alpi gene(P＜0.05).[Conclusion]250 ng/mL TNF-α stimulation could inhibit the growth of small intestinal organoids,inhibit the proliferation of intestinal stem cells and the differentiation of various functional cells,which could provide a reference for future clinical applications.
万方数据
维普
