Establishment method of premature ovarian failure rat animal model based on enterovirus group transplantation

Abstract

The invention discloses a premature ovarian failure rat animal model building method based on enterovirus group transplantation, and particularly relates to the technical field of animal model building. According to the invention, the enterovirus group of a premature ovarian failure patient is subjected to in-vitro extraction and purification, and then is transplanted into a female rat, so that a new experimental model is provided for researching the interaction between the enterovirus group and the premature ovarian failure; meanwhile, a new way is opened up for further revealing the research on the complex relation between intestinal microorganisms and female reproductive health. In addition, the model is expected to provide an experimental basis for developing a novel treatment strategy for premature ovarian failure. The method aims to deeply explore the relationship between the enterovirus / bacteriophage group and female reproductive system diseases, especially premature ovarian failure.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically to a method for establishing a rat animal model of premature ovarian failure based on enterovirus transplantation. Background Technology

[0002] Currently, low female fertility rates have become a serious challenge globally. The pathogenesis of this phenomenon is quite complex, involving multiple factors such as disease, psychological factors, environment, and changes in sex hormones, all of which can lead to female infertility or difficulty conceiving. Furthermore, with age, women's ovarian function gradually declines, and sex hormone levels decrease, resulting in reduced egg quality and lower implantation rates, increasing the risk of infertility. Because the specific pathological mechanisms of premature ovarian failure (POF) are not yet fully understood, current treatment options mainly focus on symptom relief and hormone replacement therapy, rather than directly addressing the underlying cause. This limits treatment effectiveness and improvements in patients' quality of life.

[0003] To gain a deeper understanding of the pathogenesis of premature ovarian failure (POF) and explore effective treatments, researchers have developed various animal models, including genetically engineered models, chemically induced models, and autoimmune models. These models aim to simulate different aspects of human premature ovarian failure, providing important tools for research. However, despite the crucial role animal models play in understanding POF, significant limitations remain. Some models may not fully reproduce the complexity and multifactorial etiology of human POF, limiting the clinical relevance and translational potential of research findings. Furthermore, the high cost of establishing and maintaining specific models, and the significant demand for research resources, also limit their widespread application in POF research.

[0004] To delve deeper into the pathological mechanisms of porcine ovarian failure (POF) and develop effective treatments, the scientific community has developed numerous animal models, such as genetically engineered models, chemically induced models, and autoimmune models. However, despite their crucial role in elucidating the biological mechanisms of POF, these animal models still have significant limitations. For example, genetically engineered models typically mimic the effects of genetic factors on ovarian function by knocking out or inserting specific genes, but these models may not comprehensively cover all potential causes of POF; the reproducibility and similarity to human diseases in natural or chemically induced models remain limited. Therefore, existing animal models may not fully mimic the physiological and pathological characteristics of human POF in certain aspects, which limits the translation of research results into human clinical applications. Furthermore, the establishment and maintenance of specific models are not only costly but also require substantial experimental resources, further limiting their widespread adoption and application in POF research.

[0005] Currently, there is a lack of methods for establishing a rat model of premature ovarian failure based on enterovirome transplantation (FVT). Summary of the Invention

[0006] The purpose of this invention is to provide a method for establishing a rat model of premature ovarian failure (POF) based on enterovirome transplantation (FVT).

[0007] To address the problems of the prior art, the present invention provides the following technical solution: In a first aspect, this application provides a method for establishing a POF rat animal model based on FVT.

[0008] Secondly, this application provides a drug for establishing a POF female rat animal model.

[0009] Thirdly, this application provides a food for establishing a POF female rat animal model.

[0010] The first aspect of this application provides a method for establishing a POF rat animal model based on FVT, comprising the following steps:

[0011] (1) Collect stool samples from POF patients;

[0012] (2) Separate and purify virus-like particles (VLPs) from fecal samples to obtain supernatant containing VLPs from the intestines of POF patients;

[0013] (3) The supernatant containing VLPs in the intestine of POF patients obtained in step (2) was transplanted into the intestine of healthy female rats (FVT) to construct a POF rat animal model;

[0014] (4) Observe and evaluate the transplanted individuals to verify whether female rats exhibit POF-like behavior.

[0015] Further, in step (2), the intestinal VLPs of POF patients were isolated and purified:

[0016] ① Take 2-5g of fecal sample and suspend it in 15-35mL of SM buffer to obtain the sample suspension;

[0017] ② At 22℃, the sample suspension obtained in step ① is centrifuged at a speed of 800g for 1min to obtain the supernatant;

[0018] ③ Centrifuge the supernatant obtained in step ② at 22°C, first at 800g for 1 min, then at 8000g for 10 min, to obtain a supernatant containing VLPs;

[0019] ④ The supernatant containing VLPs obtained in step ③ is filtered sequentially through 0.45μm and 0.22μm filters to obtain a filtrate containing VLPs;

[0020] ⑤ Dissolve PEG 6000 and NaCl in SM buffer and autoclave at 121°C for 20 min; after cooling, add to the filtrate containing VLPs obtained in step ④ to prepare a mixture; the volume fraction of PEG 6000 in the mixture is 20% and the concentration of NaCl is 2.5M. Incubate overnight at 4°C to obtain the mixture.

[0021] ⑥ Centrifuge the mixture obtained in step ⑤ at 11000g and 4℃ for 15min, resuspend the precipitate with 2mL SM buffer, and filter through a 0.22μm filter to obtain the supernatant containing POF virus.

[0022] Furthermore, in step ①, the mass-to-volume ratio of fecal sample to SM buffer is 2-5 g: 15-35 mL.

[0023] Furthermore, in step (3), the means of transplantation include oral capsules, gastric tubes, nasogastric tubes, colonoscopy, gastroscopy, rectal administration, or endoscopic jejunal catheter insertion.

[0024] A second aspect of this application provides a drug for establishing a POF female rat animal model, the drug containing VLPs in the intestine of POF patients.

[0025] Furthermore, the drug includes any one of tablets, capsules, oral liquids, or lyophilized powders.

[0026] Furthermore, the drug contains a pharmaceutically effective dose of POF patient intestinal VLPs and a pharmaceutically acceptable carrier.

[0027] Furthermore, pharmaceutically acceptable carriers include one or more of the following: skim milk, lactose, glucose, sucrose, sorbitol, mannose, trehalose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, or mineral oil.

[0028] A third aspect of this application provides a food for establishing a POF female rat animal model, the food containing VLPs in the intestine of POF patients.

[0029] Beneficial effects: This invention may reduce the cost of establishing and maintaining specific models, decrease the demand for experimental resources, and enable more researchers to participate in POF research. By isolating and purifying the enterovirome extracted from POF patients in vitro and transplanting it into healthy female rats, POF-like changes were successfully induced in the female rats, providing a new and effective animal model for the study of the pathological mechanism of POF and the development of treatment strategies.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] (1) This invention not only helps researchers to deepen their understanding of the etiology and pathogenesis of POF, but also provides researchers with a new tool for studying POF by providing a novel animal model of POF. The establishment of this animal model lays the foundation for further exploration of treatment methods, interventions and prevention strategies for POF, and is expected to promote the progress of POF-related research and provide important theoretical and experimental support for future clinical treatment.

[0032] (2) This invention establishes an animal model of POF rats through FVT, which not only enriches the methodology of POF research, but also provides a new perspective for understanding the complex pathological mechanism of the disease, and has important scientific research and application value.

[0033] (3) This invention reveals the complex interaction between gut microbiota and female reproductive system diseases, especially the relationship between the enterovirome and POF, providing an important foundation for further research. Attached Figure Description

[0034] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation thereof. Hereinafter, embodiments of the invention will be described in detail with reference to the accompanying drawings.

[0035] Figure 1 This is a bar chart comparing the daily number of vaginal plugs detected in female mice in the blank control group and female mice in the POF patient enterovirus transplantation group during the cohabitation period.

[0036] Figure 2 This is a bar chart comparing the total vaginal plug detection rate during co-hospitalization between female mice in the blank control group and female mice in the POF patient enterovirus transplantation group of the present invention.

[0037] Figure 3 This is a bar chart comparing the lordosis index of female mice in the blank control group and female mice in the POF patient enterovirus transplantation group during the cohabitation period.

[0038] Figure 4This is a graph showing the estrous cycle detection of female mice in the blank control group and female mice in the POF patient enterovirus transplantation group of the present invention;

[0039] Figure 5 This is a bar chart comparing the pregnancy rates of female mice in the blank control group and female mice in the POF patient enterovirus transplantation group.

[0040] Figure 6 This is a bar chart comparing the serum sex hormone levels of female mice in the blank control group and female mice in the POF patient enterovirus transplantation group of the present invention.

[0041] Figure 7 This is a bar chart comparing the mature follicle counts of female mice in the blank control group and female mice in the POF patient enterovirus transplantation group.

[0042] Figure 8 This is a flowchart illustrating the construction process of the animal model of the present invention. Detailed Implementation

[0043] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0044] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0045] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0046] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0047] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0048] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0049] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0050] The first aspect of this application provides a method for establishing a POF rat animal model based on FVT, comprising the following steps:

[0051] (1) Collect stool samples from POF patients;

[0052] (2) Separate and purify VLPs from fecal samples to obtain supernatant containing VLPs from the intestines of POF patients;

[0053] (3) The supernatant containing VLPs in the intestine of POF patients obtained in step (2) was transplanted into the intestine of healthy female rats (FVT) to construct a POF rat animal model;

[0054] (4) Observe and evaluate the transplanted individuals to verify whether female rats exhibit POF-like behavior.

[0055] In some embodiments, in step (2), the intestinal VLPs of POF patients are isolated and purified:

[0056] ① Take 2-5g of fecal sample and suspend it in 15-35mL of SM buffer to obtain the sample suspension;

[0057] ② At 22℃, the sample suspension obtained in step ① is centrifuged at a speed of 800g for 1min to obtain the supernatant;

[0058] ③ Centrifuge the supernatant obtained in step ② at 22°C, first at 800g for 1 min, then at 8000g for 10 min, to obtain a supernatant containing VLPs;

[0059] ④ The supernatant containing VLPs obtained in step ③ is filtered sequentially through 0.45μm and 0.22μm filters to obtain a filtrate containing VLPs;

[0060] ⑤ Dissolve PEG 6000 and NaCl in SM buffer and autoclave at 121°C for 20 min; after cooling, add to the filtrate containing VLPs obtained in step ④ to prepare a mixture; the volume fraction of PEG 6000 in the mixture is 20% and the concentration of NaCl is 2.5M. Incubate overnight at 4°C to obtain the mixture.

[0061] ⑥ Centrifuge the mixture obtained in step ⑤ at 11000g and 4℃ for 15min, resuspend the precipitate with 2mL SM buffer, and filter through a 0.22μm filter to obtain the supernatant containing POF virus.

[0062] In some embodiments, in step ①, the mass-to-volume ratio of the fecal sample to the SM buffer is 2-5 g: 15-35 mL.

[0063] In step (3), the means of transplantation include oral capsule, gastric tube, nasogastric tube, colonoscopy, gastroscopy, rectal administration or endoscopic jejunal catheter insertion.

[0064] The second aspect of this application provides a drug for establishing a POF female rat animal model, the drug containing VLPs in the intestine of POF patients.

[0065] In some embodiments, the drug includes any one of tablets, capsules, oral liquids, or lyophilized powders.

[0066] In some embodiments, the drug contains a pharmaceutically effective dose of POF patient intestinal VLPs and a pharmaceutically acceptable carrier.

[0067] In some embodiments, pharmaceutically acceptable carriers include one or more of skim milk, lactose, glucose, sucrose, sorbitol, mannose, trehalose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, or mineral oil.

[0068] A third aspect of this application provides a food for establishing a POF female rat animal model, the food containing VLPs in the intestine of POF patients.

[0069] Example 1

[0070] The present invention provides a method for establishing a POF rat animal model based on FVT, comprising the following steps:

[0071] (1) Collect stool samples from POF patients;

[0072] (2) Separate and purify VLPs from fecal samples to obtain supernatant containing VLPs from the intestines of POF patients;

[0073] Isolation and purification of VLPs from the intestines of POF patients:

[0074] ① The fecal sample was suspended in SM buffer to obtain a sample suspension; the mass-to-volume ratio of fecal sample to SM buffer was 2g:15mL;

[0075] ② At 22℃, the sample suspension obtained in step ① is centrifuged at a speed of 800g for 1min to obtain the supernatant;

[0076] ③ Centrifuge the supernatant obtained in step ② at 22°C, first at 800g for 1 min, then at 8000g for 10 min, to obtain a supernatant containing VLPs;

[0077] ④ The supernatant containing VLPs obtained in step ③ is filtered sequentially through 0.45μm and 0.22μm filters to obtain a filtrate containing VLPs;

[0078] ⑤ Dissolve PEG 6000 and NaCl in SM buffer and autoclave at 121°C for 20 min; after cooling, add to the filtrate containing VLPs obtained in step ④ to prepare a mixture; the volume fraction of PEG 6000 in the mixture is 20% and the concentration of NaCl is 2.5M. Incubate overnight at 4°C to obtain the mixture.

[0079] ⑥ Centrifuge the mixture obtained in step ⑤ at 11000g and 4℃ for 15min, resuspend the precipitate with 2mL SM buffer, and filter through a 0.22μm filter to obtain the supernatant containing POF virus.

[0080] (3) The supernatant containing VLPs in the intestine of POF patients obtained in step (2) was transplanted into the intestine of healthy female rats (FVT) to construct a POF rat animal model; the transplantation methods include oral capsule, gastric tube, nasogastric tube, colonoscopy, gastroscopy, rectal administration or jejunal catheter insertion under gastroscopy.

[0081] (4) Observe and evaluate the transplanted individuals to verify whether female rats exhibit POF-like behavior.

[0082] The present invention provides a drug for establishing a POF female rat animal model, the drug containing VLPs in the intestine of POF patients.

[0083] The medication is in tablet form. It contains a pharmaceutically effective dose of POF patient intestinal VLPs and a pharmaceutically acceptable carrier.

[0084] Pharmaceutically acceptable carriers are combinations of fourteen of the following: skim milk, sucrose, sorbitol, mannose, trehalose, starch, gum arabic, calcium phosphate, alginate, gelatin, methylparaben, propylparaben, talc, and mineral oil.

[0085] The present invention provides a food for establishing a POF female rat animal model, the food containing VLPs in the intestine of POF patients.

[0086] Example 2

[0087] The difference between Example 2 and Example 1 is as follows:

[0088] In step (2), VLPs in the fecal sample are separated and purified to obtain a supernatant containing VLPs from the intestines of POF patients;

[0089] Isolation and purification of VLPs from the intestines of POF patients:

[0090] ① The fecal sample was suspended in SM buffer to obtain a sample suspension; the mass-to-volume ratio of fecal sample to SM buffer was 3g:25mL.

[0091] The drug is in capsule form. Pharmaceutically acceptable carriers are combinations of four of the following: lactose, glucose, sucrose, magnesium stearate, and mineral oil.

[0092] Example 3

[0093] The difference between Example 3 and Example 1 is as follows:

[0094] In step (2), VLPs in the fecal sample are separated and purified to obtain a supernatant containing VLPs from the intestines of POF patients;

[0095] Isolation and purification of VLPs from the intestines of POF patients:

[0096] ① The fecal sample was suspended in SM buffer to obtain a sample suspension; the mass-to-volume ratio of fecal sample to SM buffer was 5g:35mL.

[0097] The drug is a lyophilized powder. Calcium phosphate is a pharmaceutically acceptable carrier.

[0098] Example 4

[0099] The difference between Example 4 and Example 1 is that the drug is an oral liquid. Pharmaceutically acceptable carriers are combinations of five of the following: glucose, sucrose, methylparaben, propylparaben, and magnesium stearate.

[0100] Example 5

[0101] The POF patients involved in the examples are those who have been clinically diagnosed with POF for the first time and have not received any drugs or other treatments.

[0102] Stool samples, reagents and materials used, and their sources:

[0103] 1. Specimen

[0104] The stool samples from POF patients used in the examples were all collected from Qilu Hospital of Shandong University.

[0105] 2. Main Reagents and Materials

[0106] 0.45μm filter (Millipore); 0.22μm filter (Millipore); 0.02μm filter (Whatman); PEG6000 (Solarbio); NaCl (Biosharp); MgSO4 (Sigma-Aldrich); Tris (Biosharp); Gelatin (HuShi); Automated biochemical analyzer (Roche); SYBR-Gold staining solution (Invitrogen).

[0107] The composition of SM buffer is as follows (per 1000 mL system):

[0108] 100mM NaCl, 8mM MgSO4, 50mM Tris (pH 7.5) and 0.002% gelatin (wt / vol) were added, and the mixture was autoclaved at 121°C for 20 min.

[0109] Isolation and purification of VLPs from the intestines of POF patients:

[0110] ① Take 2-5g of fecal sample and resuspend it in 15-35mL of SM buffer to obtain a sample suspension;

[0111] ② At 22℃, the sample suspension obtained in step ① is centrifuged at a speed of 800g for 1min to remove impurities and large fragments from the feces. The supernatant is then collected to obtain the supernatant liquid.

[0112] ③ Centrifuge the supernatant obtained in step ② at 22°C, at a speed of 8000g, for 10 minutes. Repeat centrifugation until no precipitate is visible after centrifugation to separate bacteria and VLPs communities in feces. The precipitate is bacteria, and the supernatant is VLPs communities, resulting in a supernatant containing VLPs.

[0113] ④ The supernatant containing VLPs obtained in step ③ is filtered sequentially through 0.45μm and 0.22μm filters to remove residual bacteria in the supernatant, thus obtaining a supernatant containing VLPs.

[0114] ⑤ Dissolve PEG 6000 and NaCl in SM buffer and autoclave at 121℃ for 20 min; after cooling, add to the supernatant containing VLPs obtained in step ④ to prepare a mixture. The volume fraction of PEG 6000 in the mixture is 20% and the concentration of NaCl is 2.5M. Incubate overnight at 4℃ to precipitate VLPs and obtain the mixture.

[0115] ⑥ Centrifuge the mixture obtained in step ⑤ at 11000g and 4℃ for 15min; discard the supernatant, resuspend the precipitate in 2mL of SM buffer, and filter it again through a 0.22μm filter to obtain the filtrate;

[0116] ⑦ Fix the filtrate obtained in step ⑥ with 1% formaldehyde for 15 min; filter the fixed VLPs in triplicate onto a 0.02 μm filter membrane and stain with SYBR-Gold. After staining, observe and count the VLPs under an epifluorescence microscope to obtain a sufficient amount of fecal VLPs for treatment.

[0117] Experimental Example 1

[0118] The intestinal VLPs from POF patients obtained in Example 5 were transplanted into healthy female rats aged 8-48 weeks via gavage or other methods (FVT).

[0119] Each gavage administration to female rats shall contain at least 1 x 10 VLPs. 9 The dose was 1 mL / gavage per female mouse, administered once daily for 14 days.

[0120] Experimental Example 2

[0121] The vaginal plug rate, number of vaginal plugs, mating behavior, pregnancy rate, number of mature ovarian follicles, serum estradiol (E2), progesterone (P), FSH, LH and other indicators were observed in female rats that received FVT.

[0122] Figure 1This is a bar chart comparing the daily number of vaginal plugs detected in female mice in the control group (Control) and female mice in the POF-FVT group (POF-FVT group) during the co-breeding period. The testing method was as follows: Age- and weight-indistinguishable fertile SD male mice were mated with female mice at a 1:1 ratio. Male and female mice were co-breeded at 6 PM each evening and separated at 8 AM the following morning. Before separation, vaginal plugs were checked and counted. Female mice with plugs were moved to a new metabolic cage, and day 1 of pregnancy was recorded. Female mice without plugs were randomly replaced with male mice for mating the following day. Results showed that the daily number of vaginal plugs detected in female mice in the POF-FVT group was significantly lower than that in the control group.

[0123] Figure 2 This is a bar chart comparing the total vaginal plug detection rate during cohabitation between female mice in the blank control group (Control) and female mice in the POF-FVT group (POF-FVT group). The test method is the same as described above. Figure 1 The results showed that, compared with the blank control group (Control), the detection rate of total vaginal plugs was significantly lower in female mice that received POF enterovirus transplantation (POF-FVT).

[0124] Figure 3 This is a bar chart comparing the lordosis index of female mice in the blank control group (Control) and female mice in the POF-FVT group during cohabitation. The test method is: Lordosis quotient = number of lordosis responses in female mice / number of mounting attempts by male mice, reflecting the mating intention and mating behavior of female mice. The results show that compared with female mice in the blank control group (Control), female mice in the POF-FVT group had a lower lordosis index, reflecting a significantly reduced mating intention in this group of female mice.

[0125] Figure 4This image shows estrous cycle data for female mice in the control group (Control) and female mice in the POF-FVT group (POF-FVT group). The testing method was as follows: Age- and weight-indiscriminate, fertile SD male mice were mated with female mice at a 1:1 ratio. Estrogenic cycles were detected by vaginal exfoliated cell smears at 6 PM each night. The male and female mice were then kept together, and separated at 8 AM the following morning for the same estrous cycle detection in the female mice. For the smear method of vaginal exfoliated cell detection, after fixing the rats and fully exposing the vagina, 100-200 μL of sterile saline was pipetted into the female mouse's vagina, and the liquid was aspirated 3-5 times. The aspirated liquid was then evenly dropped onto a glass slide, allowed to dry naturally, and fixed with anhydrous ethanol for 10 minutes. HE staining was then performed according to the following steps, and the slides were mounted with neutral resin. Under an optical microscope, the estrous cycle was divided into stages based on cell type: (1) Proestrus: predominantly nucleated epithelial cells; (2) Estrus: full field of view filled with keratinized epithelial cells; (3) Anaestrus: nucleated cells and keratinized cells are roughly equal; (4) Interestrus: abundant leukocytes and a small amount of mucus. Results showed that compared with the control group, female mice receiving POF-FVT transplantation had significantly prolonged estrous cycles.

[0126] Figure 5 This is a bar chart comparing the pregnancy rates of female mice in the control group (Control) and female mice in the POF-FVT group (POF-FVT). The testing method involved euthanizing all pregnant mice at 20 days of gestation and assessing the presence of embryos in the uterus to determine pregnancy status. The results showed that the pregnancy rate of female mice in the POF-FVT group was significantly lower than that in the control group.

[0127] Figure 6 This is a bar chart comparing serum hormone levels in female mice from the control group (Control) and female mice that received enterovirus transplantation from POF patients (POF-FVT). From left to right, the levels are estrogen, progesterone, gonadotropin (FSH), and luteinizing hormone (LH). The testing method was as follows: serum was collected from female mice at 20 days of gestation and the hormone levels were analyzed using a fully automated biochemical analyzer (Roche). The results showed that compared with female mice from the control group (Control), the serum estrogen and progesterone levels in female mice that received enterovirus transplantation from POF patients (POF-FVT) were significantly lower, while the FSH and LH levels were significantly higher.

[0128] Figure 7This is a bar chart comparing the number of mature follicles in ovarian tissues of female mice in the control group (Control) and female mice in the POF-FVT group after HE staining. The results showed that the number of mature follicles in female mice in the POF-FVT group was significantly reduced compared with that in the control group.

[0129] Figure 8 This is a flowchart illustrating the construction of an animal model according to the present invention.

[0130] In summary, transplantation of intestinal VLPs from POF patients can disrupt the estrous cycle in rats, reduce mating behavior in female rats, decrease the thrombus incidence rate, and reduce the number of mature follicles, significantly reducing fertility. Furthermore, intestinal VLPs from POF patients decrease serum E2 and P levels in female rats, while increasing FSH and LH levels. The key role of the intestinal VLPs of this invention in the POF female rat model can be utilized to prepare drugs, pharmaceutical compositions, foods, and food additives containing intestinal VLPs from POF patients. These drugs, pharmaceutical compositions, foods, and food additives can be used to construct a premature ovarian failure (POF) female rat model through any one or more pharmaceutically feasible methods, including but not limited to oral capsules, gastric tubes or nasogastric tubes, colonoscopy or gastroscopy, rectal administration, or endoscopic jejunal catheter insertion.

[0131] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A method for establishing a rat model of premature ovarian failure based on enterovirus transplantation, characterized in that... Includes the following steps: (1) Collect stool samples from patients with premature ovarian failure (POF); (2) Virus-like particles (VLPs) in fecal samples were separated and purified to obtain a supernatant containing intestinal virus-like particles (VLPs) from POF patients. (3) The supernatant containing VLPs in the intestine of POF patients obtained in step (2) was transplanted into the FVT in the intestine of healthy female rats to construct a rat animal model of POF premature ovarian failure. (4) Observe and evaluate the transplanted individuals to verify whether female rats exhibit POF-like behavior.

2. The method for establishing according to claim 1, characterized in that: In step (2), the intestinal VLPs of the POF patients are isolated and purified: ① Take 2-5g of fecal sample and suspend it in 15-35mL of SM buffer to obtain a sample suspension; ② At 22℃, the sample suspension obtained in step ① is centrifuged at a speed of 800g for 1min to obtain the supernatant; ③ Centrifuge the supernatant obtained in step ② at 22°C, first at 800g for 1 min, then at 8000g for 10 min, to obtain a supernatant containing VLPs; ④ The supernatant containing VLPs obtained in step ③ is filtered sequentially through 0.45μm and 0.22μm filters to obtain a filtrate containing VLPs; ⑤ Dissolve PEG 6000 and NaCl in SM buffer and autoclave at 121°C for 20 min; After cooling, it is added to the filtrate containing VLPs obtained in step ④ to prepare a mixture; the volume fraction of PEG 6000 in the mixture is 20%, the concentration of NaCl is 2.5M, and it is incubated overnight at 4°C to obtain the mixture. ⑥ Centrifuge the mixture obtained in step ⑤ at 11000g and 4℃ for 15min, resuspend the precipitate with 2mL SM buffer, and filter through a 0.22μm filter to obtain the supernatant containing the intestinal VLPs of the POF patient.

3. The method for establishing according to claim 2, characterized in that: In step ①, the mass-to-volume ratio of the fecal sample to the SM buffer is 2-5 g: 15-35 mL.

4. The method for establishing according to claim 1, characterized in that: In step (3), the means of transplantation include oral capsules, gastric tubes, nasogastric tubes, colonoscopy, gastroscopy, rectal administration, or endoscopic jejunal catheter insertion.

5. A drug for establishing a female rat model of premature ovarian failure (POF), characterized in that: The drug contains the intestinal VLPs of the POF patient as described in claim 1.

6. The drug according to claim 5, characterized in that: The drug includes any one of tablets, capsules, oral liquids, or lyophilized powders.

7. The drug according to claim 6, characterized in that: The drug contains a pharmaceutically effective dose of POF patient enterovirus particles and a pharmaceutically acceptable carrier.

8. The drug according to claim 7, characterized in that: The pharmaceutically acceptable carriers include one or more of the following: skim milk, lactose, glucose, sucrose, sorbitol, mannose, trehalose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, or mineral oil.

9. A food product for establishing a female rat model of premature ovarian failure (POF), characterized in that: The food contains the enterovirus-like particles (VLPs) of POF patients as described in claim 1.

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