An in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids, and its preparation method and application

Abstract

The present invention discloses an in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids, and a preparation method and application thereof, and relates to the technical field of aquaculture cell culture. The method comprises the following steps: preparing zebrafish intestinal bacterial metabolites rich in short-chain fatty acids; collecting zebrafish intestinal organoids; in vitro co-culture; and preparing an in vitro co-culture model. The present invention can simulate the in vivo 3D growth environment in vitro, and can more realistically reflect the body's functions, so as to study the interaction process and occurrence mechanism between zebrafish intestinal bacterial metabolites and the intestine. The co-culture product of the present invention can be used to identify a method for identifying drugs or reagents for treating diseases. It can provide accurate research tools for the study of molecular mechanisms of intestinal diseases, disease simulation, in vitro evaluation of nutrients, discovery of drug targets, and screening of drug efficacy, and has broad application prospects. The present invention solves the problem of the lack of an ideal in vitro research model of zebrafish intestinal organoids in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture cell culture, and in particular to an in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids, and a preparation method and application thereof. Background Art

[0002] In recent years, studies have found that intestinal flora can regulate the body's immune system in many ways and play a vital role in the occurrence and development of diseases. However, the specific active substances of intestinal flora in fighting diseases are not yet fully understood. Intestinal epithelial cells are the main part of the body that comes into contact with various nutrients, drug molecules and pathogenic microorganisms. Therefore, in vitro culture and research of intestinal tissues are of great significance. However, in vitro research on the intestine has always been difficult, and there is currently no ideal in vitro research model for zebrafish intestinal organoids.

[0003] The intestinal flora decomposes and utilizes nutrients in the intestine to produce many metabolites, which may be key active small molecules that play an anti-disease role. Therefore, the method of constructing a co-culture of zebrafish intestinal bacterial metabolites and intestinal organoids in vitro can provide a precise research tool for the study of molecular mechanisms of intestinal diseases, disease simulation, in vitro evaluation of nutrients, drug target discovery and efficacy screening, and has good application value. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide an in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids and its preparation method and application, so as to solve the problem of lack of an ideal in vitro research model of zebrafish intestinal organoids in the prior art.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: a method for preparing an in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids is provided, comprising the following steps:

[0006] (1) Preparing zebrafish intestinal bacterial metabolites rich in short-chain fatty acids: enriching and isolating zebrafish intestinal bacteria that can produce short-chain fatty acids, inducing zebrafish intestinal bacteria that can produce short-chain fatty acids to prepare metabolites, centrifuging, filtering and inactivating the supernatant, and obtaining zebrafish intestinal bacterial metabolites rich in short-chain fatty acids;

[0007] (2) Collecting zebrafish intestinal organoids: Collect the isolated and cultured zebrafish intestinal organoids, add organoid digestion solution, and completely digest them at room temperature. Then centrifuge and remove the supernatant, resuspend them in PBS, and prepare zebrafish intestinal organoids;

[0008] (3) In vitro co-culture: The zebrafish intestinal bacterial metabolites rich in short-chain fatty acids prepared in step (1) and the zebrafish intestinal organoids prepared in step (2) are mixed, resuspended in matrix gel, inoculated on a 24-well plate, and then co-cultured in vitro for 3-5 days;

[0009] (4) Collecting the co-culture product: collecting the supernatant of the 24-well plate in step (3) to prepare an in vitro co-culture supernatant; collecting the culture medium of the 24-well plate in step (3), adding the organoid digestion solution, and completely digesting at room temperature, centrifuging and removing the supernatant, and resuspending with PBS as an in vitro co-cultured intestinal tissue product, mixing the in vitro co-culture supernatant with the in vitro co-cultured intestinal tissue product to prepare an in vitro co-culture product, and preparing an in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids.

[0010] On the basis of the above technical solution, the present invention can also be improved as follows:

[0011] In step (1), the zebrafish intestinal bacteria include at least one of Lactobacillus, Bacillus, Enterobacter, Enterococcus, Pseudomonas and Aspergillus.

[0012] Furthermore, in step (1), the zebrafish intestinal bacteria include at least one of Lactobacillus, Enterobacter, Pseudomonas and Aspergillus.

[0013] Furthermore, in step (1), the enrichment and separation culture method is: feeding zebrafish with indigestible polysaccharides, collecting zebrafish intestinal contents under sterile conditions, inoculating them on culture medium, inverting culture, and then separating and purifying, and selecting purified bacteria for 16S rDNA sequence determination and identification.

[0014] Furthermore, indigestible polysaccharides were fed for 13-15 days.

[0015] Furthermore, indigestible polysaccharides were fed for 14 days.

[0016] Further, the non-digestible polysaccharide is lignin.

[0017] Further, the culture medium is LB plate, 4wt% NaCl nutrient agar, TCBS agar, SS agar or EMB selection medium.

[0018] Further, the culture was inverted and cultured at 28-30°C for 20-25h.

[0019] Furthermore, the cells were incubated upside down at 29°C for 24 h.

[0020] Furthermore, single colonies with different morphological characteristics were picked and inoculated onto LB plates, and single colonies were streaked under sterile conditions for separation and purification.

[0021] Furthermore, in step (1), non-digestible polysaccharides are used as fermentation substrates to induce zebrafish intestinal bacteria that can produce short-chain fatty acids to prepare metabolites.

[0022] The beneficial effect of adopting the above further technical solution is: using indigestible polysaccharides as fermentation substrates can induce intestinal bacteria to produce metabolites rich in short-chain fatty acids.

[0023] Further, the culture was carried out at 28-30°C for 20-25h to complete the induction process.

[0024] Furthermore, in step (1), filtration is performed using a 0.22 μm needle filter.

[0025] Furthermore, in step (1), the mixture is inactivated by wet heat at 75-85°C for 8-12 minutes.

[0026] Furthermore, in step (1), the mixture is inactivated by wet heat at 80°C for 10 min.

[0027] Furthermore, in step (1), the zebrafish intestinal bacterial metabolites rich in short-chain fatty acids are culture supernatant of zebrafish intestinal bacteria inactivated by heat and humidity.

[0028] Furthermore, in step (2), the isolated and cultured zebrafish intestinal organoids are primary intestinal organoids.

[0029] Furthermore, in step (2), the mixture is resuspended in PBS containing 1 wt% of five antibodies, wherein the five antibodies are penicillin, streptomycin, gentamicin, amphotericin and ciprofloxacin.

[0030] Furthermore, in step (2), 1 uL of the resuspension solution is placed on a microscope to count the organoids.

[0031] Furthermore, in step (3), the volume ratio of the zebrafish intestinal bacterial metabolites rich in short-chain fatty acids obtained in step (1) to the zebrafish intestinal organoids obtained in step (2) is 1 mL: 8-12.

[0032] The beneficial effect of adopting the above further technical solution is: according to the above volume number ratio of 1mL:8-12, it is conducive to the in vitro culture and growth of intestinal organoids.

[0033] Furthermore, in step (3), the volume ratio of the zebrafish intestinal bacterial metabolites rich in short-chain fatty acids obtained in step (1) to the zebrafish intestinal organoids obtained in step (2) is 1 mL:10.

[0034] Furthermore, in step (3), the droplet inoculation density is 2-4 drops / well, 45-55 µL / drop.

[0035] Furthermore, in step (3), the droplet inoculation density is 3 drops / well, 50 µL / drop.

[0036] Furthermore, in step (3), the cells are co-cultured in vitro at 35-40°C and 4-6vt% CO2 for 3-5 days.

[0037] Furthermore, in step (3), the cells are co-cultured in vitro at 37°C and 5vt% CO2 for 3-5 days.

[0038] The present invention also provides an in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids prepared by the above method.

[0039] The present invention also provides an application of the above-mentioned zebrafish intestinal bacterial metabolites and intestinal organoid in vitro co-culture model in the study of intestinal bacterial metabolites regulating diseases.

[0040] The present invention has the following beneficial effects:

[0041] 1. The co-culture method of the present invention can simulate the in vivo 3D growth environment in vitro, which can more realistically reflect the body's functions, thereby studying the interaction process and mechanism between zebrafish intestinal bacterial metabolites and the intestine. The co-culture collected by the present invention can be used to identify drugs or reagents for treating diseases. It can provide accurate research tools for the study of molecular mechanisms of intestinal diseases, disease simulation, in vitro evaluation of nutrients, drug target discovery and drug efficacy screening, etc., and has broad application prospects.

[0042] 2. The zebrafish intestinal bacterial metabolites of the present invention are rich in short-chain fatty acids.

[0043] 3. The co-cultivation method of the present invention can improve the efficiency of in vitro co-cultivation of zebrafish intestinal bacterial metabolites and intestinal organoids, and can meet the needs of further research on different metabolites of different bacteria.

[0044] 4. The invented co-culture method provides a preparation method with high success rate, low cost and easy operation through a series of optimization steps, which provides an important tool for the in vitro culture and research of zebrafish intestinal bacterial metabolites and intestinal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a schematic diagram of the enrichment of intestinal bacteria rich in short-chain fatty acids in Example 1;

[0046] Figure 2 This is a schematic diagram of the isolation and culture of zebrafish intestinal bacteria in Example 1;

[0047] Figure 3 This is a SEM image of the in vitro co-culture model of Example 1;

[0048] Figure 4 This is the sequencing map of the in vitro co-culture product of Example 1;

[0049] Figure 5 The in vitro co-culture model of Example 1 regulates the expression level of intestinal epithelial tight junction ZO-1;

[0050] Figure 6 The in vitro co-culture model of Example 1 regulates the expression level of Occludin in intestinal epithelial tight junctions;

[0051] Figure 7 This is a 40X microscope image of the in vitro co-culture model of Comparative Example 1. DETAILED DESCRIPTION

[0052] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0053] Embodiment 1:

[0054] An in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids, the preparation method of which comprises the following steps:

[0055] (1) Preparation of zebrafish intestinal bacterial metabolites rich in short-chain fatty acids: Zebrafish intestinal bacteria (Lactobacillus, Enterobacter, Pseudomonas, and Aspergillus) that can produce short-chain fatty acids were enriched and isolated and cultured (see Figure 2 ), the specific method is to feed zebrafish with indigestible polysaccharide lignin for 14 days, collect zebrafish intestinal contents under sterile conditions, and inoculate them on identification culture LB plates, invert and culture them at 29°C for 24 hours, pick out single colonies with different colony morphological characteristics, inoculate them on LB plates, pick out single colonies under sterile conditions and streak them multiple times for separation and purification, and select purified bacteria for 16S rDNA sequence determination and identification based on the colony morphology, color, size, etc. of zebrafish intestinal strains on different plates; then use indigestible polysaccharide lignin as a fermentation substrate, culture at 29°C for 24 hours, induce zebrafish intestinal bacteria that can produce short-chain fatty acids to prepare metabolites, centrifuge, take the supernatant, filter it with a 0.22μm needle filter, inactivate it with wet heat at 80°C for 10 minutes, and store it at 4°C to obtain zebrafish intestinal bacterial metabolites rich in short-chain fatty acids (wet heat inactivated zebrafish intestinal bacterial culture supernatant);

[0056] (2) Collecting zebrafish intestinal organoids: Collect the isolated and cultured primary intestinal organoids of zebrafish, add organoid digestion solution, and completely digest them at room temperature, then centrifuge and remove the supernatant, resuspend them in PBS containing 1wt% of five antibodies (penicillin, streptomycin, gentamicin, amphotericin, and ciprofloxacin) to prepare zebrafish intestinal organoids, and place 1μL of the resuspension on a microscope to count the organoids;

[0057] (3) In vitro co-culture: The zebrafish intestinal bacterial metabolites rich in short-chain fatty acids prepared in step (1) and the zebrafish intestinal organoids prepared in step (2) were mixed at a volume ratio of 1 mL:10, resuspended in matrix gel, and inoculated on a 24-well plate at a density of 3 drops / well, 50 µL / drop, and then co-cultured in vitro at 37°C and 5vt% CO2 for 4 days;

[0058] (4) Collecting the co-culture product: collecting the supernatant of the 24-well plate in step (3) to prepare an in vitro co-culture supernatant; collecting the culture medium of the 24-well plate in step (3), adding the organoid digestion solution, and completely digesting at room temperature, centrifuging and removing the supernatant, and resuspending with PBS as an in vitro co-cultured intestinal tissue product, mixing the in vitro co-culture supernatant with the in vitro co-cultured intestinal tissue product to prepare an in vitro co-culture product, and preparing an in vitro co-culture model.

[0059] Embodiment 2:

[0060] An in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids, the preparation method of which comprises the following steps:

[0061] (1) Preparation of zebrafish intestinal bacterial metabolites rich in short-chain fatty acids: The zebrafish intestinal bacteria (Lactobacillus, Enterobacter, Pseudomonas and Aspergillus) that can produce short-chain fatty acids were enriched and isolated and cultured. The specific method was to feed zebrafish with indigestible polysaccharides for 13 days. Under sterile conditions, the zebrafish intestinal contents were collected and inoculated on 4wt% NaCl nutrient agar. The agar was inverted and cultured at 28°C for 25 hours. Single colonies with different colony morphological characteristics were picked and inoculated on LB plates. Single colonies were picked and streaked multiple times under sterile conditions for separation and purification. According to the colony morphology, color, size, etc. of zebrafish intestinal strains on different plates, the purified bacteria were selected for 16S rDNA sequence determination and identification; then, non-digestible polysaccharides were used as fermentation substrates, cultured at 28°C for 25 hours, and zebrafish intestinal bacteria that can produce short-chain fatty acids were induced to prepare metabolites, centrifuged, and the supernatant was taken and filtered with a 0.22μm needle filter, inactivated by wet heat at 75°C for 12 minutes, and stored at 4°C to obtain zebrafish intestinal bacterial metabolites rich in short-chain fatty acids (wet heat inactivated zebrafish intestinal bacterial culture supernatant);

[0062] (2) Collecting zebrafish intestinal organoids: Collect the isolated and cultured primary intestinal organoids of zebrafish, add organoid digestion solution, and completely digest them at room temperature, then centrifuge and remove the supernatant, resuspend them in PBS containing 1wt% of five antibodies (penicillin, streptomycin, gentamicin, amphotericin, and ciprofloxacin) to prepare zebrafish intestinal organoids, and place 1μL of the resuspension on a microscope to count the organoids;

[0063] (3) In vitro co-culture: The zebrafish intestinal bacterial metabolites rich in short-chain fatty acids prepared in step (1) and the zebrafish intestinal organoids prepared in step (2) were mixed at a volume ratio of 1 mL:8, resuspended in matrix gel, and inoculated on a 24-well plate at a density of 2 drops / well, 55 µL / drop, and then co-cultured in vitro at 35°C and 6vt% CO2 for 3 days;

[0064] (4) Collecting the co-culture product: collecting the supernatant of the 24-well plate in step (3) to prepare an in vitro co-culture supernatant; collecting the culture medium of the 24-well plate in step (3), adding the organoid digestion solution, and completely digesting at room temperature, centrifuging and removing the supernatant, and resuspending with PBS as an in vitro co-cultured intestinal tissue product, mixing the in vitro co-culture supernatant with the in vitro co-cultured intestinal tissue product to prepare an in vitro co-culture product, and preparing an in vitro co-culture model.

[0065] Embodiment 3:

[0066] An in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids, the preparation method of which comprises the following steps:

[0067] (1) Preparation of zebrafish intestinal bacterial metabolites rich in short-chain fatty acids: The zebrafish intestinal bacteria (Lactobacillus, Enterobacter, Pseudomonas and Aspergillus) that can produce short-chain fatty acids were enriched and isolated and cultured. The specific method was to feed zebrafish with indigestible polysaccharides for 15 days. Under sterile conditions, the zebrafish intestinal contents were collected and inoculated on TCBS agar. The agar was inverted and cultured at 30°C for 20 hours. Single colonies with different colony morphological characteristics were picked and inoculated on LB plates. Single colonies were picked and streaked multiple times under sterile conditions for separation and purification. According to the colony morphology, color, size, etc. of zebrafish intestinal strains on different plates, the purified bacteria were selected for 16S rDNA sequence determination and identification; then, non-digestible polysaccharides were used as fermentation substrates, cultured at 30°C for 20 hours, and zebrafish intestinal bacteria that can produce short-chain fatty acids were induced to prepare metabolites, centrifuged, and the supernatant was taken and filtered with a 0.22μm needle filter, inactivated by wet heat at 85°C for 8 minutes, and stored at 4°C to obtain zebrafish intestinal bacterial metabolites rich in short-chain fatty acids (wet heat inactivated zebrafish intestinal bacterial culture supernatant);

[0068] (2) Collecting zebrafish intestinal organoids: Collect the isolated and cultured primary intestinal organoids of zebrafish, add organoid digestion solution, and completely digest them at room temperature, then centrifuge and remove the supernatant, resuspend them in PBS containing 1wt% of five antibodies (penicillin, streptomycin, gentamicin, amphotericin, and ciprofloxacin) to prepare zebrafish intestinal organoids, and place 1μL of the resuspension on a microscope to count the organoids;

[0069] (3) In vitro co-culture: The zebrafish intestinal bacterial metabolites rich in short-chain fatty acids prepared in step (1) and the zebrafish intestinal organoids prepared in step (2) were mixed at a volume ratio of 1 mL:12, resuspended in matrix gel, and inoculated on a 24-well plate at a density of 4 drops / well, 45 µL / drop, and then co-cultured in vitro at 40°C and 4vt% CO2 for 5 days;

[0070] (4) Collecting the co-culture product: collecting the supernatant of the 24-well plate in step (3) to prepare an in vitro co-culture supernatant; collecting the culture medium of the 24-well plate in step (3), adding the organoid digestion solution, and completely digesting at room temperature, centrifuging and removing the supernatant, and resuspending with PBS as an in vitro co-cultured intestinal tissue product, mixing the in vitro co-culture supernatant with the in vitro co-cultured intestinal tissue product to prepare an in vitro co-culture product, and preparing an in vitro co-culture model.

[0071] Comparative Example 1:

[0072] An in vitro co-culture model of zebrafish intestinal bacteria and intestinal organoids, the preparation method of which comprises the following steps:

[0073] The intestinal bacteria obtained by enrichment and separation and culture in step (1) were used to carry out steps (2) to (4). As a result, the zebrafish intestinal organoids were contaminated by the isolated zebrafish intestinal bacteria and could not be cultured and survived. Figure 7 )

[0074] Test example

[0075] 1. The zebrafish intestinal bacteria that can produce short-chain fatty acids and are enriched and isolated and cultured in Example 1 are subjected to metagenomic sequencing, and the intestinal bacteria that are not enriched and isolated and cultured are used as the control group to obtain intestinal bacteria rich in short-chain fatty acids. Figure 1 .

[0076] Depend on Figure 1 It can be seen that the zebrafish intestinal bacteria that can produce short-chain fatty acids and are enriched and isolated and cultured in the present application are Lactobacillus, Enterobacter, Aspergillus and Pseudomonas.

[0077] 2. Example 1: The process of isolating and culturing zebrafish intestinal bacteria that can produce short-chain fatty acids. The results are shown in Figure 2 .

[0078] Depend on Figure 2 It can be seen that the required intestinal bacteria (Lactobacillus, Aspergillus, Pseudomonas, Enterobacter) can be isolated using the identification culture medium.

[0079] 3. The in vitro co-culture model of Example 1 was observed, and the results are shown in Figure 3 .

[0080] Depend on Figure 3 It can be seen that the in vitro co-culture model can successfully culture in vitro until intestinal organoids sprout.

[0081] 4. The in vitro co-cultured products prepared in Example 1 were subjected to metabolomics sequencing, and the metabolites of zebrafish intestinal bacteria rich in short-chain fatty acids that were not co-cultured in Example 1 were used as the control group. The results are shown in Figure 4 .

[0082] Depend on Figure 4 It can be seen that metabolomics sequencing can obtain the main components of zebrafish intestinal bacterial metabolites rich in short-chain fatty acids.

[0083] 5. The in vitro co-culture model of Example 1 was subjected to Western blot to verify the expression levels of zebrafish intestinal epithelial tight junction proteins ZO-1 and Occludin, and the zebrafish intestinal organoids in step (2) were used as the control group. The results are shown in Figure 5-6 .

[0084] Depend on Figure 5-6 It can be seen that the in vitro co-culture model of Example 1 was verified by Western blot that the zebrafish intestinal bacterial metabolites can regulate the expression levels of intestinal epithelial tight junction proteins ZO-1 and Occludin.

[0085] 6. The results of comparative example 1 were tested under a 40X microscope. Figure 7 .

[0086] Depend on Figure 7 It can be seen that zebrafish intestinal organoids are contaminated by isolated zebrafish intestinal bacteria and cannot be cultured and survive.

[0087] In summary, the preparation of the in vitro co-culture model of the present invention has good application prospects.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids, characterized in that: The following steps are involved: (1) Preparing zebrafish intestinal bacterial metabolites rich in short-chain fatty acids: enriching and isolating zebrafish intestinal bacteria that can produce short-chain fatty acids, inducing zebrafish intestinal bacteria that can produce short-chain fatty acids to prepare metabolites, centrifuging, filtering and inactivating the supernatant, and obtaining zebrafish intestinal bacterial metabolites rich in short-chain fatty acids; (2) Collecting zebrafish intestinal organoids: Collect the isolated and cultured zebrafish intestinal organoids, add organoid digestion solution, and completely digest them at room temperature. Then centrifuge and remove the supernatant, resuspend them in PBS, and prepare zebrafish intestinal organoids; (3) In vitro co-culture: The zebrafish intestinal bacterial metabolites rich in short-chain fatty acids prepared in step (1) and the zebrafish intestinal organoids prepared in step (2) are mixed, resuspended in matrix gel, inoculated on a 24-well plate, and then co-cultured in vitro for 3-5 days; (4) Collecting the co-culture product: collecting the supernatant of the 24-well plate in step (3) to prepare an in vitro co-culture supernatant; collecting the culture medium of the 24-well plate in step (3), adding the organoid digestion solution, performing complete digestion at room temperature, centrifuging and removing the supernatant, resuspending with PBS, and using the in vitro co-cultured intestinal tissue product as the in vitro co-cultured intestinal tissue product; mixing the in vitro co-culture supernatant with the in vitro co-cultured intestinal tissue product to prepare an in vitro co-culture product, and preparing an in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids; In step (1), the zebrafish intestinal bacteria include at least one of Lactobacillus, Enterobacter, Pseudomonas and Aspergillus; In step (1), the enrichment and separation culture method is: feeding zebrafish with indigestible polysaccharides, collecting zebrafish intestinal contents under sterile conditions, inoculating them on a culture medium, inverting the culture medium, and then separating and purifying them, selecting purified bacteria for 16S rDNA sequence determination and identification; the indigestible polysaccharide is lignin; In step (1), indigestible polysaccharides are used as fermentation substrates to induce zebrafish intestinal bacteria that can produce short-chain fatty acids to prepare metabolites; the indigestible polysaccharides are lignin.

2. The method for preparing an in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids according to claim 1, characterized in that: In step (1), the zebrafish intestinal bacterial metabolites rich in short-chain fatty acids are culture supernatants of zebrafish intestinal bacteria inactivated by heat and humidity.

3. The method for preparing an in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids according to claim 1, characterized in that: In step (2), the isolated and cultured zebrafish intestinal organoids are primary intestinal organoids.

4. The method for preparing an in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids according to claim 1, characterized in that: In step (3), the volume ratio of the zebrafish intestinal bacterial metabolites rich in short-chain fatty acids obtained in step (1) to the zebrafish intestinal organoids obtained in step (2) is 1 mL: 8-12.

5. The method for preparing an in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids according to claim 1, characterized in that: In step (3), the cells are co-cultured in vitro at 35-40°C and 4-6vt% CO2 for 3-5 days.

6. An in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids, characterized in that: The method is prepared by the preparation method according to any one of claims 1 to 5.

7. Application of the in vitro co-culture model of zebrafish intestinal bacterial metabolites and intestinal organoids as described in claim 6 in the study of intestinal bacterial metabolites regulating diseases.

Images