Method for establishing helicobacter pylori infected mouse model

Abstract

The invention belongs to the technical field of microbiology and experimental zoology, and particularly relates to a construction method of a helicobacter pylori infected mouse model, which is suitable for helicobacter pylori pathogenic mechanism research, vaccine development and drug screening. The method disclosed by the invention is simple and convenient to operate, high in infection rate (up to more than 80%), good in repeatability and relatively high in application value.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, and specifically relates to a method for establishing a mouse model of Helicobacter pylori infection. Background Technology

[0002] Helicobacter pylori (Hp) is widely recognized as a major causative agent of gastritis, peptic ulcers, and gastric cancer, and humans are the only susceptible host for Hp infection. In developed countries, the Hp infection rate is approximately 50%, while in developing countries it exceeds 60%. Therefore, researchers both domestically and internationally are establishing animal models of Hp infection to study its pathogenic mechanisms, specific prevention and treatment methods, and the efficacy of certain drugs or probiotics.

[0003] Existing technologies have been optimized for establishing *Helicobacter pylori* (Hp) models, mainly including the following categories: 1. Screening and optimizing strains: Using specific culture media to screen for *Hp* strains with low pathogenicity, natural transformation ability, and the ability to colonize the mouse gastric mucosa without host adaptation, thereby improving the stability and infection rate of the model; 2. Animal infection optimization: Utilizing optimized gavage infection methods, such as reducing the number of gavages and shortening the modeling time, to reduce mouse mortality and complication rates, while improving the success rate of modeling; 3. Selection of novel animal models: Based on rodents (such as Balb / c mice), and considering their body size characteristics, exploring modified models that can be examined endoscopically or observed for extended periods, in order to study the dynamic process, pathogenic mechanism, and treatment methods of *Hp* infection.

[0004] ZL01105112.4 reports a method for screening and optimizing strains of *Helicobacter pylori* (Hp) using a selective medium. The main components and concentrations of this medium are: vancomycin 5–12 mg / L, amphotericin B 2–4 mg / L, nalidixinone acid 10–20 mg / L, polymyxin B 2000–2800 IU / L, and 2,3,5-triphenyltetrazolium chloride 35–45 mg / L. However, while this antibiotic combination can inhibit the growth of other bacteria, long-term use can easily induce drug resistance in *Helicobacter pylori*, leading to a decrease in isolation efficiency. Furthermore, high concentrations of some antibacterial components can inhibit the growth of *Hp* itself. Vancomycin works by inhibiting bacterial cell wall synthesis. Although *Hp* is a Gram-negative bacterium, high concentrations of vancomycin and polymyxin B can penetrate the integrity of *Hp* cells, leading to decreased *Hp* strain viability and metabolic disorders. Consequently, the selected strains are prone to failure during subsequent colonization of mouse gastric mucosa due to insufficient activity, affecting their viability and colonization ability.

[0005] Therefore, there is an urgent need in this field to develop a modeling method that is easy to operate, has a high infection rate, and is highly reproducible, so as to provide a foundation for studying the Hp infection mechanism, vaccine development, and drug screening. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an improved method for establishing a mouse model of Helicobacter pylori infection, which has the advantages of high infection rate, good stability, and simple operation.

[0007] To address the problems in the existing technology, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for constructing an animal model of Helicobacter pylori infection, characterized by comprising the following steps:

[0009] 1. Screening for Helicobacter pylori with high viability and purity

[0010] To screen for highly viable and pure Helicobacter pylori, a culture medium was designed that meets the nutritional requirements of Helicobacter pylori and inhibits the growth of other bacteria. The formula is as follows:

[0011]

[0012] Weigh each ingredient according to the formula, add ultrapure water to dissolve, adjust the pH, autoclave; cool, add linezolid, voriconazole and imipenem that have been filtered and sterilized, mix well and pour into plates, and store in a sealed container at 4°C.

[0013] The Helicobacter pylori strain was revived to avoid a decrease in strain viability; the revived bacterial solution was taken with a sterile inoculation loop, streaked on a modified medium plate, and incubated in a microaerophilic environment at 37°C for 72 hours to isolate the HP-M1 strain.

[0014] 2. Animal infection

[0015] Experimental animals: SPF grade C57BL / 6 mice (4 weeks old, weighing 11-15g, half male and half female), adapted to the barrier environment for 7 days, with free access to food and water (normal diet, antibiotic-free).

[0016] Pretreatment (targeted improvement of the colonization microenvironment):

[0017] (1) Gastric acid neutralization pretreatment: 2 hours before infection, each mouse was given a sodium bicarbonate solution by gavage to reduce the killing effect of gastric acid on SS1 strain.

[0018] (2) Antibiotic pretreatment: Two days before infection, each mouse was given 0.5 mL of antibiotic solution by gavage to clear the original dominant bacteria in the mouse stomach.

[0019] Gradual concentration staged infection: Bacterial solution was administered once on days 1, 3, 5, and 8, for a total of 4 times; feeding resumed 4 hours after gavage, and the animals were kept alive until day 30.

[0020] Post-infection care: Feed normally for 30 days, changing drinking water and feed daily to avoid cross-contamination, and do not use any antibiotics or feed.

[0021] According to an embodiment of the present invention, the culture medium has the following formulation:

[0022] Element content Brain and heart infusion culture medium 37g / L Yeast extract 5g / L glucose 1g / L NaCl 5g / L <![CDATA[K2HPO4]]> 2.5g / L Linezolid 7mg / L Voriconazole 3mg / L Imipenem 4mg / L

[0023] According to an embodiment of the present invention, in step (1), the pH is adjusted to 7.2-7.4.

[0024] According to an embodiment of the present invention, in step (2), each mouse is gavaged with 0.5 mL of 5% sodium bicarbonate solution to raise the pH in the stomach from 1.5-2.0 to 3.0-3.5.

[0025] According to an embodiment of the present invention, the antibiotic solution in step (2) is azithromycin 1.2 mg / mL, ampicillin 2.5 mg / mL, and gentamicin 0.6 mg / mL.

[0026] According to an embodiment of the present invention, the gradient concentration staged infection step in step (2) is as follows:

[0027] (1) Day 1 (adaptation period): 0.2 mL 5×10 8 CFU / mL bacterial culture allows the strain to initially adapt to the gastric microenvironment of mice, reducing stress response;

[0028] (2) Day 3 (transplanting period): 0.2 mL 1×10 9 CFU / mL bacterial solution, high concentration of bacterial strain to enhance binding with gastric mucosal epithelial cells and establish initial colonization;

[0029] (3) Days 5 / 8 (consolidation period): 0.2 mL 1×10 9 CFU / mL bacterial solution was added to increase the number of bacterial strains and ensure the formation of a stable colony in the gastric mucosa.

[0030] Secondly, the present invention provides the application of the constructed animal model in screening drugs against Helicobacter pylori infection or in experimental research on Helicobacter pylori infection.

[0031] Validation Model

[0032] On day 30 post-infection, mice were sacrificed, and half of each stomach tissue (antrum and body) was harvested for four-dimensional validation to comprehensively evaluate HP-M1 strain colonization and pathological damage:

[0033] (1) Urease test: Under aseptic conditions, take the stomach tissue of the mouse, scrape and dip the tissue surface of the pyloric antrum with the detection cotton swab of the Helicobacter pylori urease rapid test strip, place it on the indicator of the test strip, and observe the color change. If it turns red, it is positive.

[0034] (2) Pathological examination: The gastric tissue paraffin sections were stained with MGG and observed under a microscope to be positive for spiral Helicobacter pylori cells in the lamina propria of the gastric mucosa.

[0035] A positive result for both urease and pathological tests indicates a successful infection.

[0036] The beneficial effects of this invention are:

[0037] This invention establishes a complete standardized operating procedure (SOP) from strain screening, animal pretreatment, infection procedures to result determination, facilitating reproducibility and ensuring the consistency and reliability of model construction. By optimizing the strain screening and infection protocol, this model achieves a stable and reproducible high infection success rate, providing a reliable animal model for in vivo screening of anti-Hp drugs and vaccines, and accurately evaluating the therapeutic effects of drugs / vaccines. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of typical colony morphology of Helicobacter pylori on selective culture medium in Example 1.

[0039] Figure 2 This is a comparison chart of HP colonization (urease and pathological double positive) results between the triple therapy group and the control group.

[0040] Figure 3 This is a schematic diagram of a pathological section of mouse gastric tissue infected with Helicobacter pylori (MGG staining).

[0041] Figure 4 This is a statistical chart showing the positive rates of HP colonization urease and pathological colonization in mouse gastric tissue at the infection time point (30 days). Detailed Implementation

[0042] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. After reading this invention, any modifications of the present invention in various equivalent forms, or direct or indirect applications in other related technical fields, made by those skilled in the art, shall fall within the scope defined by the appended claims.

[0043] Example 1: Screening of HP-M1 strain with high colonization ability

[0044] Ten clinical strains of Helicobacter pylori isolated from patients with clinical Helicobacter pylori infection were cultured on HP selective medium. The main components and contents of the selective medium are shown in the table below:

[0045] Element content Brain and heart infusion culture medium 37g / L Yeast extract 5g / L glucose 1g / L NaCl 5g / L <![CDATA[K2HPO4]]> 2.5g / L Linezolid 7mg / L Voriconazole 3mg / L Imipenem 4mg / L

[0046] Culture medium preparation method: Weigh each component according to the formula, add ultrapure solution to dissolve, adjust pH to 7.2-7.4, autoclave at 121℃ for 15 min; when cooled to 50℃, add filtered sterilized linezolid, voriconazole, and imipenem, mix well, pour into plates, and store in a sealed container at 4℃ (use within 14 days).

[0047] Helicobacter pylori strains were removed from -80℃ cryovials and rapidly revived in a 37℃ water bath (1-2 min) to prevent loss of bacterial viability. 50 μL of the revived bacterial solution was streaked onto a modified culture medium plate using a sterile inoculation loop and incubated at 37℃ in a microaerophilic environment (5% O2, 10% CO2, 85% N2) for 72 h. The HP-M1 strain (translucent, pinhead-sized, with neat edges, and corresponding appendages) was isolated. Figure 1 Microscopic examination confirmed that the strain had a spiral proportion of ≥95%, was actively motile (significant flagellar movement), and was free from contamination by other bacteria (Gram staining showed a purity of ≥99%).

[0048] Example 2: Construction of the HP-M1 infection model

[0049] Ten 4-week-old male C57BL / 6 mice, weighing 11±2g, were acclimatized for one week, and a suspension of Helicobacter pylori HP-M1 was prepared. Two days before infection, each mouse was administered 0.5mL of antibiotic solution (azithromycin 1.2mg / mL, ampicillin 2.5mg / mL, gentamicin 0.6mg / mL) by gavage to eliminate the dominant gut flora. Two hours before infection, each mouse was administered 0.5mL of 5% sodium bicarbonate solution by gavage to raise the gastric pH from 1.5-2.0 to 3.0-3.5, reducing the bactericidal effect of gastric acid on the HP-M1 strain.

[0050] Mice were fasted for 24 hours, and then administered HP-M1 bacterial suspension by gavage at a dose of 0.5 mL / mice / time (bacterial density adjusted to 1×10⁻⁶). 8 CFU / mL),

[0051] Gradient concentration staged infection:

[0052] (1) Day 1 (adaptation period): 0.2 mL 5×10 8 CFU / mL bacterial culture allows the strain to initially adapt to the gastric microenvironment of mice, reducing stress response;

[0053] (2) Day 3 (transplanting period): 0.2 mL 1×10 9 CFU / mL bacterial solution, high concentration of bacterial strain to enhance binding with gastric mucosal epithelial cells and establish initial colonization;

[0054] (3) Days 5 / 8 (consolidation period): 0.2 mL 1×10 9 CFU / mL bacterial solution was added to increase the number of bacterial strains and ensure the formation of a stable colony in the gastric mucosa.

[0055] Post-infection care: Feed normally for 30 days, changing drinking water and feed daily to avoid cross-contamination, and do not use any antibiotics or feed.

[0056] Example 1 (Modeling Evaluation)

[0057] Urease test: Under aseptic conditions, take mouse stomach tissue, scrape and dab the tissue surface of the pyloric antrum with a cotton swab from the Helicobacter pylori urease rapid test strip, place it on the indicator of the test strip, and observe the color change.

[0058] The results are as follows Figure 2 As shown, the positive rate of urease reaction in the sampled mice was 90%.

[0059] Example 2 (Pathological Examination)

[0060] Pathological examination: Gastric tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and serially sectioned to a thickness of 6 μm. MGG staining was performed, and the tissue was observed under a microscope.

[0061] MGG staining revealed that Helicobacter pylori colonized the superficial layer, gastric pits, and glands. Figure 3 The middle arrow points to Helicobacter pylori (MGG staining, magnification, ×400).

[0062] Based on gastric tissue pathological examination, Helicobacter pylori colonization was observed in 80% of the randomly selected C57BL / 6 mice. Positive urease and pathological tests indicated successful infection.

[0063] Example 3: Efficacy Evaluation

[0064] Sixty 4-week-old male C57BL / 6 mice, weighing 11±2g, were acclimatized for one week. Experimental groups included: a blank control group (uninfected), a model control group (infected but untreated), and a triple treatment group. Each group consisted of 20 mice. Mice were fasted for 24 hours prior to inoculation, and then administered HP-M1 bacterial suspension (bacterial density adjusted to 1×10⁻⁶) via gavage at a dose of 0.5 mL / mouse / time. 8 The mice were administered the drug (CFU / mL) once every other day for a total of 4 times. Eight hours after administration, the mice were allowed to resume feeding and were fed for another 30 days. Three mice from the model control group were randomly selected, and their gastric pyloric end tissue was taken for HP urease detection, with a positive rate of 100%.

[0065] After successful infection assessment in mice, a triple therapy regimen of omeprazole-clarithromycin-amoxicillin was administered for 7 consecutive days: omeprazole (3.5 mg / kg), clarithromycin (87.5 mg / kg), and amoxicillin (175 mg / kg). Four weeks after the end of treatment, stomach tissue from mice was collected for urease detection and pathological examination, and the urease positivity rate was calculated.

[0066] The results are as follows Figure 4 As shown, the urease positivity rate in the model group mice was higher than 80%, indicating that the mouse infection model was successfully established. The urease positivity rate in the triple-drug group mice was 20%, which was significantly lower than that in the model group, suggesting that the triple-drug regimen can effectively clear *Helicobacter pylori* infection in the mouse stomach. The mouse *Helicobacter pylori* infection model constructed in this invention can be used for drug efficacy evaluation.

[0067] Comparative Example 1

[0068] Prepare the patented ZL01105112.4 culture medium and the culture medium of the present invention, and inoculate HP-M1, ATCC43504 and SS1 strains onto the culture medium of the present invention and the ZL01105112.4 culture medium, respectively. According to the model construction method and index determination of the present invention, the infection success rate of each group after 8 weeks is compared (see Table 1 below).

[0069] Table 1

[0070]

[0071] The results are shown in Table 1. All four experimental groups showed a certain success rate in infection after 8 weeks. Among them, the experimental group using this invention had the highest infection success rate; and among the three groups using ZL01105112.4 medium, the HP-M1 strain had the highest infection success rate. Therefore, it is clear that the medium and screening strain HP-M1 of this invention have significant advantages for model construction and can improve the success rate of model construction using traditional mediums and strains.

Claims

1. A method for constructing an animal model of Helicobacter pylori infection, characterized in that, Includes the following steps: (1) Screening for Helicobacter pylori with high activity and purity The culture medium that meets the nutritional needs of Helicobacter pylori and inhibits the growth of other bacteria has the following formula: Weigh each ingredient according to the formula, add ultrapure water to dissolve, adjust the pH, autoclave; cool, add linezolid, voriconazole, and imipenem (filtered and sterilized), mix well, pour into plates, and store in a sealed container at 4°C. The Helicobacter pylori strain was revived, and the revived bacterial solution was taken with a sterile inoculation loop, streaked on a modified medium plate, and cultured in a microaerophilic environment at 37°C for 72 hours to isolate the HP-M1 strain. (2) Animal infection a. Preprocessing Gastric acid neutralization pretreatment: 2 hours before infection, each mouse was administered sodium bicarbonate solution by gavage; Antibiotic pretreatment: Two days before infection, each mouse was administered 0.5 mL of antibiotic solution by gavage; b. Gradient concentration staged infection The bacterial solution was administered once on days 1, 3, 5, and 8, for a total of 4 times. (3) Feeding after infection Four hours after gavage with the bacterial solution, the animals resumed eating and continued to be fed.

2. The method for constructing an animal model according to claim 1, characterized in that: The culture medium has the following formula:

3. The method for constructing an animal model according to claim 1, characterized in that: In step (1), adjust the pH to 7.2-7.

4.

4. The method for constructing an animal model according to claim 1, characterized in that: In step (2), each mouse was given 0.5 mL of 5% sodium bicarbonate solution by gavage to raise the pH in the stomach from 1.5-2.0 to 3.0-3.

5.

5. The method for constructing an animal model according to claim 1, characterized in that: In step (2), the antibiotic solution consists of azithromycin 1.2 mg / mL, ampicillin 2.5 mg / mL, and gentamicin 0.6 mg / mL.

6. The method for constructing an animal model according to claim 1, characterized in that: In step (2), during the staged infection with gradient concentrations, 0.2 mL of 5×10⁻⁶ gavage was administered on day 1. 8 CFU / mL bacterial culture, 0.2mL of 1×10⁻⁶ bacteria was administered by gavage on day 3. 9 CFU / mL bacterial culture, 0.2mL of 1×10⁻⁶ bacteria administered by gavage on days 5 and 8. 9 CFU / mL bacterial culture 7. The method for constructing an animal model according to claim 1, characterized in that: The feeding time in step (3) is 30 days.

8. The application of the animal model constructed by the method for constructing an animal model according to any one of claims 1 to 7 in screening drugs against Helicobacter pylori infection or in experimental research on Helicobacter pylori infection.

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