Effects of Akkermansia on AOM/DSS Inflammatory-associated Colorectal Cancer / 肿瘤防治研究

Objective To investigate the effects of Akkermansia muciniphila (AKK) on azomethane-oxide (AOM)/glucan sodium sulfate (DSS)-induced inflammatory colorectal cancer mouse model and intestinal stem cells. Methods AOM/DSS-induced mouse models of inflammatory-associated colorectal cancer were randomly divided into three groups, namely, model, AKK and aspirin groups, based on different administration of drugs by gavage. The tumor number, size, distribution, and burden were observed 10 weeks after intervention. Immunohistochemical method was used to analyze the expressions of Ki67 and Lgr5 proteins, which are utilized to characterize tumor malignancy and stem cells. The mRNA expressions of Lgr5, CD133, Nanog, and ALDH1 were detected by qRT-PCR. Results Compared with those of the model group, the tumor number, size, and burden of the AKK group were significantly reduced (P < 0.01). The expressions of Ki67 and Lgr5 in the AKK group of tumor tissues were significantly decreased (P < 0.05), and the mRNA expressions of CD133, Nanog and ALDH1 were significantly down-regulated. Conclusion AKK is effective against AOM/DSS-induced colitis-associated colorectal cancer in mice, and its mechanism of action may be closely related to colorectal stem cell activity.

Establishment and Evaluation of Intestinal Injury Model of Mouse Acute Graft Versus Host Disease Based on An Organoid Technology / 中国实验血液学杂志

OBJECTIVE@#To establish an intestinal organoid model that mimic acute graft versus host disease (aGVHD) caused intestinal injuries by using aGVHD murine model serum and organoid culture system, and explore the changes of aGVHD intestine in vitro by advantage of organoid technology.@*METHODS@#20-22 g female C57BL/6 mice and 20-22 g female BALB/c mice were used as donors and recipients for bone marrow transplantation, respectively. Within 4-6 h after receiving a lethal dose (8.0 Gy) of γ ray total body irradiation, a total of 0.25 ml of murine derived bone marrow cells (1×107/mice, n=20) and spleen nucleated cells (5×106/mice, n=20) was infused to establish a mouse model of aGVHD (n=20). The aGVHD mice were anesthetized at the 7th day after transplantation, and the veinal blood was harvested by removing the eyeballs, and the serum was collected by centrifugation. The small intestinal crypts of healthy C57BL/6 mice were harvested and cultivated in 3D culture system that maintaining the growth and proliferation of intestinal stem cells in vitro. In our experiment, 5%, 10%, 20% proportions of aGVHD serum were respectively added into the organoid culture system for 3 days. The formation of small intestinal organoids were observed under an inverted microscope and the morphological characteristics of intestinal organoids in each groups were analyzed. For further evaluation, the aGVHD intestinal organoids were harvested and their pathological changes were observed. Combined with HE staining, intestinal organ morphology evaluation was performed. Combined with Alcian Blue staining, the secretion function of aGVHD intestinal organoids was observed. The distribution and changes of Lgr5+ and Clu+ intestinal stem cells in intestinal organoids were analyzed under the conditions of 5%, 10% and 20% serum concentrations by immunohistochemical stainings.@*RESULTS@#The results of HE staining showed that the integrity of intestinal organoids in the 5% concentration serum group was better than that in the 10% and 20% groups. The 5% concentration serum group showed the highest number of organoids, the highest germination rate and the lowest pathological score among experimental groups, while the 20% group exhibited severe morphological destruction and almost no germination was observed, and the pathological score was the highest among all groups(t=3.668, 4.334,5.309,P<0.05). The results of Alican blue staining showed that the secretion function of intestinal organoids in serum culture of aGVHD in the 20% group was weaker than that of the 5% group and 10% of the organoids, and there was almost no goblet cells, and mucus was stainned in the 20% aGVHD serum group. The immunohistochemical results showed that the number of Lgr5+ cells of intestinal organoids in the 5% group was more than that of the intestinal organoids in the 10% aGVHD serum group and 20% aGVHD serum group. Almost no Clu+ cells were observed in the 5% group. The Lgr5+ cells in the 20% group were seriously injuried and can not be observed. The proportion of Clu+ cells in the 20% group significantly increased.@*CONCLUSION@#The concentration of aGVHD serum in the culture system can affect the number and secretion function of intestinal organoids as well as the number of intestinal stem cells in organoids. The higher the serum concentration, the greater the risk of organoid injury, which reveal the characteristics of the formation and functional change of aGVHD intestinal organoids, and provide a novel tool for the study of intestinal injury in aGVHD.

Establishment of mini-guts organoid and research on intestinal disease from the new perspective / 中华胃肠外科杂志

Intestinal organoids, also named "mini-guts", reconstitute sophisticated three-dimensional architecture recapitulating diversified intestinal epithelial cell types and physiology, which is driven by the proliferative and self-assembling characteristics of crypt stem cells. The initiation of organoids study relies on the identification of Lgr5+ crypt stem cells from different intestinal segments and the key role of EGF, Wnt, BMP/TGF-β, Notch signal pathways within the microenvironment during the cultivation process. Besides constituting polarized crypt-villus structures, these "mini-guts" exhibit various effective functions of intestinal epithelium. Since 2009 when the culture system of small intestinal organoids was established by Sato et al, intestinal organoids excel conventional intestinal models depending on genetical mutation in multiple aspects and thus have become the hotspot among the research on intestinal diseases. Combined with genomics, material science and engineering, "mini-guts" have been widely applied to the research on intestinal development, intestinal transport physiology, epithelial barrier, pathogen-host interaction and the study on cystic fibrosis, infectious diarrhea, ulcerative colitis, Crohn's disease, intestinal cancer, etc. In this review, we summarize the new insights introduced by organoid into the research on intestinal diseases, and related research advances and applications.

Stem cell-based therapy for inflammatory bowel disease

Inflammatory bowel disease (IBD) is an idiopathic, multi-etiological disease characterized by inflammation and mucosal destruction of the gastrointestinal tract. Despite the remarkable advance in immunomodulating therapies, there still remains a certain population of patients who are refractory to conventional as well as biologic therapies and fail to achieve mucosal healing. To improve the prognosis of those patients, at least 2 types of stem cells have been tested for their potential therapeutic use. Transplantation of hematopoietic stem cells or mesenchymal stem cells have been tested in several clinical studies, but their beneficial effect still remains controversial. In this review, we would like to overview the recent clinical challenges of stem cell-based therapies in IBD and also introduce our new therapeutic plan of intestinal stem cell transplantation for IBD, based on our ex vivo intestinal organoid culture technique.

Establishment of an intestinal organoid-based method to investigate the radiation mitigation effect of Epiregulin / 中华放射医学与防护杂志

Objective To establish an intestinal organoid-based assay to investigate the radiation mitigation effect of epiregulin in vitro. Methods Intestinal crypts were released from tissue incubated with EDTA. Intestinal crypts seeded in 3D matrigel were irradiated at 24 h after plating. The radiation mitigation effect of epiregulin was evaluated by measuring the survival rate, size and budding numbers of the organoid after irradiation, and the basic FGF was used as a positive control of epiregulin. Results Radiation-induced lethality and dose-dependent survival curve of the intestinal organoid were consistent with in vivo data. Treatment with epiregulin (400 ng/ml) at 24 h post-radiation significantly increased survival rate of 8 Gy X-ray irradiated intestinal organoid in comparison with non-treated group [(12.56 ± 1.02)％vs. (4.73 ± 0.38)％, t=12.43,P<0.05]. Conclusions Epiregulin has radiation mitigation effect on intestinal organoid and could serve as a potential medical countermeasure to mitigate gastrointestinal toxicity.

Radio-protective effect of sulfated polysaccharide purified from Ecklonia cava against small intestinal stem cells of gamma-ray irradiated mice / 동물의과학연구지

Our previous research on sulfated polysaccharide purified from Ecklonia cava, a brown alga found in Jeju island, Korea, showed that sulfated polysaccharides modulate the apoptotic threshold of intestinal cells, thereby preventing intestinal damage caused by ionizing radiation. In this study, we investigated the ability of sulfated polysaccharide to augment restoration of small intestinal stem cells from gamma-ray-induced damage. In our results, sulfated polysaccharide treatment increased the numbers of Ki-67-positive cells as well as inducible nitric oxide synthase (iNOS)-expressing cells in the small intestine compared with those of irradiated only mice. Meanwhile, exposure to irradiation increased the number of paneth cells, which are frequently associated with intestinal inflammation, whereas sulfated polysaccharide treatment reduced the number of paneth cells in the small intestinal crypt. Conclusively, our data suggest that reduction of iNOS-expressing cells and paneth cells in sulfated polysaccharide-treated mice contributes to the inhibition of radiation-induced intestinal inflammation.