Construction method and application of model with fusobacterium nucleatum damaged mouse intestinal organs

Abstract

The invention belongs to the technical field of biology, and particularly relates to a construction method and application of a model with fusobacterium nucleatum damaged mouse intestinal tract organoid, and the construction method comprises the following steps: separating mouse intestinal tract organoid; obtaining a culture supernatant with fusobacterium nucleatum; obtaining a cell-free extract with fusobacterium nucleatum; viable bacteria with fusobacterium nucleatum are obtained; and treating the mouse intestinal organ with the fusobacterium nucleatum culture supernatant or the fusobacterium nucleatum cell-free extract or the fusobacterium nucleatum viable bacteria to obtain the model of the mouse intestinal organ damaged by the fusobacterium nucleatum. The construction method of the organ-like model with the fusobacterium nucleatum damaged intestinal tract is simple, operability and convenience can be increased for further research on a regulation mechanism of the fusobacterium nucleatum damaged intestinal tract, and a reliable and accurate research tool is provided for gastrointestinal diseases such as inflammatory bowel diseases and colorectal cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for constructing a mouse intestinal organoid model damaged by Fusobacterium nucleatum and an application thereof. Background Art

[0002] Fusobacterium nucleatum Fusbacterium nucleatum Fn (Fn) is a Gram-negative anaerobic bacterium and a member of the human microbiome. Recent research results have shown that an increase in Fn load is closely related to the development and prognosis of colorectal cancer (CRC), so Fn has been studied more deeply among colorectal cancer-related pathogens. Fn infection in the intestine is closely related to the occurrence and development of CRC, but its specific mechanism of action has not yet been fully clarified. Relevant studies have shown that Fn promotes the occurrence and development of gastrointestinal diseases represented by colorectal cancer through multiple mechanisms in regulating the body's immune response, epigenetics, and host metabolism. The mechanisms that may be involved include: Fn induces intestinal epithelial-mesenchymal transition with the help of its membrane surface virulence factors, activates inflammation-related signaling pathways to promote the body's micro-inflammatory environment, induces immunosuppression and affects the body's anti-tumor immunity, and induces disease through the toxic effects of secreted metabolites. At present, the models that are widely used in research include cell lines and animal xenotransplantation, but cell lines are difficult to show histological characteristics, and animal xenotransplantation models are still controversial at the ethical level. Based on the limitations of animal models and cell line models, it is urgent to provide an effective and reliable in vitro model to conduct relevant research on the early occurrence of the disease, so as to better and more accurately reflect the changes in disease indicators and discover key molecules for drug development.

[0003] Organoids are a type of model that has self-assembly characteristics and self-renewal capabilities, and are 3D in vitro research models that are closer to the physiological or pathological state in vivo. The morphological characteristics of organoids are closely related to the state of intestinal development and the degree of damage. They can not only be used as a research model for the pathological effects of a wider range of pathogenic bacteria infecting the intestinal epithelium, but also provide new clues for revealing the involvement of intestinal pathogens in the early development of CRC cancer. At present, there is no systematic in vitro model for simulating intestinal damage caused by Fusobacterium nucleatum, so the current biomedical field urgently needs a method that can stably, efficiently, comprehensively and conveniently establish a method for simulating the pathological effects of intestinal damage caused by Fusobacterium nucleatum. In view of this, it is necessary to develop a method for constructing an organoid model of intestinal damage caused by Fusobacterium nucleatum. Summary of the invention

[0004] In view of the above defects or improvement needs of the prior art, the present invention provides a method for constructing a mouse intestinal organoid model damaged by Fusobacterium nucleatum and its application. The construction method is based on the isolated and prepared mouse intestinal organoids. After adding the culture supernatant / cell-free extract / live bacteria of Fusobacterium nucleatum for co-cultivation, the mouse small intestinal organoid model is damaged in vitro, showing poor growth of the small intestinal organoids and a slightly decreased trend in spheroidization and budding proliferation capabilities. In other words, a mouse intestinal organoid model damaged by Fusobacterium nucleatum is obtained, which increases the operability and convenience for further studying the regulatory mechanism of Fusobacterium nucleatum damage to the intestine, and provides a reliable and accurate research tool for gastrointestinal diseases such as inflammatory bowel disease and colorectal cancer.

[0005] In order to achieve the above object, the present invention provides a method for constructing a mouse intestinal organoid model of Fusobacterium nucleatum damage, comprising: Isolation of mouse intestinal organoids; Obtaining the culture supernatant of Fusobacterium nucleatum; Obtaining a cell-free extract of Fusobacterium nucleatum; Obtaining live Fusobacterium nucleatum bacteria; The mouse intestinal organoids are treated with the culture supernatant of Fusobacterium nucleatum, the cell-free extract of Fusobacterium nucleatum, or the live bacteria of Fusobacterium nucleatum to obtain a mouse intestinal organoid model damaged by Fusobacterium nucleatum.

[0006] Furthermore, in the above technical solution, the method for isolating mouse intestinal organoids includes: washing, chopping, digesting and filtering the mouse small intestinal tissue in sequence, centrifuging to obtain mouse intestinal crypts, and culturing the mouse intestinal organoids with complete medium to obtain mouse intestinal organoids. Specifically, the method includes the following steps: (1) After killing the mice, they were disinfected by soaking in 75% alcohol, and the small intestinal segments were cut and placed in pre-cooled PBS buffer to remove the mesentery and fat on the surface of the small intestinal segments. (2) Use a syringe to draw PBS solution to repeatedly flush out the contents of the intestinal segment, and cut the intestine longitudinally in the PBS solution to expose the intestinal villi; (3) Flatten the intestine with the villi facing upward, scrape off the intestinal villi, and wash the intestinal segment with ice-cold PBS solution until the PBS solution becomes clear; (4) Prepare EDTA solution, cut the intestinal segments into small segments of 2-5 mm, mix with EDTA solution, and shake gently on a shaker; (5) After shaking, discard the waste liquid, aspirate the EDTA solution, add ice-cold PBS solution, invert several times and let the intestinal segment settle, then discard the waste liquid, aspirate the PBS solution and pour the intestinal segment into a clean centrifuge tube, add ice-cold PBS solution, vortex for 5 seconds, and repeatedly vortex the intestinal segment to separate the crypts until the PBS becomes turbid; (6) Install a cell strainer at the mouth of the centrifuge tube, pass the mixed solution in step (5) through the cell strainer to remove excess villi and larger tissue fragments, and collect the filtered mixed solution; (7) Take the filtered mixed solution in step (6) for microscopic examination and crypt counting. After counting, aspirate the mixed solution containing the required number of crypts, centrifuge for 2 min, discard the supernatant, mix with an appropriate amount of Matrigel, and resuspend without generating bubbles. (8) Take out the 24-well cell culture plate that has been preheated in the incubator, add 30 μL of the resuspension in step (7) to each well to form three-dimensional droplets, and place it in a 37°C cell culture incubator for 10 min to allow the Matrigel to solidify. When the Matrigel solidifies, take out the culture plate, add 300 μL of small intestinal organoid culture medium to each well, and add 500 μL of PBS solution to the remaining wells to maintain humidity. Place it in a 37°C, 5% CO incubator. 2 Cultured in a cell culture incubator to obtain mouse intestinal organoids.

[0007] Furthermore, in the above technical solution, the mouse intestinal organoid complete culture medium includes the following components: Advanced DMEM / F12 culture medium 12mL, Glutamax 120μL, 4-hydroxyethylpiperazineethanesulfonic acid buffer 120μL, Y-27632 supplement 50μL, N 2 supplement 120μL, B27 supplement 120μL, Noggin protein 100ng / mL, R-spondin1 protein 500ng / mL, EGF protein 50ng / mL.

[0008] Furthermore, in the above technical scheme, the culture method of Fusobacterium nucleatum is as follows: 10 μL of frozen strains of Fusobacterium nucleatum are picked from a -80°C refrigerator and activated in 10 mL of BHI liquid culture medium, and then cultured in an anaerobic box at 37°C for 24 h. The activated bacterial solution is inoculated into BHI solid culture medium and cultured at 37°C for 48 h; then a monoclonal colony is picked and placed in BHI liquid culture medium, and cultured for 24 h until the logarithmic growth phase, the cultured bacteria are centrifuged at 2000 r / min and 4°C for 10 min, the bacteria are collected, washed once with PBS, and resuspended in antibiotic-free DMEM / F12 culture medium, and then the absorbance value at 600 nm is measured using an ultraviolet spectrophotometer, and the MOI turbidity of the bacterial suspension is adjusted to 1×10 according to the OD value. 8 CFU / mL is reserved.

[0009] Furthermore, in the above technical solution, when the culture supernatant of Fusobacterium nucleatum is used to treat mouse intestinal organoids, the added concentration of the culture supernatant of Fusobacterium nucleatum is 1-5%, and the preferred concentration is 1%.

[0010] Furthermore, in the above technical solution, when the Fusobacterium nucleatum cell-free extract is used to treat mouse intestinal organoids, the protein concentration of the Fusobacterium nucleatum cell-free extract is 10-1000 μg / mL, and the preferred concentration is 100-1000 μg / mL.

[0011] Furthermore, in the above technical solution, when the mouse intestinal organoids were treated with live Fusobacterium nucleatum, the number of live Fusobacterium nucleatum was 1×10 8 CFU, MOI was 100 / 200.

[0012] The present invention also provides a mouse intestinal organoid model damaged by Fusobacterium nucleatum constructed by the above construction method.

[0013] The present invention also provides an application of the above-mentioned Fusobacterium nucleatum damaged mouse intestinal organoid model in evaluating the effect of Fusobacterium nucleatum on the proliferation and differentiation of intestinal stem cells in vitro.

[0014] The present invention also provides an application of a Fusobacterium nucleatum-damaged mouse intestinal organoid model in research related to inflammatory bowel disease, intestinal injury regeneration, and colorectal cancer.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention is based on the study of the effect of Fusobacterium nucleatum on the pathological damage of intestinal epithelium. Based on the isolated and prepared mouse intestinal organoids, the culture supernatant / cell-free extract / live bacteria of Fusobacterium nucleatum were added for co-culture to construct a mouse intestinal organoid model damaged by Fusobacterium nucleatum. The model can be used to study the effects of pathogenic bacteria on the growth and development of mouse intestinal organoids, and to explore the effects of Fusobacterium nucleatum on the proliferation and differentiation of intestinal stem cells in vitro and its mechanism of action. This lays a foundation for subsequent related research and drug development on the effects of Fusobacterium nucleatum on various intestinal diseases such as inflammatory bowel disease, intestinal damage regeneration, and colorectal cancer. At the same time, it has broad application prospects in subsequent in vitro research on Fusobacterium nucleatum, and has reference value for the construction of pathogenic bacteria damage and bacterial infection models of other organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 It is a flow chart of the method for constructing a mouse intestinal organoid model damaged by Fusobacterium nucleatum of the present invention; Figure 2 This is a light microscopic observation of the intestinal organoids damaged by the culture supernatant of Fusobacterium nucleatum of the present invention (objective lens 10×); Figure 3 This is a light microscopic observation of the intestinal organoids damaged by the cell-free extract of Fusobacterium nucleatum of the present invention (objective lens 10×); Figure 4 This is a light microscopic observation picture (objective lens 10×) of intestinal organoids damaged by co-culture with live Fusobacterium nucleatum of the present invention. DETAILED DESCRIPTION

[0018] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials involved in the following examples are common commercial products and can be purchased from the market unless otherwise specified.

[0019] The above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution.

[0020] The raw materials involved in the embodiments of the present invention are either existing commercially available products or can be prepared according to existing methods.

[0021] The present invention provides a method for constructing a mouse intestinal organoid model of Fusobacterium nucleatum damage, comprising: Isolation of mouse intestinal organoids; Obtaining the culture supernatant of Fusobacterium nucleatum; Obtaining a cell-free extract of Fusobacterium nucleatum; Obtaining live Fusobacterium nucleatum bacteria; The mouse intestinal organoids are treated with the culture supernatant of Fusobacterium nucleatum, the cell-free extract of Fusobacterium nucleatum, or the live bacteria of Fusobacterium nucleatum to obtain a mouse intestinal organoid model damaged by Fusobacterium nucleatum.

[0022] Among them, the method for isolating mouse intestinal organoids includes: taking mouse small intestinal tissue, washing, chopping, digesting and filtering in sequence, centrifuging to obtain mouse intestinal crypts, and culturing with mouse intestinal organoid complete culture medium to obtain mouse intestinal organoids.

[0023] The complete medium for mouse intestinal organoids is composed of 12 mL of Advanced DMEM / F12 medium, 120 μL of Glutamax, 120 μL of 4-hydroxyethylpiperazineethanesulfonic acid buffer, 50 μL of Y-27632 supplement, and 50 μL of N 2 supplement 120μL, B27 supplement 120μL, Noggin protein 100ng / mL, R-spondin1 protein 500ng / mL, EGF protein 50ng / mL.

[0024] In addition, the culture media involved in the follow-up are: BHI liquid medium: tryptone 10.0g / L, dehydrated calf brain extract powder 12.5g / L, dehydrated beef heart extract powder 5.0g / L, sodium chloride 5.0g / L, glucose 2.0g / L, disodium hydrogen phosphate 2.5g / L, pH 7.4. BHI solid medium is the above medium with 1.5% agar added.

[0025] DMEM / F12 culture medium is produced by Gibco, where DMEM F12 medium is improved by mixing DMEM medium and Ham's F-12 medium in a ratio of 1:1. In addition to glucose, amino acids and vitamins in DMEM medium, DMEM / F12 also contains other ingredients in Ham's F-12, including zinc, putrescine, hypoxanthine and thymidine.

[0026] The following is further described with reference to specific embodiments.

[0027] Example 1: Isolation and preparation of mouse intestinal organoids The isolation and preparation of mouse intestinal organoids includes the following steps: (1) After killing the mouse, soak it in 75% alcohol for 2 minutes for disinfection. Use dissecting scissors to cut the mouse's abdominal skin and peritoneum, cut out a 15 cm small intestinal segment, put it in pre-cooled PBS buffer, and carefully remove the mesentery and fat on the surface of the small intestinal segment. (2) Use a syringe to draw PBS solution to repeatedly flush out the contents of the intestinal segment, discard the waste liquid, and cut the intestine longitudinally in the PBS solution to expose the intestinal villi; (3) Flatten the intestinal surface with the villi facing upward, use a glass coverslip to gently scrape off the intestinal villi, and wash the intestinal segment with ice-cold PBS solution until the PBS solution becomes clear; (4) Add 1 mL of 0.5 M EDTA solution to 100 mL of ice-cold PBS to prepare a 5 mM EDTA solution. Cut the intestinal segment into 2-5 mm small segments, mix with 25 mL of the prepared EDTA solution, and gently shake the intestinal segment on a shaker until it is completely shaken for 30 min. (5) After shaking, discard the waste liquid, aspirate the EDTA solution, add 25 mL of ice-cold PBS solution, gently invert several times and let the intestinal segment settle, then discard the waste liquid, carefully aspirate the PBS solution and pour the intestinal segment into a clean centrifuge tube, add 25 mL of ice-cold PBS solution, vortex on a vortex oscillator for 5 seconds, and repeatedly vortex the intestinal segment to separate the crypts until the PBS becomes turbid; (6) Place a 70 μm cell strainer on the mouth of a 50 mL centrifuge tube, pass the mixture from step (5) through the 70 μm cell strainer to remove excess villi and larger tissue fragments, and collect the filtered mixture; (7) Take 20 μL of the filtered mixture in step (6) for microscopic examination and crypt counting. After counting, aspirate the mixture containing the required number of crypts and centrifuge it for 2 min using a handheld centrifuge. Discard the supernatant and mix with an appropriate amount of Matrigel and resuspend without generating bubbles. (8) Take out the 24-well cell culture plate that has been preheated in the incubator, add 30 μL of the resuspension in step (7) to each well to form three-dimensional droplets, and place it in a 37°C cell culture incubator for 10 min to allow the Matrigel to solidify. When the Matrigel solidifies, take out the culture plate, add 300 μL of small intestinal organoid culture medium to each well, add 500 μL of PBS solution to the remaining wells to maintain humidity, and place it in a 37°C, 5% CO incubator. 2 Cultured in a cell culture incubator to observe the status of small intestinal organoids.

[0028] Example 2: Establishment of a mouse intestinal organoid model of Fusobacterium nucleatum injury 1. Establishment of the intestinal organoid model of Fusobacterium nucleatum-damaged mice, including the following steps: (1) The pathogenic bacterium Fusobacterium nucleatum (BNCC361670) was purchased from Beina Biotech. 10 μL of the frozen strain of Fusobacterium nucleatum was picked from a -80°C refrigerator and activated in 10 mL of BHI liquid medium. After culturing in an anaerobic chamber at 37°C for 24 hours, the activated bacterial solution was inoculated into BHI solid medium and cultured at 37°C for 48 hours. During the experiment, a single clone colony was picked and placed in BHI liquid medium and cultured for 24 hours until the logarithmic growth phase for use in the experiment. The cultured bacteria were centrifuged at 2000 r / min and 4°C for 10 minutes, the bacteria were collected, washed once with PBS, and resuspended in DMEM / F12 culture medium without antibiotics. The absorbance at 600 nm was then measured using a UV spectrophotometer, and the MOI turbidity of the bacterial suspension was adjusted to 1×10 according to the OD value. 8 CFU / mL for future use; (2) Fn culture supernatant (CFS) treatment: The culture supernatant collected by centrifugation after culturing the above-mentioned Fn strain was added to the complete medium of the organoid at a concentration of 1% and 5%, respectively. A blank control and a negative control group (PBS and sterile BHIS treatment groups) were set up to observe the morphology of the organoids. (3) Fn cell-free extract (CFE) treatment: The Fn bacterial suspension was ultrasonically disrupted for 5-10 min. The obtained bacterial lysate was used to measure the protein concentration using a BCA kit and then used for later use. When used, it was diluted to different protein concentrations (10, 100, 1000 μg / mL) and added to the complete culture medium of mouse intestinal organoids. A blank control group was set up to observe the morphology of the organoids. (4) Fn live bacteria co-culture treatment: Intestinal organoids that have been isolated and cultured for 3-4 days and have good growth (starting to sprout) are replaced with 1×10 8 The cells were cultured in complete medium containing CFU of Fusobacterium nucleatum (multiplicity of infection MOI = 100 / 200, ratio of bacteria to organoids = 100 / 200), and co-cultured in a cell culture incubator at 37°C for 3 h. A sterile blank control group was set up to observe the morphology of the organoids.

[0029] 2. Morphological observation of organoids damaged by Fusobacterium nucleatum Observe the organoids treated with Fn culture supernatant / cell-free extract / live bacteria under an optical inverted microscope, select culture wells with clear background and complete structure, observe the morphology of organoids with complete structure and take photos for statistics. The observation indicators and their calculation methods are as follows.

[0030] (1) Spheroidization: The morphology of intestinal crypts was observed under a microscope on the first day after treatment with Fusobacterium nucleatum, and the number of crypts that spheroidized in each well was counted. The spheroidization rate of intestinal crypts in each well was calculated according to the following formula: Crypt spheroidization rate (%) = number of crypt spheroidization / total number of crypts × 100 (2) Budding: The morphology of intestinal crypts was observed under a microscope 4 and 6 days after treatment with Fusobacterium nucleatum, and the number of budding organoids in each well was counted; (3) Surface area: The morphology of intestinal crypts was observed under a microscope 4 and 6 days after treatment with F. nucleatum, and the area of ​​organoids in each well was measured and counted using ImageJ software.

[0031] After the intestinal organoid model of Fusobacterium nucleatum-injured mice was established according to the above method, the morphology of intestinal organoids at different treatment times was tracked and observed. The results are as follows Figures 2 to 4 shown.

[0032] according to Figure 2 The results show that when CFS with a gradient concentration of 1% and 5% was co-cultured with intestinal organoids, the number of organoids formed on the first day was 22.33 and 23.00, and the sphere formation rate was 30.05% and 31.79%, respectively. The number of organoids sprouted on the fourth day was 2.67 and 2.00, and the surface area was 571.02 and 563.96, respectively. The number of organoids sprouted on the sixth day was 2.67 and 3.00, and the surface area was 654.50 and 577.16, respectively. Compared with the blank control group, 1% and 5% CFS significantly reduced the sphere formation and sprouting proliferation ability of organoids, affecting the growth and development of organoids, and the inhibitory effect of 1% CFS was more significant.

[0033] according to Figure 3As shown in the results, treatment of mouse intestinal organoids with CFE at a final concentration of 10, 100, and 1000 μg / mL can cause damage to the morphological development of intestinal organoids. After 1, 4, and 6 days of treatment, the number of spheres, sphere formation rate, budding rate, and surface area of ​​mouse organoids treated with 100 and 1000 μg / mL CFE were significantly reduced compared with the control group. Among them, the surface area was not significantly different from the control group on the 6th day of treatment with 100 μg / mL, and reached the lowest value after 6 days of treatment with 1000 μg / mL.

[0034] according to Figure 4 As shown in the results, treatment of mouse intestinal organoids with Fn live bacteria at a multiplicity of infection ratio (MOI) of 100 and 200 can cause a significant decrease in the budding rate and surface area of ​​intestinal organoids, thereby impairing their growth and development.

[0035] Based on the above results, it was shown that the mouse small intestinal organoid model was damaged in vitro after co-culture with the addition of Fn culture supernatant / cell-free extract / live bacteria based on the isolated and prepared mouse intestinal organoids, showing poor growth of the small intestinal organoids and a slightly decreased trend in spheroidization and budding proliferation ability. In other words, a mouse intestinal organoid model damaged by Fusobacterium nucleatum was obtained, which can be used to establish research on the effects of pathogenic bacteria on the proliferation and differentiation of intestinal stem cells and their mechanism of action.

[0036] The intestinal organoid model of Fusobacterium nucleatum damaged in mice constructed in the present invention can lay the foundation for subsequent related research and drug development on the effects of Fusobacterium nucleatum on various intestinal diseases such as inflammatory bowel disease, intestinal damage regeneration, and colorectal cancer; at the same time, this model has broad application prospects in subsequent in vitro research on Fusobacterium nucleatum, and has reference value for the construction of pathogenic bacteria damage and bacterial infection models of other organoids.

[0037] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing a mouse intestinal organoid model of Fusobacterium nucleatum damage, characterized in that: include: Isolation of mouse intestinal organoids; Obtaining the culture supernatant of Fusobacterium nucleatum; Obtaining a cell-free extract of Fusobacterium nucleatum; Obtaining live Fusobacterium nucleatum bacteria; The mouse intestinal organoids are treated with the culture supernatant of Fusobacterium nucleatum, the cell-free extract of Fusobacterium nucleatum, or the live bacteria of Fusobacterium nucleatum to obtain a mouse intestinal organoid model damaged by Fusobacterium nucleatum.

2. The method for constructing a mouse intestinal organoid model of Fusobacterium nucleatum damage according to claim 1, characterized in that: The method for isolating mouse intestinal organoids includes: taking mouse small intestinal tissue, washing, chopping, digesting and filtering in sequence, centrifuging to obtain mouse intestinal crypts, and culturing with mouse intestinal organoid complete culture medium to obtain mouse intestinal organoids.

3. The method for constructing a mouse intestinal organoid model of Fusobacterium nucleatum damage according to claim 2, characterized in that: The mouse intestinal organoid complete culture medium includes the following components: 12 mL of Advanced DMEM / F12 culture medium, 120 μL of Glutamax, 120 μL of 4-hydroxyethylpiperazineethanesulfonic acid buffer, 50 μL of Y-27632 supplement, 120 μL of N2supplement, 120 μL of B27 supplement, 100 ng / mL of Noggin protein, 500 ng / mL of R-spondin1 protein, and 50 ng / mL of EGF protein.

4. The method for constructing a mouse intestinal organoid model of Fusobacterium nucleatum damage according to claim 1, characterized in that: The culture method of the Fusobacterium nucleatum is as follows: 10 μL of frozen strains of Fusobacterium nucleatum are picked from a -80°C refrigerator and activated in 10 mL of BHI liquid culture medium, and then cultured in an anaerobic box at 37°C for 24 h, and then the activated bacterial solution is inoculated into a BHI solid culture medium, and cultured at 37°C for 48 h; then a monoclonal colony is picked and placed in a BHI liquid culture medium, and cultured at 37°C for 24 h until the logarithmic growth phase, and the cultured bacteria are centrifuged at 2000 r / min and 4°C for 10 min, the bacteria are collected, washed once with PBS, and resuspended in an antibiotic-free DMEM / F12 culture medium, and then the absorbance value at 600 nm is measured using an ultraviolet spectrophotometer, and the MOI turbidity of the bacterial suspension is adjusted to 1×10 according to the OD value. 8 CFU / mL is reserved.

5. The method for constructing a mouse intestinal organoid model of Fusobacterium nucleatum damage according to claim 1, characterized in that: When the culture supernatant of Fusobacterium nucleatum is used to treat mouse intestinal organoids, the added concentration of the culture supernatant of Fusobacterium nucleatum is 1-5%, and the preferred concentration is 1%.

6. The method for constructing a mouse intestinal organoid model of Fusobacterium nucleatum damage according to claim 1, characterized in that: When the Fusobacterium nucleatum cell-free extract is used to treat mouse intestinal organoids, the protein concentration of the Fusobacterium nucleatum cell-free extract is 10-1000 μg / mL, preferably 100-1000 μg / mL.

7. The method for constructing a mouse intestinal organoid model of Fusobacterium nucleatum damage according to claim 1, characterized in that: When live Fusobacterium nucleatum was used to treat mouse intestinal organoids, the number of live Fusobacterium nucleatum was 1×10 8 CFU, MOI was 100 / 200.

8. A mouse intestinal organoid model damaged by Fusobacterium nucleatum constructed by the construction method described in any one of claims 1 to 7.

9. Use of the intestinal organoid model of Fusobacterium nucleatum damaged mice as described in claim 8 in evaluating the effects of Fusobacterium nucleatum on the proliferation and differentiation of intestinal stem cells in vitro.

10. An application of the intestinal organoid model of Fusobacterium nucleatum-damaged mice as described in claim 8 in research related to inflammatory bowel disease, intestinal damage regeneration, and colorectal cancer.

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