Use of intestinal organoids in the preparation of a medicament for the treatment of intestinal diseases

Abstract

This invention discloses the application of intestinal organoids in the preparation of drugs for treating intestinal diseases. Based on the self-renewal capacity and colonization ability of intestinal organoids in damaged intestinal epithelial regions, this invention utilizes intestinal organoid transplantation for the treatment of infectious and non-infectious inflammatory bowel diseases. Results show that the intestinal organoids, when colonized in damaged intestinal epithelial regions, effectively repair intestinal mucosal damage, significantly reduce the pathogenic bacterial load in colonic tissue during infectious inflammatory bowel diseases, and effectively inhibit the dissemination of pathogenic bacteria to distal organs such as the liver and spleen. This invention overcomes the adverse reactions such as toxic side effects and antibiotic resistance associated with existing drugs for treating intestinal diseases. By utilizing the self-renewal and colonization characteristics of organoids to repair intestinal barrier function and restore intestinal homeostasis, it effectively treats both infectious and non-infectious inflammatory bowel diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of intestinal organoids in the preparation of drugs for treating intestinal diseases. Background Technology

[0002] Inflammatory bowel diseases (IBDs) can be classified into infectious and non-infectious types based on their etiology. Infectious IBDs are mainly caused by pathogenic bacteria such as Salmonella, Shigella, and Escherichia coli. Non-infectious IBDs mainly include inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis) and irritable bowel syndrome (IBS), whose pathogenesis is closely related to non-infectious factors such as immune system dysregulation, symbiotic flora imbalance, or gut-brain axis dysfunction. Although the etiologies and pathogenesis of infectious and non-infectious IBDs differ significantly, both can lead to intestinal barrier dysfunction, intestinal microecological imbalance, and systemic inflammatory responses, thus severely impacting patients' intestinal health and quality of life. Currently, treatment strategies for infectious and non-infectious IBDs still face many challenges and limitations. For infectious inflammatory bowel diseases, antibiotics remain the primary treatment, but their clinical application has several limitations: First, antibiotic overuse accelerates the evolution of multidrug-resistant bacteria (such as extended-spectrum β-lactamase-producing Escherichia coli and methicillin-resistant Staphylococcus aureus), posing a serious threat to global public health security; second, antibiotics are ineffective in eliminating facultative intracellular bacteria such as Salmonella, leading to prolonged or recurrent infections; furthermore, while broad-spectrum antibiotics kill pathogens, they also indiscriminately kill intestinal commensal bacteria, disrupting the intestinal microecological balance and potentially causing serious consequences such as secondary Clostridium difficile infection; thirdly, some antibiotics (such as aminoglycosides and vancomycin) have dose-dependent nephrotoxicity or hepatotoxicity, limiting their use in specific patient groups; finally, for severely infected patients with severely impaired intestinal barrier function (such as elderly people with weakened immune function or immunosuppressed patients), although antibiotics can control the pathogen load to some extent, they cannot effectively promote intestinal epithelial regeneration or repair mucus layer function, resulting in limited treatment efficacy and poor prognosis. For non-infectious inflammatory bowel diseases, clinical treatment mainly relies on drug-mediated immune response modulation, such as glucocorticoids, immunosuppressants, and biologics. However, the efficacy of existing drugs varies significantly among individuals, and some patients do not respond well to treatment. Furthermore, long-term use of immunomodulatory drugs may cause adverse reactions such as opportunistic infections and metabolic abnormalities, and it is difficult to achieve substantial repair of intestinal barrier function. Therefore, there is an urgent need to provide a new method that can effectively repair the intestinal barrier and restore intestinal microecological balance without over-reliance on drug therapy, thereby improving treatment efficacy and promoting long-term recovery of intestinal health.

[0003] Organoid technology is a cutting-edge biomedical research technique that involves constructing three-dimensional organoids that mimic the structure and function of in vivo tissues by culturing primary or induced blast stem cells in vitro. These organoids not only simulate real tissue environments but also possess the ability to self-renew and differentiate, finding wide application in disease model building, drug screening, and regenerative medicine. Intestinal organoids, cultured from intestinal stem cells, can mimic the epithelial tissue, glands, and microvilli of the intestine. They are widely used to study intestinal diseases, the interaction between the microbiome and the host, and the effects of drugs on the intestine. However, the role of intestinal organoid transplantation in the treatment of infectious and non-infectious inflammatory bowel diseases requires further investigation. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of intestinal organoids in the preparation of drugs for treating infectious and non-infectious intestinal inflammatory diseases.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution:

[0006] This invention utilizes conventional methods for constructing intestinal organoids to isolate colonic crypts from the mouse colon and then culture them in three dimensions using a complete organoid culture medium to obtain intestinal organoids. The aim is to explore the potential of organoid transplantation in treating infectious and non-infectious intestinal inflammatory diseases. Organoid transplantation involves transplanting a donor organoid into a damaged intestinal region of a recipient. Infectious intestinal inflammatory diseases, represented by infections caused by intestinal pathogens such as Shigella dysenteriae, refer to diseases caused by pathogenic microorganisms such as pathogenic bacteria and opportunistic pathogens invading the host's intestine. Non-infectious intestinal inflammatory diseases, represented by inflammatory bowel diseases such as ulcerative colitis, refer to diseases caused by non-infectious factors such as abnormalities in the host's immune system and impaired intestinal barrier function. To comprehensively verify the therapeutic effects of organoid transplantation, mouse models of both infectious and non-infectious intestinal inflammatory diseases were constructed. In infectious intestinal inflammatory disease models, this invention constructed a mouse model of Shigella dysenteriae infection. Results showed that organoid transplantation not only significantly reduced the pathogenic bacterial load in colonic tissue but also effectively inhibited the spread of pathogens to distal organs such as the liver and spleen. Simultaneously, the infection symptoms in mice (such as weight loss, diarrhea, and decreased activity) were significantly improved. In non-infectious intestinal inflammatory disease models, this invention used sodium dextran sulfate (DSS) to construct a lethal colitis model. Experimental results showed that after transplantation of intestinal organoids, the survival rate of mice was significantly improved, the colonic inflammation score was significantly reduced, and intestinal mucosal damage was significantly repaired. These experimental results fully demonstrate that the intestinal organoids described in this invention possess the characteristics of being able to colonize damaged areas of the intestinal epithelium and have the ability to self-renew, thereby effectively repairing intestinal mucosal damage caused by intestinal infection or non-infectious factors. This invention achieves precise repair and functional reconstruction of the intestine through organoid transplantation technology, providing an innovative and effective solution for the treatment of various intestinal inflammatory diseases. It achieves substantial repair of the intestinal barrier function while avoiding adverse consequences such as opportunistic infections, metabolic abnormalities, and antibiotic resistance that may be caused by current treatment regimens. Therefore, intestinal organ transplantation has great potential in the treatment of infectious and non-infectious intestinal inflammatory diseases, and provides experimental evidence and technical support for the development of new intestinal disease treatment strategies.

[0007] Therefore, the present invention first provides the application of intestinal organoids in the preparation of medicaments for treating intestinal diseases, which are infectious or non-infectious intestinal diseases.

[0008] Furthermore, the infectious inflammatory bowel disease is a bacterial intestinal disease.

[0009] Preferably, the bacteria include one or more of Salmonella, Shigella, and Escherichia coli.

[0010] Preferably, the bacteria is Shigella dysenteriae.

[0011] Furthermore, the drug treats infectious intestinal inflammatory diseases by repairing intestinal mucosal damage, reducing the number of pathogenic bacteria in the intestine, and inhibiting the spread of pathogenic bacteria.

[0012] Furthermore, the non-infectious intestinal inflammatory disease is inflammatory bowel disease.

[0013] Preferably, the inflammatory bowel disease is fatal colitis.

[0014] Furthermore, the drug treats non-infectious inflammatory bowel diseases by repairing intestinal mucosal damage and reducing inflammation. The intestinal organoids of the present invention are functional three-dimensional structures obtained through in vitro three-dimensional culture of intestinal stem cells, obtained using conventional techniques in the art or commercially available. As one possible implementation, the intestinal organoids are obtained by isolating colonic crypts and then performing three-dimensional culture.

[0015] Preferably, the method for preparing the intestinal organoids includes isolating colonic crypts, performing three-dimensional culture in organoid complete culture medium to obtain colon-derived organoids, and passaged the colon-derived organoids every 3 to 5 days for 7 to 15 times.

[0016] Preferably, the method for preparing the intestinal organoids includes isolating colonic crypts, performing three-dimensional culture in a complete organoid culture medium to obtain colon-derived organoids, and passaged the intestinal organoids every 3 to 5 days for 10 passages.

[0017] Furthermore, the drug contains 2,000 to 3,000 intestinal organoids.

[0018] Preferably, the drug contains 2,500 intestinal organoids.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention provides the application of intestinal organoids in the preparation of drugs for treating inflammatory bowel diseases, including infectious and non-infectious inflammatory bowel diseases. Based on the self-renewal capacity and colonization ability of intestinal organoids in damaged intestinal epithelial regions, this invention utilizes intestinal organoid transplantation for the treatment of both infectious and non-infectious inflammatory bowel diseases. Results show that the intestinal organoids, when colonized in damaged intestinal epithelial regions, effectively repair intestinal mucosal damage caused by infectious or non-infectious factors, significantly reduce the pathogenic bacterial load in colonic tissue during infectious bowel diseases, and effectively inhibit the dissemination of pathogenic bacteria to distal organs such as the liver and spleen. This invention overcomes the adverse reactions such as toxic side effects and antibiotic resistance associated with existing drugs for treating inflammatory bowel diseases. By utilizing the self-renewal and colonization characteristics of organoids to repair intestinal barrier function and restore intestinal homeostasis, it effectively treats both infectious and non-infectious inflammatory bowel diseases. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of a mouse model of Shigella-induced intestinal infection treated with organoid transplantation.

[0022] Figure 2 A diagram showing the results of damage to the intestinal epithelial region induced by bacterial infection during organoid colonization.

[0023] Figure 3 The figure shows the effect of organoid transplantation on the body weight of mice with intestinal infection.

[0024] Figure 4 The figure shows the effect of organoid transplantation on the reproduction and spread of pathogenic bacteria in mice.

[0025] Figure 5 This is a schematic diagram of a DSS-induced lethal colitis mouse model treated with organoid transplantation.

[0026] Figure 6 This is a diagram showing the results of damaged intestinal epithelial regions induced by organoid colonization colitis.

[0027] Figure 7 The figure shows the effect of organoid transplantation on the survival rate of mice with lethal colitis.

[0028] Figure 8 The figure shows the effect of organoid transplantation on the body weight of a lethal colitis model mouse.

[0029] Figure 9 The graph shows the results of the disease activity index score in a mouse model of lethal colitis after organoid transplantation. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0031] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0032] Example 1

[0033] This embodiment establishes a mouse model system for treating intestinal pathogen-induced intestinal infection by intestinal organoid transplantation, and verifies the repair effect of intestinal organoid transplantation on infectious intestinal injury. The specific methods and results are as follows:

[0034] (1) Establish a mouse intestinal infection model system for treating intestinal pathogenic bacteria induced by organoid transplantation.

[0035] Establishment of a mouse model of intestinal infection induced by intestinal pathogens through intestinal organoid transplantation. Figure 1 As shown. Approximately 1–2 months prior to transplantation, colonic crypts were isolated from 4–8 week old C57BL / 6-ZsGreen mice (constitutive expression of green fluorescent protein in whole-body tissue cells) and cultured in three dimensions in organoid complete culture medium to obtain colon-derived organoids. Organoids were passaged every 3–5 days, and organoids passaged 10 times were suitable for transplantation. 24 hours before infection (day -1), mice were fasted for 4 hours, followed by pretreatment with 20 mg streptomycin via gavage for 20 hours; 4 hours before infection, mice were fasted for 1 × 10⁻⁶ mg streptomycin via gavage. 8 An infection model was established using colony-forming units (CFUs) of Shigella dysenteriae. Day 2 was chosen as the transplantation time point. Before transplantation, organoids were disassembled and counted, with 2500 organoids transplanted to each mouse. On day 5, mice were dissected and tissue samples were collected. Paraffin sections of the colon tissue were prepared, and viable bacteria were counted in the colon, liver, and spleen. The successful colonization of organoids in the bacterial infection-induced damaged intestinal epithelial region is shown in the results. Figure 2 As shown, green fluorescently labeled intestinal epithelial cells are visible in the paraffin sections, indicating that the organoids derived from the donor C57BL / 6-ZsGreen mouse have been successfully colonized in the intestinal epithelial region of the recipient C57BL / 6 wild-type mouse.

[0036] (2) Demonstrates that intestinal organoid transplantation can effectively repair intestinal damage in infectious intestinal diseases.

[0037] Intestinal organoid transplantation significantly promoted the recovery of body weight in mice with intestinal infection models, such as... Figure 3 As shown, the mice's weight began to slowly recover after transplantation treatment. Figure 4 As shown, the bacterial load in the colon, liver, and spleen of mice that underwent intestinal organoid transplantation was significantly lower than that of control mice, indicating that intestinal organoid transplantation effectively limited the proliferation and distant dissemination of pathogenic bacteria in mice. These results demonstrate that intestinal organoid transplantation can effectively alleviate intestinal pathological damage in mice with infectious enteroinflammatory disease and significantly inhibit the proliferation and distant dissemination of pathogenic bacteria.

[0038] Example 2

[0039] This embodiment establishes a mouse model of lethal colitis induced by dextran sulfate sodium (DSS) through intestinal organoid transplantation, and verifies the repair effect of intestinal organoid transplantation on non-infectious intestinal injury. The specific methods and results are as follows:

[0040] (1) Establish a mouse model of lethal colitis induced by intestinal organoid transplantation for the treatment of dextran sulfate sodium (DSS).

[0041] Establishment of a mouse model of lethal colitis induced by DSS through intestinal organoid transplantation. Figure 5 As shown. Colitis was induced in C57BL / 6 wild-type mice by administering a 3% DSS solution. The date of starting DSS feeding was designated as day 0, and feeding continued until day 7, after which the mice were given drinking water. By day 7, the mice exhibited typical colitis symptoms such as diarrhea, bloody stools, and weight loss. The acquisition and preparation of the intestinal organoids were consistent with those described in Example 1. Day 7 was chosen as the transplantation time point, and the relevant procedures and precautions during transplantation were consistent with those described in Example 1. On day 11, the mice were dissected and tissue samples were collected. Paraffin sections of the colon tissue were prepared to examine the colonic organoid colonization. The results of successful colonization of the intestinal organoids in the colitis-induced damaged intestinal epithelial region are shown below. Figure 6 As shown, green fluorescently labeled intestinal epithelial cells are visible in the paraffin sections, indicating that the intestinal organoids derived from the donor C57BL / 6-ZsGreen mouse have been successfully colonized in the intestinal epithelial region of the recipient C57BL / 6 wild-type mouse.

[0042] (2) Demonstrates that intestinal organoid transplantation can effectively repair intestinal damage in non-infectious inflammatory bowel diseases.

[0043] The changes in mouse survival rate from day 0 to day 11 during modeling are as follows: Figure 7 As shown, intestinal organoid transplantation significantly improved the survival rate of mice. Mice in the non-transplantation group all died on day 10, while the survival rate of mice in the intestinal organoid transplantation group remained at 37.5%. Figure 8 As shown, intestinal organoid transplantation can promote the recovery of body weight in mice with colitis; the body weight of surviving mice after transplantation treatment began to slowly recover on day 10. Figure 9 As shown, intestinal organoid transplantation reduced the disease activity index in colitis model mice. Following organoid transplantation on day 7, the disease activity index gradually decreased starting on day 9. These results indicate that intestinal organoid transplantation can effectively alleviate pathological damage in mice with non-infectious intestinal inflammatory diseases and significantly improve the survival of affected mice.

Claims

1. The application of intestinal organoids in the preparation of drugs for treating intestinal diseases, characterized in that, The intestinal disease is either an infectious or non-infectious inflammatory bowel disease.

2. The application according to claim 1, characterized in that, The intestinal pathogens causing the infectious enteritis are bacteria.

3. The application according to claim 2, characterized in that, The bacteria are selected from one or more of Salmonella, Shigella, and Escherichia coli.

4. The application according to claim 1, characterized in that, The non-infectious inflammatory bowel disease mentioned is inflammatory bowel disease.

5. The application according to claim 4, characterized in that, The inflammatory bowel disease mentioned is fatal colitis.

6. The application according to claim 1, characterized in that, The drug treats infectious intestinal inflammatory diseases by repairing intestinal mucosal damage, reducing the number of pathogenic bacteria in the intestine, and inhibiting the spread of pathogenic bacteria.

7. The application according to claim 1, characterized in that, The drug treats non-infectious inflammatory bowel diseases by repairing intestinal mucosal damage and reducing inflammation.

8. The application according to claim 1, characterized in that, The intestinal organoids were obtained by three-dimensional culture after isolating the colonic crypts.

9. The application according to claim 8, characterized in that, The method for preparing the intestinal organoids includes isolating colonic crypts, performing three-dimensional culture in organoid complete culture medium to obtain colon-derived organoids, and passaged the colon-derived organoids every 3 to 5 days for 7 to 15 times.

10. The application according to claim 1, characterized in that, The drug contains 2,000 to 3,000 intestinal organoids.

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