Preparation method for constructing rat embryo implantation failure model based on streptococcus agalactiae

Abstract

The invention belongs to the technical field of animal models, and particularly relates to a preparation method for constructing a rat embryo implantation failure model based on streptococcus agalactiae. The method comprises the following steps: culturing a streptococcus agalactiae strain with the number of BNCC336970 by using a solid culture medium containing sheep blood, adjusting the concentration of a bacterial solution to 1 * 10 < 8 > CFU / mL, and carrying out vaginal perfusion on an SPF-grade female SD rat every day for 21 days continuously. And after the perfusion is finished, the female mouse is mated with the male mouse at the early oestrus stage, the detected vaginal suppository is marked as the 0.5 th day of pregnancy, the mouse is killed at the 7.5 th day of pregnancy, and the model is evaluated by counting the embryo implantation quantity and detecting the expression level of endometrial tissue receptivity factors MMP9 and VEGF (Vascular Endothelial Growth Factor). A clinical pathogen pathogenic mechanism is simulated through a genital tract infection way for the first time, and the problem of physiological status deviation caused by traditional hormone intervention, immune agonists or genetic engineering methods is solved. The constructed model can stably induce the obvious reduction of the embryo implantation number and the reduction of endometrial receptivity, and provides a standardized tool for the pathological mechanism research of infectious embryo implantation failure and the screening of therapeutic drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal models, and in particular to a preparation method for constructing a rat embryo implantation failure model based on Streptococcus agalactiae. BACKGROUND

[0002] Embryo implantation failure is an important difficulty in assisted reproductive technology and a core factor leading to pregnancy termination, with a clinical incidence rate of up to 5-10%. After 3 fresh or frozen cycles of at least 3 high-quality embryos in adult women under the age of 40, if clinical pregnancy is still not achieved, it is defined as recurrent implantation failure (RIF). The causes of embryo implantation failure are complex and the mechanism is unknown, and the construction of an animal model thereof plays an important role in studying the pathological mechanism and exploring treatment options.

[0003] Existing animal models of embryo implantation failure mainly rely on hormone intervention (such as subcutaneous injection of mifepristone to induce luteal dysfunction), immune agonist (such as intraperitoneal injection of lipopolysaccharide to induce increased endotoxin levels), and genetic engineering (Furin gene conditional knockout mediated endometrial decidualization deletion). However, these models have certain defects: they cannot simulate the pathogenic mechanism of common reproductive tract pathogens in the clinic. Streptococcus agalactiae (S. agalactiae), as a conditional pathogenic bacterium colonizing the reproductive tract of 20-30% of women, can cause postpartum infection, bacteremia, skin and soft tissue infection, and osteomyelitis. However, there is currently no standardized method for constructing an embryo implantation failure animal model based on S. agalactiae infection. Streptococcus agalactiae SUMMARY

[0004] In view of the problems in the prior art, the present application aims to provide a preparation method for constructing a rat embryo implantation failure model based on S. agalactiae. By infusing S. agalactiae into the vagina of rats, the rat pregnancy rate and embryo implantation rate can be induced to decrease, and other embryo implantation failure-related phenotypes can be induced.

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

[0006] The present application discloses an application of S. agalactiae in constructing an animal model, and the S. agalactiae has the number BNCC336970.

[0007] Further, the application is an application in constructing an embryo implantation failure animal model.

[0008] The present application further discloses a construction method of an embryo implantation failure animal model, characterized in that: the S. agalactiae of claim 1 is used to treat experimental animals by a vaginal perfusion method.

[0009] Further, the method comprises the following steps:​ S1. Culturing Streptococcus agalactiae to logarithmic growth phase; S2. Adjusting the concentration of the bacterial solution to 1×10 8 CFU / mL; S3. Continuously perfusing female SD rats with 200 μL per rat for 21 days to obtain an embryo implantation failure animal model.

[0010] Further, the culture medium in step S1 is a solid culture medium containing 15 g / L trypsin peptone, 5 g / L soy peptone, 5 g / L sodium chloride, 20 g / L agar, and 50 mL / L sterile defibrillated sheep blood, and the pH is 7.3±0.2.

[0011] The application further discloses application of the method in any of the above aspects in construction of embryo implantation failure.

[0012] The application further discloses application of the embryo implantation failure animal model in the above aspects, and the application includes application in research on pathogenesis of embryo implantation failure.

[0013] The application further discloses application of the embryo implantation failure animal model in the above aspects, and the application includes application in screening of drugs for treating embryo implantation failure.

[0014] Compared with the prior art, the application has the following beneficial effects.

[0015] The application successfully constructs a rat model of embryo implantation failure by perfusing Streptococcus agalactiae into the vagina, and the model can simulate the pathophysiological characteristics of embryo implantation failure, and avoids the dependence on hormone induction and gene conditional knockout, so that the model is consistent with the physiological state. The application establishes an embryo implantation failure model based on disorder of the reproductive tract flora, simulates the pathophysiological state of embryo implantation failure by interfering with the reproductive tract flora, and provides a new experimental tool for research on the pathogenesis and treatment of embryo implantation failure. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Effect of Streptococcus agalactiae on the number of embryo implantation of rats on the 7.5th day of pregnancy, wherein A is the number of embryo implantation of the right and left uteruses of female rats on the 7.5th day of pregnancy, B is the number of embryo implantation of the right uteruses of female rats on the 7.5th day of pregnancy, and C is the number of embryo implantation of the left uteruses of female rats on the 7.5th day of pregnancy.

[0017] Figure 2 Effect of Streptococcus agalactiae on embryo implantation and expression of endometrial receptivity indexes of endometrial tissue of rats, wherein A is the expression of the transcription level of VEGF of the endometrium, and B is the expression of the transcription level of MMP9 of the endometrium. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments, as all technologies implemented based on the present invention fall within the scope of the present invention.

[0019] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0020] Example 1: Streptococcus agalactiae has the effect of reducing indicators related to embryo implantation in rat endometrium.

[0021] Streptococcus agalactiae was found Streptococcus agalactiae ) has the effect of inducing embryo implantation failure phenotype in rats. The inventors introduced Streptococcus agalactiae ( Streptococcus agalactiae ) Intravaginal instillation of bacterial fluid into SD rats revealed a decrease in the number of embryo implantations and a reduction in the expression of factors associated with embryo implantation in endometrial tissue. Therefore, this strain can be used to establish an animal model of embryo implantation failure.

[0022] The present invention uses Streptococcus agalactiae ( Streptococcus agalactiae ) was purchased from Beina Biotechnology, number BNCC336970. The culture method for Streptococcus agalactiae was as follows: S. agalactiae was inoculated onto a solid culture medium and cultured aerobically at 37°C for 16 hours. A single colony was picked and inoculated into liquid culture medium and grown aerobically at 37°C for 18–24 hours.

[0023] The solid culture medium consists of 15 g / L tryptone, 5 g / L soytone, 5 g / L sodium chloride, 20 g / L agar, and 50 mL / L sterile defibrinated sheep blood, with a pH value of 7.3±0.2; the liquid culture medium consists of 17 g / L trypticase, 3 g / L soy papain hydrolysate, 5 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, and 2.5 g / L glucose, with a pH value of 7.3±0.2.

[0024] Example 2: Construction of animal model.

[0025] The experimental animals used in the application are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., the temperature is 20-24℃, the humidity is 50-60%, the illumination is 12h (8:00-20:00), the sound insulation is good, the free feeding and drinking water are provided, and the experiment is carried out after one week of adaptation. The required reagents and instruments are: anhydrous ethanol (Beijing Chemical Plant), TRIzol (Invitrogen Company), isopropanol (Beijing Chemical Plant), chloroform (Beijing Chemical Plant), DEPC water (Solabio Technology Co., Ltd.), HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme Company), ChamQ Universal SYBR gPCR Master Mix (Vazyme Company), rat index primers (Shenguo Biological Engineering Co., Ltd.) (see Table 1), CO2 cell incubator (Thermo Company), ultrasonic cell disrupter (SONICS & MATERIALS, INC Company), optical microscope (OLYMPUS DP74), inverted optical microscope (OLYMPUS), paraffin sectioning machine (Leica Jung Company), real-time quantitative PCR instrument (Roche LightCycler 96), small tabletop centrifuge (Crystal Company), ultra-low temperature refrigerator (Thermo Scientific Company), electronic balance (Shanghai Ohaus Instrument Co., Ltd.), super-clean workbench (China Suzhou Antai Air Technology Co., Ltd.).

[0026] Table 1 Real-time PCR primers .

[0027] Method.

[0028] 1, Rat grouping and modeling: 6-week-old female SD rats, weighing 200-210g, 12 rats, were randomly divided into a control group and an experimental group, 6 rats in each group. The experimental group rats were vaginally perfused with 200μL / one of streptococcus agalactiae (1×10 8 CFU / mL), with a perfusion frequency of once a day, for 21 days. The control group was given the same amount of sterile PBS. After 21 days of vaginal perfusion, the estrous cycle was detected daily. The SD female rats were caged with SD male rats (1:1) before the proestrus of female rats (pregnancy day 0 15:00), and the next morning, the SD female rats with vaginal plugs were considered to have successfully mated and were recorded as pregnancy day 0.5, and the caging was continued for 5 days. The SD female rats were sacrificed on pregnancy day 7.5, and the number of embryo implantation was recorded.

[0029] 2, Extraction of RNA and RT-qPCR detection of embryo implantation and endometrial receptivity index MMP9 and VEGF expression in rat endometrial tissue.

[0030] result.

[0031] Compared with the control group, the number of embryo implantation on day 7.5 of pregnancy in the experimental group was significantly decreased, and the expression of indicators related to embryo implantation and endometrial receptivity was significantly decreased. Figures 1-2 .

[0032] In summary, the present invention utilizes Streptococcus agalactiae ( Streptococcus agalactiae ) established a rat embryo implantation failure model, addressing the critical issue of existing technologies in simulating the pathogenic mechanisms of common reproductive tract infections. Experimental data confirmed that this model closely matches the characteristics of embryo implantation failure at the pathological and molecular levels, providing an innovative tool for etiological research and drug screening, with significant scientific and clinical application value.

[0033] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A use of Streptococcus agalactiae in constructing an animal model, wherein the Streptococcus agalactiae is numbered BNCC336970.

2. The use according to claim 1, characterized in that The application is an application in constructing an animal model of embryo implantation failure.

3. A method for constructing an animal model of embryo implantation failure, characterized by: The Streptococcus agalactiae according to claim 1 is used to treat experimental animals by vaginal instillation.

4. The method according to claim 3, comprising the steps of: S1. Cultivate Streptococcus agalactiae until the logarithmic growth phase; S2. Adjust the bacterial concentration to 1×10 8 CFU / mL; S3. Female SD rats were given continuous vaginal infusion for 21 days at 200 μL / rat to establish an embryo implantation failure model.

5. The method according to claim 4, characterized in that: The culture medium in step S1 is a solid culture medium containing 15 g / L tryptone, 5 g / L soytone, 5 g / L sodium chloride, 20 g / L agar and 50 mL / L sterile defibrinated sheep blood, with a pH of 7.3±0.

2.

6. Use of the method according to any one of claims 3 to 5 in treating embryo implantation failure.

7. A use of the embryo implantation failure animal model according to claim 6, characterized in that: The applications include applications in the study of the pathogenesis of embryo implantation failure.

8. A use of the embryo implantation failure animal model according to claim 6, characterized in that: Application in drug screening for the treatment of embryo implantation failure diseases.

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