The Culture Of Organoids From Mouse Stomach And Small Intestine And Its Application In The Study Of Gastrointestinal Diseases

Part 1.Establishment of gastroids culture system in C57BL/6 miceObjective: To culture three-dimensional gastroids from mouse in vitro as a reliable model,which simulates gastric epithelial structure and function,for subsequent experimental research.Methods: The gastric antrum and body mucosa glands were rapidly isolated from the mouse,planted in Matrigel,and cultured in a medium containing growth factors inclduing Wnt3 a etc.The morphology of gastroids was dynamicly observed.The gastroids were passaged,frozen,and resuscitated,and the cell markers contained in the gastroids were detected by immunofluorescence and q PCR.Results: The morphology of the gastroids cultured in this experiment is consistent with the literature.It could be effectively passaged,frozen,resuscitated,and it has multiple cell markers of gastric antrum and body mucosa,such as MUC5 ac,MUC6,Pepsingogen,H+-K+ATPase,Lgr5,etc.,while without intestinal epithelial marker MUC2.The gastroids can be used for further studies.Conclusion: Gastroids from mouse stomach were successfully established in our experiment.The gastroids cultured are consistent with the literature reports on morphological characteristics and cell markers,providing a reliable model for subsequent studies in vitro.Part 2.Effects of chenodeoxycholic acid on growth,proliferation,apoptosis and differentiation and expression of ghrelin in gastroidsObjective: To investigate the effects of Chenodesoxycholic Acid(CDCA),one of the bile components,on the growth,proliferation,apoptosis and the expression of ghrelin m RNA in mouse gastroids.Methods: Different concentrations of CDCA were used to stimulate the gastroids with good growth state.The diameters and morphological changes of gastroids were observed by inverted microscope.Flow cytometry with PI staining was used to detect cell proliferation in gastroids.Flow cytometry with Annexin V/PI staining was used to detect apoptosis in gastroids.q PCR was used to detect ghrelin m RNA expression.Single cell RNA-sequencing was used to analyze the effect of CDCA on cell differentiation in gastroids.Results: When the CDCA concentration reached 200 ?mol/L,the gastroids became smaller and died.When the concentration of CDCA was between 10 ~100 ?mol/L,the proliferation activity of gastroids did not change significantly with the increase of concentration.The proportion of cells in early apoptosis and late apoptotic state were increased when CDCA concentration was 30 or 100 ?mol/L.Stimulation of CDCA at 10 ?mol/L for 24 h resulted in a change in the proportion of pit cells in gastroids,promoting apoptosis of subpopulation in cell division cycle.As the concentration of CDCA increased,the expression level of ghrelin m RNA in gastroids gradually decreased.Conclusion: Primary bile acid CDCA could promote the apoptosis of pit cells subgroup in the cell division cycle and inhibit the renewal of pit cells.Meanwhile,CDCA could down-regulate the expression of ghrelin m RNA in endocrine cells in gastroids.This may partly explain the proliferation of epithelial cells in gastric mucosa caused by bile acid stimulation and the disorder of gastrointestinal hormone levels in patients with bile reflux gastritis.Part 3.Establishment of enteroids culture system in C57BL/6 miceObjective: To culture three-dimensional enteroids from mouse in vitro as a reliable model,which simulates intestinal epithelial structure and function,and for subsequent experimental studies.Methods: The small intestinal mucosa of male C57BL/6 mice was rapidly isolated,planted in Matrigel,and cultured in a medium containing growth factors Supplement 1 and 2.The morphology of enteroids were dynamicly observed.The enteroids were passaged,frozen,and resuscitated.The cell types contained in the enteroids were verified by immunofluorescence.Results: The morphology of the enteroids cultured in this experiment is consistent with the literature.It can be effectively passaged,frozen,resuscitated.Epithelial cells,paneth cells,proliferating cells,tuft cells could be observed by immunofluorescence staining.The enteroids can be used for further studies.Conclusion: The enteroids from the small intestine crypt of mice were successfully established in our experiment.The morphological characteristics and the cells contained in the enteroids were consistent with the literature,providing a reliable model for subsequent study in vitro.Part 4.Bifidobacterium longum regulates intestinal epithelial proliferation activity and improves visceral hypersensitivity in rats of IBS model induced by WASObjective: To observe the intestinal epithelial proliferation activity,the secretion function of Paneth cells and the intestinal microbiome in rats with irritable bowel syndrome(IBS)induced by water-avoidance stress(WAS).The effects of Bifidobacterium longum(B.longum)on the intestinal epithelial proliferation activity,Paneth cell function and intestinal microbiome of IBS rats induced by WAS were investigated.Methods: The IBS model of rats was established by WAS for 10 days.HE staining was used to observe the crypt-villi structure of the terminal ileum.Scanning electron microscopy was used to observe the colonization of B.longum.FITC-D 4000 was used to observe intestinal permeability.Western-blot was used to detect the expression of tight junction protein Occludin.Immunofluorescence and q PCR were used to observe the activity of intestinal epithelial proliferation activity.The number of paneth cells and the expression of lysozyme were observed by immunofluorescence,q PCR and western blot.The changes of intestinal microbiome were analyzed by 16 S r RNA sequencing.The effect of B.longum on the intestinal epithelial proliferation activity,lysozyme expression by Paneth cell and intestinal microbiome in rats induced by WAS was observed.B.longum powder was inoculated,cultured,passaged and identified.Enteroids with good growth status were co-cultured with 1.0 ~1.5×108 CFU of B.longum.After co-culture for 4 h,the m RNA of the Control group and Bifido.group was extracted for high-throughput m RNA sequencing.The differential genes were screened and further verified by q PCRResults: WAS induced visceral hypersensitivity in rats mimicing the pathogenesis of IBS-in human.It caused decreased intestinal epithelial proliferation activity with shortened villus/crypt length of terminal ileum and increased intestinal permeability.Decreased expression of lysozyme content were also observed.WAS caused intestinal dysbiosis with decreased ?-diversity and ?-diversity.B.longum could be effectively colonized on the epithelium of the terminal ileum,increasing the proliferative activity of intestinal epithelial cells and the expression of lysozyme.B.longum could improve the intestinal microbiome dysbiosis caused by WAS at the level of phylum and genus.According to the results of differential gene expression in Control group and Bifido.group,the pathway annotation was obtained.Wnt3 A signaling pathway,EGF signaling pathway and TGF-? signaling pathway were found to be up-regulated in Bifido.Group.The m RNA expression of Wnt3 A and TGF-? in Bifido.group was significantly increased by q PCR.Conclusion: WAS caused visceral hypersensitivity in rats.Intervention of B.longum could effectively enhance proliferative activity of intestinal epithelial cells and improve intestinal barrier function.Also,B.longum could regulate intestinal microbiome dysbiosis,and alleviate visceral hypersensitivity in rats.