Preparation method of chronic endometritis rat model

Abstract

The invention discloses a method for constructing a chronic endometritis rat model based on Hungata (preservation number: DSM 13479), and belongs to the technical field of animal models. The bacterial strain is subjected to anaerobic culture to prepare a bacterial solution, 150 mu L vagina perfusion is performed twice every 3 days for 3 weeks, rat endometrial inflammation reaction is induced, and endometrial atrophy and endometrial inflammation cell infiltration are taken as pathological characteristics. According to the invention, the problems of high infection, high death rate and the like caused by traditional injection of lipopolysaccharide or pathogenic bacteria through the uterus horn are avoided, and an efficient experimental tool is provided for mechanism research and drug development of chronic endometritis.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and specifically to a method for preparing a rat model of chronic endometritis. Background Art

[0002] Chronic endometritis (CE) is a significant pathological factor leading to infertility in women of reproductive age. The establishment of animal models of CE plays a crucial role in studying its pathological mechanisms and exploring treatment options. The principle behind establishing CE animal models is to simulate certain key characteristics of CE, such as thinning of the endometrium, plasma cell infiltration in the stroma, and increased inflammatory factors.

[0003] Currently, the main methods used to construct animal models of cerebrovascular accidents (CE) are lipopolysaccharide (LPS) induction and pathogenic bacteria induction. The former constructs a model based on the outcome of the disease rather than its cause, which does not reflect the physiological state of human CE; the latter requires intrauterine injection of pathogenic bacteria to observe CE-like symptoms, but carries a high risk of postoperative infection and mortality. Currently, no research has found evidence to support this view. Hungatella hathewayi It can lead to CE (Certainty Emissions). Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to provide a method based on... Hungatella hathewayi Methods for inducing a rat model of chronic endometritis. This was achieved by instilling vaginal fluid into the rats. Hungatella hathewayi It can induce CE-related pathological phenotypes.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0006] This invention discloses Hungatella hathewayi Application of the strain in constructing a rat model of chronic endometritis, the Hungatella hathewayi The strain has the accession number DSM 13479.

[0007] Furthermore, the animal model is a rat model.

[0008] This invention also discloses a method for constructing an animal model of chronic endometritis, characterized by comprising the following steps: (a) The collection number DSM 13479 Hungatella hathewayi Cultivate under anaerobic conditions; (b) Adjust the bacterial concentration to 1×10⁻⁶ 9 CFU / mL; (c) Instill 150 μL of the bacterial solution obtained in step (b) into the vagina of the experimental animals.

[0009] Furthermore, the experimental animal was a female SD rat.

[0010] Further, the culture in step (a) uses a liquid culture medium comprising: 5.0 g / L casein peptone, 5.0 g / L vesicular enzyme digest, 10.0 g / L yeast extract, 0.0005 g / L sodium resazurin, and 0.5 g / L L-cysteine ​​hydrochloride.

[0011] The present invention also discloses an animal model of chronic endometritis, characterized in that it is constructed by any of the methods described above.

[0012] This invention also discloses the application of the above-described animal model in the study of the pathogenesis of chronic endometritis.

[0013] The present invention also discloses the application of the above-described animal model in screening drugs for the treatment of chronic endometritis.

[0014] The present invention also discloses the application of the above-described animal model in evaluating the effectiveness of drugs for treating chronic endometritis.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0016] This invention is achieved through Hungatella hathewayi A rat model of cystic urethritis (CE) was successfully established via vaginal instillation. This model can simulate the pathophysiological characteristics of CE, avoiding the inconsistency between the model and the physiological state caused by reliance on lipopolysaccharide induction, and also avoiding the risks of high postoperative infection and mortality rates in animals due to transabdominal intrauterine instillation of pathogenic bacteria. This invention establishes a CE model based on reproductive tract microbial dysbiosis, simulating the pathophysiological state of CE by regulating the reproductive tract flora, providing a new experimental tool for studying the pathogenesis and treatment of CE. Attached Figure Description

[0017] Figure 1 vaginal irrigation Hungatella hathewayi Histopathological changes of rat endometrial tissue under light microscopy.

[0018] Figure 2 Immunofluorescence observation Hungatella hathewayi Inducing chronic endometritis in rats.

[0019] Figure 3 Hungatella hathewayi Promotes the expression of cytokine proteins in rats with chronic endometrial inflammation. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0021] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0022] Example 1 Hungatella hathewayi It has the effect of inducing endometritis.

[0023] Discover Hungatella hathewayi It has the effect of inducing the CE phenotype. The inventors will Hungatella hathewayi When the bacterial culture was administered vaginally to rats, it was found to induce endometrial atrophy, endometrial inflammatory cell infiltration, and increased inflammatory factors. Therefore, the strain can be used to construct a CE animal model.

[0024] This invention uses Hungatella hathewayi Collection number DSM 13479. Hungatella hathewayi The cultivation method is as follows: Hungatella hathewayi Inoculate onto a plate solid culture medium and culture at 30°C for 3 days under anaerobic conditions. After 3 days, pick single clones and inoculate them into liquid culture medium, where they grow at 30°C for 2-3 days under anaerobic conditions.

[0025] The plate solid culture medium consists of 5.0 g / L casein peptone, 5.0 g / L nitroglycerin digest, 10.0 g / L yeast extract, 0.0005 g / L sodium resazurin, 0.5 g / L L-cysteine ​​hydrochloride (components: 0.01 g / L CaCl2·2H2O, 0.02 g / L MgSO4·7H2O, 0.04 g / L K2HPO4, 0.04 g / L KH2PO4, 0.4 g / L NaHCO3, 0.08 g / L NaCl), and 20.0 g / L agar, with a pH of 6.8-7.0; the liquid culture medium consists of 5.0 g / L casein peptone, 5.0 g / L nitroglycerin digest, 10.0 g / L yeast extract, 0.0005 g / L sodium resazurin, and 0.5 g / L... L-cysteine ​​hydrochloride (composed of 0.01 g / L CaCl2·2H2O, 0.02 g / L MgSO4·7H2O, 0.04 g / L K2HPO4, 0.04 g / L KH2PO4, 0.4 g / L NaHCO3, and 0.08 g / L NaCl) has a pH of 6.8-7.0.

[0026] Example 2: Animal Model Construction.

[0027] The experimental animals used in this invention were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The environment was maintained at 20-24℃, constant humidity at 50-60%, with 12 hours of light (8:00-20:00), soundproof, and with free access to food and water. Experiments were conducted after one week of acclimatization. The following antibodies were used: hematoxylin (Solepro Science & Technology Co., Ltd.), eosin (Solepro Science & Technology Co., Ltd.), rabbit anti-IL-1β polyclonal antibody (Abixin (Shanghai) Biotechnology Co., Ltd.), and mouse anti-TNF-α monoclonal antibody (Proteintech).

[0028] Methods: Twelve 8-week-old female SD rats, weighing 180-250g, were randomly divided into (1) a normal control group and (2) an experimental group. Hungatella hathewayi 1×10 9 CFU / mL; 6 rats per group. The control group was given 150 μL of anaerobic PBS. The rats were given 150 μL of bacterial solution via vaginal irrigation twice every 3 days for 3 weeks. Uterine tissue was harvested 21 days after modeling, and uterine sections were used to observe pathological changes. The expression levels of inflammatory factor proteins were detected by Western blotting.

[0029] Results: Experimental group: 150 μL of bacteria perfusion, twice every 3 days, for 3 weeks.

[0030] Compared with the control group, Hungatella hathewayi It can significantly induce endometrial atrophy and inflammatory cell infiltration in the endometrial stroma. The number of CD138 and CD38 positive cells is significantly higher than that in the control group. Western blot results show that the protein expression levels of TNF-α and IL-1β in the endometrium of rats in the experimental group are increased. See the results below. Figures 1 - 3 .

[0031] In summary, this invention utilizes for the first time... Hungatella hathewayi A rat model of cerebrovascular accident (CE) was constructed to simulate the natural progression of the disease, addressing key issues of existing models such as deviation from physiological states, high infection rates, and high mortality rates. Experimental data confirmed that this model highly closely matches the characteristics of CE in terms of pathology and immune microenvironment, providing an innovative tool for etiological research and drug screening, and possessing significant scientific and clinical application value.

[0032] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Hungatella hathewayi Application of the strain in constructing a rat model of chronic endometritis, the Hungatella hathewayi The strain has the accession number DSM 13479 and is deposited at the German Center for the Preservation of Microbial Cultures.

2. The application according to claim 1, characterized in that, The animal model is a rat model.

3. A method for constructing an animal model of chronic endometritis, characterized in that, The following steps are involved: (a) The collection number DSM 13479 Hungatella hathewayi Cultivate under anaerobic conditions; (b) Adjust the bacterial concentration to 1×10⁻⁶ 9 CFU / mL; (c) Instill 150 μL of the bacterial solution obtained in step (b) into the vagina of the experimental animals.

4. The method according to claim 3, characterized in that, The experimental animals were female SD rats.

5. The method according to claim 3, characterized in that, The culture in step (a) uses a liquid culture medium consisting of: 5.0 g / L casein peptone, 5.0 g / L vesicular enzyme digest, 10.0 g / L yeast extract, 0.0005 g / L sodium resazurin, and 0.5 g / L L-cysteine ​​hydrochloride.

6. An animal model of chronic endometritis, characterized in that, It is constructed by the method described in any one of claims 3-5.

7. The application of the animal model described in claim 6 in the study of the pathogenesis of chronic endometritis.

8. The use of the animal model of claim 6 in screening drugs for the treatment of chronic endometritis.

9. The use of the animal model of claim 6 in evaluating the effectiveness of drugs for treating chronic endometritis.

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