| Intestinal epithelial cells have powerful self-organizing and self-renewal capacity.Intestinal stem cells are the source of various types of intestinal cells and drive their proliferation,regeneration,and differentiation.The new cells produced by intestinal stem cells maintain the fresh vitality of the intestine and maintain intestinal homeostasis.Nowadays,the incidence rate of inflammatory bowel disease(IBD)is on the rise year by year in the world,and the regeneration and repair of intestinal mucosa epithelium is been regarded as a key treatment goal to IBD.In recent years,the emergence of intestinal organoid technology provided an ideal in vitro research model for exploring biomedical applications such as intestinal proliferation and development,disease treatment,regeneration and repair.Latexin(LXN),a 222 amino acid protein,was discovered in the lateral neocortex of the developing rat brain.As the only known caroboxypeptidase inhibitor in mammals,LXN has multiple biological functions.It is closely related to inflammation response,tumor inhibition,cell differentiation,and regulation of stem cells with a negative correlation.Research has found that LXN is involved in the occurrence of intestinal inflammation,and LXN deficiency accelerates the process of dextran sulfate sodium(DSS)-induced colitis in mice.However,the role of LXN in intestinal regeneration and repair is still unclear.Therefore,by constructing a colitis and repair model and a 3D intestinal organoid model,we explore the role of LXN in intestinal proliferation and development,as well as intestinal epithelial cell regeneration and repair.The main content of this article are as follows:In the first chapter,we briefly introduce inflammatory bowel disease,intestinal regeneration and repair and related signaling pathways,intestinal organoids and their research progress,Latexin and related fields,and clarify the research significance and ideas of this paper.In the second chapter,we first established DSS-induced colitis and repair model in WT and LXN-/-mice,and analyzed the clinical symptoms of IBD in mice by observing and recording indicators such as body weight changes,stools situation,and DAI index.The results showed that during the recovery period of colitis,the clinical symptoms of IBD in LXN-/-mice were significantly reduced.Then,by measuring the length of mouse colon and H&E staining and Alcian blue staining of tissue sections,it was found that LXN-/-mice had higher recovery rate of intestinal length,more complete crypts of intestinal epithelium,higher growth rate of goblet cell,and faster recovery of intestinal epithelium during the recovery of colitis.Finally,in vivo Ed U-labeled technology was used to trace proliferative cells in the intestine.The results showed that under steady-state conditions,there were more proliferating cells in the intestine after LXN deficiency,and during the recovery period of colitis,the proliferation of cells in the colon of LXN-/-mice was faster.The above results indicate that LXN is involved in the process of intestinal regeneration and repair,and when LXN is absent,it can promote the regeneration and repair of intestinal epithelium.In the third chapter,we have preliminarily explored the mechanism by which LXN affects intestinal regeneration and repair.Firstly,Western Blot and RT-PCR experiments were used to detect the expression of Lgr5 in intestinal stem cells of WT and LXN-/-,and immunofluorescence and immunohistochemistry experiments were used to verify the upregulation of Lgr5 expression in the intestine after LXN deficiency.And through double immunofluorescence labeling experiments on LXN and Lgr5,it was found that LXN and Lgr5 have co localization,indicating that LXN and Lgr5 have a close relationship.In order to further explore the impact of LXN on the intestine,we established in vitro 3D intestinal organoids model of WT and LXN-/-type intestines,and observed and analyzed the growth of the two types of intestinal organoids.Ed U-labeled experiments were conducted on intestinal organoids.The results indicate that intestinal organoids with LXN deficiency grow faster and have more proliferative cells.RNA-Seq analysis of WT and LXN-/-intestinal organoids during growth showed that cell cycle and DNA replication signaling pathways in LXN-/-intestinal organoids were significantly up-regulated compared with WT group.These results indicate that LXN deficiency can promote the growth of intestinal organoids.Next,we used Western Blot and RT-PCR experimental methods to detect the expression of Lgr5 in intestinal organoids,and validated it with immunofluorescence staining.The results showed that LXN deficiency increased the number of Lgr5 positive cells in intestinal organoids.And immunohistochemical staining was performed on the colon during regeneration and repair,and it was also found that the LXN-/-group had a higher number of Lgr5 positive cell growth in the colon.Further analysis of intestinal organoids RNA-Seq revealed that YAP signaling closely related to tissue growth,development,and organ size were activated and upregulated.The expression level of YAP1 was verified through Western Blot and RT-PCR experiments,and the results showed that YAP1 and its target genes were more significantly upregulated in LXN-/-intestinal organoids.In addition,Lgr5 as the target gene for the Wnt signaling pathway,and Wnt signaling is a key signaling pathway for intestinal development.We also focused on the situation of Wnt signaling in intestinal organoids and found that the Wnt signaling pathway target gene is significantly upregulated in LXN-/-intestinal organoids.Finally,immunohistochemical staining was performed on the colon during regeneration and repair to detect the nuclear localization of YAP1 and β-catenin which is the key factors in the Wnt pathway.The results showed that during the regeneration and repair period of LXN-/-type mouse colon,YAP and Wnt signaling were activated,leading to YAP1 and β-Catenin has stronger nuclear localization,better triggering transcription,and promoting the proliferation and repair of intestinal epithelial cells.In summary,this study found that LXN deficiency can activate YAP and Wnt signaling to trigger transcriptional upregulation of intestinal stem cells marker Lgr5,thereby promoting intestinal development and regeneration and repair after injury. |
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