KR102233990B1 - Development of endometriosis animal model through Ureaplasma urealyticum infection - Google Patents

Abstract

The present invention relates to a technology for developing an endometriosis animal model through induction of infection and inflammation by injecting bacteria into the abdominal cavity of a mouse. More specifically , about an animal model in which a lesion similar to human endometriosis occurs when Ureaplasma urealyticum is infected in the abdominal cavity of a mouse to induce an inflammatory response, and then the uterine tissue is crushed and injected into the abdominal cavity. will be.

Description

Development of endometriosis animal model through Ureaplasma urealyticum infection}

The present invention relates to a technique for developing an endometriosis animal model through induction of infection and inflammation by injecting bacteria into the abdominal cavity of a mouse.

Endometriosis is the abnormal presence of endometrial tissue in organs other than the uterus. It is a common disease that occurs in about 10-15% of women of childbearing age. Endometriosis can occur in women who menstruate, that is, at all ages from menarche to menopause, and the main symptoms involved include severe menstrual pain, lower abdominal pain, and infertility, and it is a very frequent disease. Although the cause of endometriosis has not been accurately identified, the most promising hypothesis is the regurgitation of menstrual blood and the hypothesis of heterozygous. Most of the menstrual blood is discharged through the vagina, but it is hypothesized that some of the menstrual blood flows back through the fallopian tube and enters the abdominal cavity, resulting in endometriosis (Sampson, 1927). In addition, it has been reported that the inflammatory environment in the abdominal cavity is closely related to the occurrence of endometriosis.

Recently, studies on the relationship between bacterial infection and endometriosis have also been reported (Kodati, 2008). A large amount of Escherichia coli is detected in the menstrual blood of patients with endometriosis, and as the menstrual blood that has entered the pelvic cavity and E. coli are also introduced, macrophages cause an inflammatory reaction, creating an environment in which endometrial tissue can settle in the abdominal cavity. There is a report (Khan, 2018). In addition, there are studies that show that bacterial toxins damage adhesion factors between peritoneal cells, so that endometrial cells are located in the peritoneum, which causes endometriosis (Khan, 2018).

The development of endometriosis includes uncontrolled proliferation of endometrial cells, resistance to apoptosis (anti一 apoptosis), angiogenesis, migration and invasion, and changes in the local immune system. in local immune system), which are very similar to the process of cancer cell growth (Farquhar, 2007; Fazleabas et al., 2004; Garai et al., 2006; Hastings and Fazleabas, 2003; Hastings and Fazleabas, 2006; ; Wu et al., 2007). In addition, in the lesion of endometriosis, there is overexpression/overproduction of estrogen, prostaglandin, cytokine, and metalloproteinase (Bulun, 2009) (see FIG. 2).

Ureaplasma Ureaplasma urealyticum ) is Mycoplasmataceae, which does not have a cell wall, and is a bacterium with proliferation ability in artificial medium, and mainly inhabits mucous membranes of humans and animals. Since this bacterium is an important opportunistic pathogen commonly found in sexually active female reproductive organs, it is known to cause pelvic inflammatory disease, premature birth, chorionitis, and postpartum endometritis. The prevalence ranges from 60% to 80% worldwide.

Therefore, the research team estimated that ureaplasma urearithicum induces inflammation in the peritoneum and contributes to endometrial evaporation. To prove this, an animal model of endometriosis caused by bacterial infection in the abdominal cavity was newly developed. Until now, there has been no animal model in which endometriosis is triggered by bacterial infection. When ureaplasma urearithicum is infected in the peritoneal mesothelial cells, various inflammatory cytokines and chemokines are produced, the expression of adhesion factors is also increased, and the connection of cells is loosened. The present invention newly developed an animal model in which endometriosis occurs using this mechanism.

Korean Patent Registration No. 10-1357119 Korean Patent Registration No. 10-1873527

(Research Paper 0001) Brown J1. & Farquhar C, Endometriosis: an overview of Cochrane Reviews. Cochrane Database Syst Rev. 2014 Mar 10;(3):CD009590

The present invention provides an animal model of endometriosis and a method for manufacturing the same by inducing infection and inflammation of the peritoneum by injecting bacteria into the abdominal cavity of a mouse. In addition, another object of the present invention is to provide a screening method for preventing and treating endometriosis using the animal model.

In order to achieve such an object, the present invention can provide a method of manufacturing an animal model of endometriosis, including the step of infecting the peritoneum with ureaplasma urearithicum.

In the present invention, the " ureaplasma urealyticum " bacteria is Mycoplasmataceae, which does not have a cell wall, has a proliferation ability in an artificial medium, and mainly lives in the mucous membrane of humans or animals. Since this bacterium is an important opportunistic pathogen commonly found in sexually active female reproductive organs, it is known to cause pelvic inflammatory disease, premature birth, chorionitis, and postpartum endometritis.

In one embodiment of the present invention, the method of manufacturing an animal model of endometriosis comprises the steps of: (a) preparing an animal providing uterine tissue and an animal receiving uterine tissue injection; (b) injecting ureaplasma ureariticum into the abdominal cavity of the animal receiving the uterine tissue injection; (c) removing the uterus of the animal providing the uterine tissue and pulverizing it into small tissues; And (d) injecting the pulverized uterine tissue into the abdominal cavity of an animal receiving the uterine tissue. It characterized in that it comprises a.

In one embodiment of the present invention, the animal model may be all mammalian animals except humans, such as horses, sheep, pigs, goats, dogs, mice, rats, guinea pigs, hamsters, and the like. Particularly preferably, it may be a rodent such as a mouse, rat, guinea pig, or hamster, and most preferably, it may be a rat. For the production of an animal model according to the present invention, ICR, DDY, BALB/cA, C57BL/6N, C3H/HeN, DBA/2N, BDF1 (C57BL/6 x DBA/2), CDF1 (CBA/N x DBA/2) ) Or B6C3F1 (C57BL6 x C3H/HeN) can be used as long as they are used as experimental mice, and in the present invention, 6-week-old female mice (C57BL/6) were used.

In one embodiment of the present invention, the timing of injecting the ureaplasma urearithicum intraperitoneally into the abdominal cavity of the animal receiving the uterine tissue can be infected 7 to 1 days before the injection of the uterine tissue, and the present invention At 3 days prior to infection, uterine tissue was injected.

In one embodiment of the present invention, the mouse providing the uterine tissue can be excised the uterus after injecting the follicular maturation hormone into the abdominal cavity 1 to 3 days before the uterine excision, and in the present invention, the follicular maturation hormone 2 days ago. Was injected.

In one embodiment of the present invention, it includes additionally infecting the ureaplasma ureaticum 3 days before the injection of the uterine tissue and once every 3 days after the injection.

In the animal model injected with the uterine tissue, the lesion is removed to confirm endometriosis and observed with the naked eye, or by performing H & E staining (Hematoxylin and eosin stain, H & E stain), or immunohistochemistry (IHC).

In another embodiment of the present invention, the method comprising: administering a candidate substance for treating endometriosis to an animal model for endometriosis prepared in the present invention; And it can provide an endometriosis treatment screening method comprising the step of observing the endometriosis lesion of the animal model.

In the present invention, the administration of the candidate substance for treating endometriosis may be oral, parenteral, intraarterial, intradermal, transdermal, intramuscular, intraperitoneal, intravenous or subcutaneous administration.

In the present invention, the production of cytokines and chemokines, which are inflammation indicators, in mesothelial cells of mice infected with ureaplasma urearithicum, increased expression of adhesion molecule factors, and the onset of endometriosis is induced through a TLR2-dependent signaling mechanism. Confirmed. Accordingly, the endometriosis-inducing animal model of the present invention is very excellent for use as an animal model for endometriosis research.

The present invention provides an endometriosis induction model with excellent endometriosis induction effect by infecting ureaplasma urearithicum, using this to study the inflammatory response that causes endometriosis, and screening for endometriosis treatment to prevent endometriosis and It is expected to contribute to the development of therapeutic agents.

1 shows that ureaplasma urearithicum induces the production of cytokines and chemokines in peritoneal mesothelial cells.
Fig. 2 shows that Ureaplasma urearithicum induces the production of cytokines and chemokines in peritoneal mesothelial cells through a TLR2-dependent signaling mechanism.
3 shows ureaplasma urearithicum activates MAPKs through a TLR2-dependent signaling mechanism in peritoneal mesothelial cells.
4 shows that Ureaplasma urearithicum increases the expression of adhesion molecule factors in peritoneal mesothelial cells through TLR2-dependent signaling mechanisms.
5 shows that Ureaplasma urearithicum increases the activity of MMP2 through a TLR2-dependent signaling mechanism in peritoneal mesothelial cells.
6 shows an increase in the incidence of endometriosis in mice infected with ureaplasma urearithicum through a TLR2-dependent signaling mechanism.

The present invention will be described in detail based on the following examples. The terms, examples, etc. used in the present invention are merely exemplified to explain the present invention in more detail and to aid the understanding of those skilled in the art, and the scope of the present invention should not be interpreted as being limited thereto.

Technical terms and scientific terms used in the present invention represent the meanings commonly understood by those of ordinary skill in the art, unless otherwise defined.

1. Mouse Infection Model

The experimental animals to be used in this study were purchased from Daehan Biolink using normal C57BL/6 female mice. Tlr2-deficient female mice were purchased from Jackson Laboratories. These mice were placed in a room at a constant temperature (22 ± 24 ℃) in the laboratory animal room, and a dark cycle was added with a brightness of 14 hours and a dark color of 10 hours. Food and water were free to use. Mice were acclimatized in the laboratory animal room for a week before the experiment. Mice were sacrificed by cervical dislocation, and animal experiments were conducted with approval according to the regulations of the Animal Care and Use Committee of Konyang University (IACUC; approved protocol number: P-17-09-E-01).

2. Ureaplasma urealyticum culture

Ureaplasma urealyticum was purchased from ATCC (ATCC 27618). The medium consisted of a hydrogen based medium and an oxygen additive solution. The basal medium was prepared by adding 3.5 g of PPLO broth, 10 g of casein, 5 g of gelatin, and 500 mL of distilled water, and sterilized at 121°C for 15 minutes and used. Sterile medium that needs filtration is 10x CMRL 1066 medium 50 mL, yeast extract 35 mL, TC yeast 20 mL, FBS (Fetal bovine serum, calf serum) 170 mL, 0.1% phenol red solution 20 mL, urea 1 g, 205 mL It was made with distilled water and filtered through 0.22 μm. The bacteria were added to a mixed medium in which the above two mediums were mixed at a ratio of 1:1, and cultured at 37 degrees under anaerobic conditions. As the bacteria proliferated, the color of the medium changed from yellow to red. Medium change ^{meant 1} ×10 4 CCU/mL. In order to confirm the more accurate number of bacteria, the number of bacteria was calculated using Mycoplasma IST 2, and the cells and mice were infected.

3. Isolation and culture of mouse mesothelial cells

The peritoneum, liver, spleen, and kidney were removed from normal and Tlr2-deficient mice, and mesothelial cells were isolated. After that, organs were put in a 0.25% trypsin/IDTA solution and stored at 37°C for 50 minutes. Centrifugation was performed at 500 g for 5 minutes, and the supernatant was removed. The pellet was released with a nutrient medium containing 15% FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin in DMEM medium, and incubated in a T25 flask at 37°C for 12 hours. After 12 hours, the supernatant was removed, washed twice with PBS, and mesothelial cells were obtained. The obtained mesothelial cells were subcultured twice and used in the experiment.

4. Endometriosis mouse model

To make a mouse model of endometriosis, a mouse that provides uterine tissue and a mouse that receives uterine tissue infusion (recipient mouse) are needed. Six week old female mice (C57BL/6) were used. First, ureaplasma urearithicum was injected into the abdominal cavity 3 days before the uterine tissue was injected into the recipient mice to infect them. Donor mice were injected with follicular maturation hormone PMSG intraperitoneally 2 days ago. After 2 days, the uterus of the donor mice was excised, crushed into small tissues, and injected into the abdominal cavity of the recipient mice. Afterwards, the ureaplasma ureariticum was infected once every 3 days in the abdominal cavity of the recipient mice, and the occurrence of endometriosis was confirmed through autopsy on the 30th day after uterine tissue injection. To confirm endometriosis, lesions were excised, and the size and number of lesions, H&E staining (Hematoxylin and eosin stain, H&E stain), and immunochemical staining (IHC) were performed.

As a result , the production of cytokines and chemokines, which are inflammation indicators, was increased in mice infected with Ureaplasma urealyticum (Figs. 1 and 2), and MAPKs are activated through a Tlr2-dependent signaling mechanism in peritoneal mesothelial cells. It could be confirmed that it became (FIG. 3). In addition, when Ureaplasma urealyticum was infected in normal mice than in Tlr2-deficient mice, it was confirmed that the expression of the adhesion molecule factor was increased, and the activity of MMP2 was also increased (FIGS. 4 and 5).

From these results, it was confirmed that Tlr2-dependent inflammatory reactions and adhesion molecule factors were highly expressed in mice infected with Ureaplasma urealyticum, resulting in endometriosis (FIG. 6).

Claims (9)

In the method for producing an animal model of endometriosis, comprising the step of infecting the peritoneum with Ureaplasma urealyticum,
(a) preparing a non-human mammalian animal to provide uterine tissue and a non-human mammalian animal to receive the uterine tissue;
(b) injecting ureaplasma urearithicum into the abdominal cavity of the animal receiving the uterine tissue injection;
(c) removing the uterus of the animal providing the uterine tissue and pulverizing it into small tissues; And
(d) injecting the pulverized uterine tissue into the abdominal cavity of an animal receiving the uterine tissue;
Characterized in that it comprises a, endometriosis (endometriosis) method for producing an animal model.
delete The method of claim 1,
The method for producing an endometriosis animal model comprising the step of further injecting ureaplasma urearithicum into the abdominal cavity of the animal receiving the uterine tissue after step (d).
The method of claim 3,
Urea Plasma ureariticum is characterized in that it comprises an additional injection into the abdominal cavity once every 3 days, endometriosis (endometriosis) animal model manufacturing method.
The method of claim 1,
(e) The animal providing the uterine tissue of step (c) is an animal injected with follicular maturation hormone before hysterectomy.
The method of claim 1,
The method for producing an animal model of endometriosis, characterized in that the animal is a rat.
An animal model of endometriosis produced by the method of any one of claims 1, 3 to 6.
Administering a candidate substance for treating endometriosis to the animal model of claim 7; And
Steps to observe endometriosis lesions in animal models
Containing, endometriosis therapeutic agent screening method.
The method of claim 8,
The administration of the candidate therapeutic agent is oral, parenteral, intraarterial, intradermal, transdermal, intramuscular, intraperitoneal, intravenous or subcutaneous administration.

Images