WO2007058021A1

WO2007058021A1 - Animal model and method for producing the same

Info

Publication number: WO2007058021A1
Application number: PCT/JP2006/319415
Authority: WO
Inventors: Tetsuo Maruyama; Hirotaka Masuda; Yasunori Yoshimura; Hideyuki Okano; James Hirotaka Okano; Yumi Matsuzaki
Original assignee: Keio University; Chugai Pharmaceutical Co., Ltd.; Central Institute For Experimental Animals
Priority date: 2005-11-16
Filing date: 2006-09-29
Publication date: 2007-05-24
Also published as: JPWO2007058021A1; JP5288395B2

Abstract

It is intended to provide an animal model which can reflect a lesion site of human endometriosis more faithfully, a method for producing the same, an animal model which enables noninvasive observation of a transplanted cell, and a method for producing the same. The animal model which has a uniform endometriosis lesion can be produced by introducing an expression vector containing a gene encoding an observation marker such as luciferase into a cell isolated from human, transplanting a cell which has come to express the observation marker under the renal capsule of an immunodeficient mouse such as an NOG mouse, and isolating the cell to be transplanted from the endometrium or a focal site of endometriosis.

Description

Specification

Animal model and production method

Technical field

[0001] The present invention relates to an animal model and a method for producing the same.

Background art

Endometriosis is an ectopic occurrence of endometrial cells in various organs other than the uterus, particularly in the peritoneum, ovary, and eclampsia, where it proliferates in response to normal female hormone secretion. · It is an estrogen-dependent disease that bleeds, and its main symptoms are various pains and infertility, including menstrual pain, lower abdominal pain, and back pain. This disease is one of the most common diseases in which the number of patients is as high as 15% of women of reproductive age, and the incidence is increasing in recent years. And this disease is a typical disease that damages women's quality of life (QOL), and disability at the time of women's social advancement also contributes to the era of low birthrates, so it cannot be overlooked socially. It is one of the diseases.

[0003] Organic lesions of eclampsia include blueberry spots, ovarian chocolate cysts, gonococcal adenomyosis, and Douglas fistula endometriosis. Treatments include pharmacotherapy and surgery depending on the situation. Treatment is selected. For drug therapy, GnRH (gonadotropic hormone-releasing hormone) agonist is the first-line drug, but its long-term administration is difficult due to its side effects. In addition, analgesics and low-dose oral contraceptives (LOC) have been shown to be effective for this disease, but they are only symptomatic. As surgical treatment, lesion removal surgery by laparoscopic surgery is common, but complete removal of microlesions is difficult. In this way, radical treatment has been established for the treatment of V and misalignment! As a result, this disease recurs until the patient's menopause. Therefore, establishment of early diagnosis and treatment is expected.

[0004] In addition to the effects of sex hormones that change periodically with menstruation, they are also induced by changes in the genetic structure due to the effects of stress, environmental hormones, the breakdown of the defense system, or the action of environmental hormones themselves However, the cause is not uniform, and there are many unclear points about the mechanism of occurrence.

[0005] On the other hand, endometriosis occurs ectopically and repeats proliferation and exfoliation. The stem cell nature of the cells has also been suggested. Fundamental research is active on its unique biological characteristics as well as its clinical background. As animals that naturally develop endometriosis are limited to some primates, in vivo models as a research tool for eclampsia have been developed as various animal models mainly for human eclampsia intimal transplantation. Yes. For example, an experiment to reconstruct a tissue similar to human endometrial tissue by xenografting a human endometrial tissue piece subcutaneously is possible. (Tu Rai, Rui Matsuura—¾awada, Takashi Murakami, Yuka uzawa, et al. Hu man Reproduction, 2005, 20 (6): 1477-1484).

Disclosure of the invention

Problems to be solved by the invention

[0006] However, in conventional animal models, the amount of transplanted yarn and tissue cells is not constant, the in vivo phenomenon is unstable to reproduce, long-term non-invasive and real-time There were problems such as difficulty in monitoring the organization. Due to the lack of such an effective in vivo model, clinical studies such as elucidation of changes in endometriosis kinetics and screening of therapeutic agents in an in vivo environment have been difficult.

[0007] In view of the above problems, the present invention provides an animal model that can more accurately reflect a lesion of human intimal disease, a method for producing the same, and an animal that enables noninvasive observation of transplanted cells. It is an object of the present invention to provide a model and a manufacturing method thereof.

Means for solving the problem

[0008] In order to solve the above problems, the inventors diligently worked on the creation of a pathological animal model that would be useful for investigation of the etiology of endometriosis and development of therapeutic agents, and luciferase was placed under the renal membrane of NOG mice. By transplanting dispersed cells derived from the human endometrium with the marker introduced, the human endometrial environment mouse that faithfully reproduces the human endometrial environment that can be observed noninvasively in vivo Successfully developed. In this way, an animal model that can more accurately reflect the lesion of human endometriosis and an animal model that enables non-invasive observation of transplanted cells were developed, and the present invention as described below was completed. It came.

[0009] That is, the animal model according to the present invention is a non-human vertebrate model having transplanted cells under the kidney capsule, and using the transplanted cell force observation marker, It is characterized by being able to distinguish vertebrate cell forces by invasive observation. The The location and z or proliferation of the transplanted cells may then be determined in real time by non-invasively observing the vertebrates. The transplanted cells may be dispersed cells. The transplanted cell may have an observation marker that allows noninvasive observation. The observation marker may be a fluorescent marker or a luminescent marker. The vertebrate may be an immunodeficient mouse, for example NO

G (NOD / SCID / y ^nuU ) mice may also be used.

c

[0010] On the other hand, the transplanted cells may be derived from human eclampsia intima or may be derived from a lesion of human eclampsia. In these cases, the present invention also includes a screening method for a therapeutic agent for endometriosis, comprising an administration step of administering a candidate substance for the therapeutic agent for endometriosis to an animal model. Alternatively, the transplanted cells may be derived from a diseased lesion other than human eclampsia. In this case, the present invention also includes a therapeutic drug screening method characterized by including an administration step of administering a therapeutic drug candidate substance for the disease to an animal model.

[0011] Next, a method for producing an animal model according to the present invention is a production method including a transplanting step of transplanting cells under the kidney coating of a vertebrate other than a human, and the cells are placed under the kidney capsule. After being transplanted, the vertebrate cells can be distinguished from the vertebrate cells by non-invasive observation. This cell location and Z or proliferation may be determined in real time by non-invasive observation of vertebrates after transplantation under the renal capsule. The cells may also be dispersed when transplanted. In addition, this production method may include an introduction step of introducing an observation marker capable of noninvasive observation into cells. The observation marker may be a fluorescent marker or a luminescent marker. Further, this observation marker may be introduced into a cell by introducing an expression vector having a gene encoding the observation marker, and the expression vector may be a viral vector. In addition, the production method of the present invention may further include a sorting step of sorting cells into which the observation marker has been introduced using the observation marker as an index before transplantation, or the cells are subjected to the cell before transplantation. The method may further include a second introduction step for introducing the selection marker and a selection step for selecting the cell into which the observation marker is introduced, using the selection marker as an index. The cells to be transplanted may be derived from human eclampsia. Alternatively, it may be derived from a diseased part of a human disease.

On the other hand, the pathological animal model of eclampsia associated with the present invention is a vertebrate model other than human, and a lesion derived from cells isolated from the human endometrium is placed under the renal capsule. It has. The vertebrate may be derived from an immunodeficient mouse, for example, a NOG mouse. The present invention also provides a screening method for a therapeutic agent for endometriosis, comprising an administration step of administering a candidate substance for the treatment of endometriosis to such an animal model of endometriosis. Including.

[0013] Further, the present invention provides a human endometrium with respect to any of the animal models of endometriosis in which cells derived from a human endometrium or a lesion of endometriosis are transplanted. Including a method of reproducing the environment, the method comprising the step of administering an exogenous steroid hormone.

[0014] [Cross-reference with related literature]

The present application claims priority based on Japanese Patent Application No. 2005-332113 filed on November 16, 2005. This document is incorporated herein by reference.

Brief Description of Drawings

FIG. 1 is a diagram showing the results of gross and histological observations of NOG mice transplanted with endometrial dispersed cells in an example of the present invention. Force of transplanted mouse Photo of the isolated uterus (first row) and kidney (second row, the transplant site is indicated by the arrowhead), and H-E staining (third row) and immunostaining of the tissue section of the transplant site An optical micrograph of (fourth column) is shown.

FIG. 2 is a diagram showing the results of immunohistological observation of the transplanted site in the examples of the present invention. The immunostained images (first and second rows) and the nuclear stained images (third row) using the antibodies listed in Table 1 are shown as a superimposed image (fourth row).

FIG. 3 is a diagram showing a method and results of cyclic hormone treatment applied to endometrial cell transplanted mice in the examples of the present invention. (A) is a schematic diagram showing the process of hormone treatment, which is a combination of constant administration of E and cyclic administration of P to mice after cell transplantation.

twenty four

Represents the creation of a hormonal environment that mimics the menstrual cycle. (B) Macroscopic findings of the kidneys of mice sacrificed 11 weeks after cell transplantation (0 and an enlarged view of the transplant site (ii), In addition, an observation image (iii) of the tissue section at the transplantation site by an optical microscope and an enlarged view (iv) of the rectangular frame portion therein are shown.

FIG. 4 is a schematic diagram showing a gene map of a marker gene expression vector introduced into a cell to be transplanted in an example of the present invention.

FIG. 5 is a diagram showing the results of observing endometrial cells cultured by introducing a marker gene in Examples of the present invention. (a) shows a fluorescence microscope image of stromal cells (upper) or glandular epithelial cells (lower) on the culture plate. (b) shows an observation image of the whole culture plate, and the intensity of luminescence by luciferase at the concentration of the scale bar on the right side of the figure.

[Fig. 6] In the example of the present invention, the transplantation site of the abdomen (a, the position of the kidney in the circle) viewed from the outside of the endometrial cell transplanted mouse, the kidney exposed by the laparotomy of the same site (b), FIG. 3 is a diagram showing the results of BLI observation of bioluminescence emitted by the left and right kidneys (c) that were removed. The image power of the luminescence intensity indicated by the density of the scale bar on the right side of each figure is superimposed on the photographic image, and the dark part near the center of the luminescent part has a particularly strong luminescence intensity (about 30000 for a). Above, b shows about 3.5 X 10 ⁵ or more, and c shows about 12000 or more).

FIG. 7 is a BLI observation image (a) of the abdomen of an endometrial cell transplanted mouse subjected to steady hormone treatment in the example of the present invention (a) and a graph showing the result of quantifying the luminescence intensity by analyzing the image (b ). The dark portion near the center of the light-emitting part of the image a shows that the light emission intensity is particularly strong.

[Fig. 8] A BLI observation image (a) of the abdomen of a mouse and hormonal and antihormone-treated mouse treated with hormones and antihormones (a) and a dra ). The dark part near the center of the light emitting part of the image a shows that the light emission intensity is particularly strong.

[Fig. 9] Schematic diagram of cyclic hormone treatment applied to endometrial cell transplanted mice in Example of the present invention (a), image of observation results by BLI of transplanted mouse abdomen (b), and results of quantification thereof. It is a graph (c) showing. The dark and colored portions near the center of the light emitting part of the image b indicate that the light emission intensity is particularly strong.

BEST MODE FOR CARRYING OUT THE INVENTION

= = = Cells to be transplanted = = = The vertebrate model according to the present invention is a vertebrate model having transplanted cells under the kidney capsule, and the transplanted cells can be easily obtained by noninvasive observation of the vertebrate using an observation marker. Can be distinguished from other cells. In this way, if the transplanted cells can be distinguished from the vertebrate recipient animals by non-invasive observation of the vertebrates, the state of the transplanted cells can be observed while the vertebrates remain alive. Will be able to.

[0017] The cells to be transplanted may be derived from any source, but a disease model animal can be prepared by using cells derived from human lesions. In addition, cell morphology is cell clumps, tissue pieces, individually dispersed cells, cells that have been isolated and cultured, cells that have been transplanted into other animals, and cells that have been isolated from animal power through a process. Any cell can be used, but dispersed cells are preferred. This is because the number of cells at the time of transplantation can be made uniform, and the reproducibility of the cell state after transplantation is improved. In addition, the number of cells to be transplanted may be a small number of 10 ⁶ or less! However, it is possible to transplant more cells!

[0018] = = = Transplant site and recipient animal = = =

Next, the site for cell transplantation according to the present invention is under the capsule of the kidney. In addition to being easy to observe as described above, (1) a small amount of cells required for transplantation (10 ⁶ or less) (2) Rejection is less likely to occur, (3) Reasonable factors such as abundant growth factors are suitable. The use of organs such as kidneys that have a pair of animals on the left and right side means that different grafts can be transplanted and compared to the transplant sites on the left and right organs of the same individual. It is also preferable in that the difference between the resulting transplantation results and the effect on the therapeutic agent can be accurately evaluated.

[0019] And, as a vertebrate that receives such transplantation, it is difficult to cause rejection, so that it is excellent in transplantability even if it is desirable to use immunodeficient animals such as nude mice and NOD-SCID mice. Use of NOG (NOD / SCID / y ^nuU ) mice with

C

However, other vertebrates other than humans can be used as recipient animals as long as they have a kidney that enables transplantation as described above.

[0020] = = = Marker for cell differentiation = = =

Observation markers that can be non-invasively observed may have cells to be transplanted or The transplanted vertebrate cell may have an observation marker. As an example of the latter, a genetically modified animal in which a gene for an observation marker is integrated into a body cell, such as a recombinant mouse into which a luminescent gene has been introduced, a so-called “shining mouse” is used as a recipient animal. Can be mentioned. However, it is more preferable to introduce a non-invasive observation marker for the cells to be transplanted, because it is possible to select an appropriate observation marker and an observation method corresponding to it in transplantation.

[0021] If a fluorescent marker such as GFP is used as this observation marker, for example, by irradiating the animal with excitation light, the site where the cells are transplanted can be easily and non-invasively optically observed. However, if a luminescent marker such as luciferase is used, it can be easily observed non-invasively by using an observation method such as bioluminescence imaging (BLI). Alternatively, proliferation can be determined in real time by non-invasive observation of the transplanted vertebrate. Others Appropriate observation methods, such as X-ray computed tomography (computerized tomography), such as radioisotopes used as contrast agents, can be used as observation markers to make the transplanted site observable. tomography, CT) 'Magnetic resonance imaging ^ MRI ·% ^ When combined with positron emission tomography (PET), non-invasive observation is possible.

[0022] As a method for introducing such an observation marker into a cell, if the observation marker is obtained by gene expression, for example, a protein, an expression vector having a gene encoding the observation marker is used in the cell. As an example, a method for infecting a virus is mentioned. As the expression vector, for example, a virus vector is suitable in terms of high infection efficiency, but other commonly used expression vectors such as a plasmid vector and a cosmid vector can also be used. Even if the observation marker is a protein as well as other substances, general introduction methods for introducing foreign substances into living cells, such as the lipofusion method and the electoral position method, can also be used to introduce observation markers. Can be used for

[0023] = = = Sorting of transplanted cells = = =

Furthermore, the transplanted cells remain in the state where donor organisms and patient force are collected, or Although it may be used in the state after the introduction of the observation marker as described above, an operation for selecting cells having the observation marker can be added at the time of transplantation. This is desirable because it allows for more precise differentiation of transplanted cells from recipient animal cells. In this case, the marker for selection may be the same as the aforementioned marker for observation, or a different marker may be used. As such a selection marker, it is preferable to use a visualization marker such as a luminescent marker or a fluorescent marker for the same reason as in the case of the marker for observation described above. For example, if a fluorescent marker is used, flow cytometry can be used for sorting. However, other substances that can be used as selection markers, such as surface antigens and magnetic beads, can also be used as a suitable selection method, such as cell panning and magnetic separation methods. Can be used in combination

[0024] The method for introducing the selection marker into the cell can be the same method as the method for introducing the observation marker described above. The step of introducing the selection marker may be performed simultaneously by the same method as the introduction of the observation marker, or may be performed separately by the same or different method. Here, as an example of performing separately, if the marker is a protein, for example, the ability to separately infect cells with two types of expression vectors having the genes of each marker separately Co-infection of both at the same time This is preferable because both markers are more likely to coexist in the same cell. On the other hand, when the introduction is performed simultaneously, for example, a single expression vector having both marker genes can be constructed and cells can be infected with it. This method is more preferable because the introduction process is simpler and the cells selected using the selection marker have an observation marker.

[0025] = = = Production and utilization of disease model animals = = =

Cell force to be transplanted If it is derived from a diseased lesion in a human or other organism, an animal model of the disease can be prepared according to the present invention. Then, by continuously breeding the created animal model and observing the transplantation site, it is possible to noninvasively follow the changes in lesions derived from the disease. For example, the details to be transplanted By using cells derived from human eclampsia as the cell, an animal model of eclampsia can be produced, and by using cells derived from other diseases such as tumors, tumor animal models can be produced. Therefore, it is possible to easily observe a lesion derived from such a disease non-invasively.

[0026] In the case of a conventional animal model, it is possible to confirm the tissue structure of the lesioned part only after performing excisional biopsy, histological observation after sacrifice, and the like. However, in the animal model of the present invention, the vertebrate cell force can be distinguished by non-invasive observation of the transplanted cell force vertebrate using the observation marker. The condition can be observed continuously and quantitatively in a non-invasive manner. Furthermore, if the lesion or the entire animal is treated, and changes in the transplantation site are traced to the treatment, analysis of the effectiveness of the treatment and basic research for investigating the cause of the disease will be conducted. Yes. For example, with respect to an endometriosis animal model which is one embodiment of the present invention, a cyclic hormone that mimics the change in hormone concentration according to the menstrual cycle by adjusting the dose and timing. By conducting drug administration experiments, the menstrual cycle-like hormonal environment can be continuously reproduced, and as a result, changes in the lesion similar to changes in the menstrual cycle in the endometrium can be analyzed. In addition, if an animal model of endometriosis drug candidates is administered to such animal models and screening is performed for candidate substances that can improve lesions, development of endometriosis drugs Can be useful.

[0027] Thus, the animal model of the present invention has the advantage of being able to observe a lesion in real time in a non-invasive manner, and thus a disease state that requires continuous tracking over a long period of time. When a model, for example, a tumor cell derived from a tumor patient is transplanted, it is extremely useful as a model of a disease state of a tumor. In addition to the animal model itself described so far, a method for producing such an animal model is also included in the scope of the present invention. It can be provided for the elucidation of the cause of the corresponding disease and the development of therapeutic methods and drugs. For example, if a tumor therapeutic drug candidate substance is administered to an animal model of a tumor prepared as described above, and screening is performed for a substance that can improve the lesion, It can be used to develop therapeutic drugs.

[0028] = = model animal for eclampsia = =

Here, the endometriosis or pathological animal model of endometriosis, which has a diseased part derived from cells from which human endometriosis or endometriotic lesion force has been isolated, under the kidney capsule, has no marker introduced. However, it can be used effectively. This pathologic model shows that by administering periodic exogenous steroid hormones, the isolated endometrial cell force reconstructed tissue shows changes similar to the endometrial menstrual cycle change, i.e. This is the first model animal to reproduce the human endometrial environment. For this animal model, exogenous steroid hormones such as estrogen and progesterone should be administered by various methods such as subcutaneous and intraperitoneal injection, dietary administration, and sustained release subcutaneous transplantation. Can do. Then, if an analysis such as a histological observation is performed on the transplantation site of an animal model that reproduces the menstrual cycle-like hormonal environment as described above by adjusting the hormone administration, it is possible to analyze eclampsia. Useful for research. Furthermore, for example, a substance that can improve the lesion by administering a candidate drug for treating endometriosis to the model animal for endometriosis, a substance that causes regression of the lesion, or a periodic By selecting a substance that becomes resistant to mon administration and does not respond to hormones, it can be used to develop endometriosis drugs.

Example

[0029] Hereinafter, the best mode for carrying out the present invention will be described in further detail using examples.

[0030] There is no particular explanation for the embodiments and examples! In the case of J. Sambrook, E. F. Fritsch &

T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold bpnng Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, A method described in a standard protocol collection such as JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd., or a modified or modified method thereof is used. In addition, when using commercially available reagent kits or measuring devices, use the protocols attached to them unless otherwise specified.

[0031] It should be noted that the object, features, advantages, and ideas of the present invention are described in the present specification by those skilled in the art. The present invention can be easily reproduced by those skilled in the art based on the description of the present specification. The embodiments and specific examples of the invention described below show preferred and embodiments of the present invention, and are shown only for illustration or explanation. It is not limited to them. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

[0032] = = = Preparation of endometrial dispersed cells (SDEC) and transplantation of NOG mice under the renal capsule = = Endometrial tissue fragments isolated from humans, 0.2% (w / v) Collagenase (Wako Pure Chemicals, Osaka), DMEM + medium containing 0.05% (w / v) DNaseI (GIBCO, California, USA) (1% antibiotic antifungal (GIBCO), 10% In Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich, Missouri, USA) containing urine fetal serum (BioWest, Florida, USA) The cells were subjected to enzymatic cell dispersion treatment by shaking at ° C for 1.5 hours. Further, the cells were dispersed to a single cell state by mechanical dispersion treatment in which the cells were passed through a 40-m pore size cell strainer (BD Biosciences, Massachusetts, USA).

[0033] In order to separate the endometrial stromal cells from the dispersed cells, the layers were layered on FicoU-Paque PLUS (Amersham Biosciences USA-U.J.) For 15 minutes at 780 X g. A single cell dispersion was recovered from the interface layer. On the other hand, resuspend the cell mass remaining on the cell strainer and pass it through the cell strainer again after enzyme treatment with 0.05% (w / v) trypsin EDTA and 0.05% (w / v) DNaseI solution. Thus, endometrial gland epithelial cells were collected as a single cell dispersion.

[0034] NOG (NOD / SCID / _y ^nuU ) mice (Laboratory Animal Central Laboratory, ^Kaoru Kawasaki) 10% bent

C

A 40 μl injection of valpital (Dainippon Pharmaceutical) -containing phosphate buffer (Sigma) was injected intraperitoneally to cause intoxication, and the ovaries were removed to remove the effects of endogenous female hormones. In this mouse, a tapered capillary pipette with an inner diameter of 1 mm was passed through one end of the kidney and the tip was stopped just below the capsule on the other end. Approximately 5 X 10 ⁵ SDECs obtained by the above method (5: Transplantation was performed by injecting L0 μl of DMEM + dispersion in medium). [0035] = = = Morphological and histological observation of the transplant site = = =

For some NOG mice that have undergone SDEC transplantation, estrogen)

Two letts (Innovative Research of America, Florida, USA) were transplanted subcutaneously by 0, 1 or 2 tablets, and different amounts of female hormones were continually administered, and the breeding was continued. After a predetermined period of time, the kidney was removed and used for the following gross findings and immunohistological observations.

[0036] First, the excised kidney was embedded in Tissue-Tek OCT compound (Sakura Finetech, Califol, U.S., USA), frozen, and 6 μm thick using a cryostat (Leica Microsystems, Germany's Wetzlar). Sliced continuously. A portion of the obtained frozen section was subjected to tissue observation with a hematoxylin 'eosin (HE) staining solution (Sigma-Aldrich), and the remaining sections obtained on the slide glass were summarized in Table 1. The primary antibodies shown in the above were used in combination, followed by immunostaining according to the most appropriate method shown below.

[0037] [Table 1] List of antibodies used in this example

Antigen clone Source Isotype for dilution ratio cytokeratin MNF1 16 ⁰ A mouse IgG 1 100 vimentin V9 ³ B Gy3-conjugated mouse IgG 1 400 vimentin V9 ³ A mouse IgG 1 400 卜 CD9 P 1/33/2 A mouse IgG 1 100 mouse CD9 M-L13 C PE-conjugated mouse IgG undiluted human CD10 SS2 / 36 A mouse IgG 1 100 mouse CD13 WM-47 A mouse IgG 1 100 human CD31 JC70A ^a A mouse IgG 1 100 mouse CD45 2B1 1 + PD7 / 26 ^a A mouse IgG 1 100 mouse CD45 T29 / 33 A PE-conjugated mouse IgG undiluted progesterone receptor (PR) PgR636 A mouse IgG 1 100 human smooth muscle actin (Qi SMA) 1 A4 ^c B Gy3 -conjugated mouse IgG 1 400-smooth muscle actin (Qi SMA) 1 A4 ^c A mouse IgG 1 400 prolactin polyclonal D rabbit antiserum 1 1000 mouse CD31 MEC13.3 ^b C rat IgG 1 100 mouse CD45 30-F1 1 ^b C rat IgG 1 100 Mouse erythrocytes TER-1 19 ^b E rat IgG 1 100 a: Antibody that reacts only with human antigen A: DakoCytomation (Grostrap, Denmark) b: Responds only to mouse antigen Corresponding antibody B: SIGMA Chemical (Missouri, USA)

c: Antibody reacting with human and mouse antigens C: BD PharMingen (California, USA).

D: Upstate Biotechnologies (New York, USA) E: e-Bioscience (California, USA) [0038] That is, a part of the section was fixed with 100% acetone on ice at 4 ° C for 10 minutes, and then washed with a phosphate buffer. The rest was fixed with 4% paraformaldehyde at room temperature for 20 minutes and then washed with phosphate buffer. These fixed sections were immersed in a 10% ushi serum albumin solution for 30 minutes for blocking treatment, and then stained for 1 hour in a predetermined diluted solution of each primary antibody at room temperature for antibody staining. . The stained sections were washed and secondary fluorescently stained with Alexa Fluor 488 (for green fluorescence) or Alexa Fluor 568 (for red fluorescence) labeled secondary antibody (Molecular Probes, Inc., Oregon, USA). Furthermore, the sections were counterstained with the nuclear stain Hoechst 33258 (Sigma) for 5 minutes. The sections after each staining were encapsulated with VECTAS HIELD (Vector Laboratories, California, USA) and examined with a DMIRE2 inverted fluorescence microscope (Leica Microsystems). Images were captured with a VB-700 CCD camera (Keyence Corp., Osaka City). I took it in.

[0039] In the kidney of the transplanted mouse thus obtained, a new lesion that can be identified with the naked eye is generated at the site where the subcapsular transplant was performed (Fig. 1, second row arrowhead). The size gradually increased with the amount of E applied (Fig. 1, upper relative force in the order of E force, 0, 1

twenty two

2 doses). In addition, from the observations of transmitted light and immunostained images of the tissue sections of the lesion, it was found that the inside also had a tissue structure similar to that of the endometrium (third column in Fig. 1 and (4th column).

[0040] Subsequently, lesions newly formed by performing immunostaining with primary antibodies specific to human stromal cells, glandular epithelial cells, smooth muscle cells and blood cells on tissue sections, respectively. It was clarified that the cells constituting it were derived from humans and the tissue structure was very similar to that of the endometrium (Fig. 2). In addition, subcutaneous transfer of E sustained release pellets

2

Lutem * 25 (Imperial Organs, Tokyo), a progesterone (P) formulation

Four

Reproduction of the menstrual cycle-like hormonal environment by applying cyclic hormone treatment to transplanted mice in combination with periodic administration by subcutaneous injection of the drug (the method is shown in Figure 3a as a schematic diagram of the administration scheme) . As a result, a macroscopic change in the transplanted site, similar to the red spot, which is a peritoneal activity lesion of endometriosis, was induced (Fig. 3b (i, ii)). it is a component force ivy that structures similar to endometriosis lesions (FIG. 3 b (iii, iv)) 0 Thus, the transplantation of a child Imperial Household membrane cells into animal models of the present invention, uterus The intimal tissue could be structurally reconstructed.

[0041] = = = Construction of marker gene expression vector and introduction into endometrial isolated cells = = = Lentivirus-derived Venus (fluorescent marker protein that is a GFP analog derived from YFP) Expression vector pCSII-EF-MCS-IRES2-Venus (Non-patent document 2: M lyoshi, H., Blomer, U., Takahashi, M., Gage, FH & Verma, IM Development of a self-inactivating lentivirus vector. J Virol. 72, 8150-8157 (1998)) to the restriction enzyme BamHI cleavage site, and the gene for the red luminescent luciferase CBR luc (a bioluminescent marker protein that is a mutant derived from the beetle luciferase) Was introduced to construct a marker gene expression vector (Fig. 4).

[0042] Endometrial dispersed cells (SDEC) prepared by the same method as described above were cultured, and SDEC on a culture plate grown to a confluency of about 60% was compared with a multiplicity of infection of 1: 1. The marker gene expression vector solution was added so as to maintain the ratio, and the culture was further continued. Here, when stromal cells and glandular epithelial cells on the culture plate were examined with an inverted fluorescence microscope, Nikon ECLIPSE TS100 (Nikon, Tokyo), both of them were observed to emit fluorescence and bioluminescence (Fig. 5). ). When the cell culture reaches confluence, the cells on the plate are collected and transferred to HBSS + medium containing 2 μg Zml of Probidium Iodine (PI). After staining only dead cells, the MoFlo cell sorter (Cytomation , Colorado, USA). Sorted cells that were negative for PI staining and positive for Venus fluorescence were used as live cells in which the marker gene had been introduced into SDEC in the following transplantation experiments.

[0043] = = = Transplantation of marker gene-introduced cells and observation of the transplant site by BLI = = =

The sorted gene-transferred cells were transplanted under the kidney capsule of ovariectomized NOG mice in the same manner as described above, and the mice were bred.

[0044] A bioluminescent substrate D-luciferin (Sumisho Bioscience, Tokyo) was intraperitoneally administered to the transplanted mice under anesthesia by administration of 2% isoflurane (Merck's Whey, Osaka) at a dose of 150mgZkg body weight. Immediately after that, the abdomen of the transplanted mouse in the living state was observed using a BLI viewing image (Xenogen-IVIS 100 cooled and CD optical macroscopic imaging system) (Sumisho Bioscience), and bioluminescence was imaged. . As a result, luminescence was detected from the site considered to correspond to the transplant site (Fig. 6a). Some transfers for reference The abdomen of the transplanted mouse was incised to expose the periphery of the kidney (Fig. 6b), and only the kidney was removed (Fig. 6c), and each was similarly observed with a BLI device. It was confirmed that only the lesioned part emitted light.

[0045] = = = BLI observation of the transplanted site with constant hormonal treatment = = =

By substituting 0, 1 or 2 E sustained-release pellets subcutaneously into a portion of the mice that have received the SDEC transplanted with the marker gene, the same as described above.

2

Hormone treatment was performed. Breeding was continued, and BLI observation of the transplanted mouse abdomen was carried out in the same manner as above (Figure 7a). For each of the obtained bioluminescence images, a specific region was determined and the luminescence intensity was quantified. As a result, the passage of time and E

It was observed that the intensity of the luminescent signal of the lesion force gradually increased with both doses of administration (Fig. 7b).

[0046] = = = BLI observation of transplanted sites treated with anti-hormones = = =

Marker gene transfer 1 tablet of sustained release E for a part of mice transplanted with SDEC

2

A constant hormonal treatment was performed by subcutaneous transplantation. In addition, an anti-hormone was administered to some of these by daily subcutaneous injection of estrogen receptor antagonist ICI 182,780 (Tocris Cookson Inc., Missouri, USA) at a dose of 100 μg / ml. Processed. As a result of BLI observation of the lesion at 1, 2, and 3 months after transplantation, a decrease in the luminescence signal was observed from the first month to the second month (Fig. 8). Thus, the effect of administration of antihormonal agents on lesions derived from the eclampsia intima was observed.

[0047] = = = BLI observation of transplanted sites with cyclic hormone treatment = = =

Marker gene transfer For some mice transplanted with SDEC, E sustained release pellet skin

2

Constant hormone treatment was performed by subtransplantation. In addition, a part of them was subjected to periodic hormonal treatment by subcutaneous injection of 1 mg dose of P preparation in the same manner as described above (Figure 9a).

Four

A schematic diagram of the chiem is shown). When BLI observation was performed, it was shifted according to the periodic administration of P administration.

Four

An increase and decrease in the luminescence signal from the planting site was observed (Figures 9b and c). Thus, endometrial lesions showed similar responses to endometrium to changes in hormone concentrations that mimic menstruation. Therefore, the menstrual cycle of the endometrium was functional using a transplanted mouse model. It was possible to reproduce it. [0048] = = = Conclusion = = =

As described above, the animal model of the present invention is particularly suitable for continuously observing the kinetics of transplanted cells over a long period of time. For example, if cell transplantation derived from the eclampsia is performed, the pathology of endometriosis It was possible to create an animal model that reproduced the above. Furthermore, it is possible to observe such an animal model non-invasively, continuously and quantitatively with the method of the present invention, elucidating the etiology of a disease from which transplanted cells are derived, and therapeutic agents. For example, in the case of an animal model of eclampsia, it is clear that it is possible to provide an animal model that can track changes in the pathology according to the administration of hormones and antihormonal agents. became.

Industrial applicability

[0049] According to the present invention, there are provided an animal model that can more accurately reflect a lesion of human endometriosis, a method for producing the same, an animal model that enables noninvasive observation of transplanted cells, and a method for producing the animal model. Can be provided.

Claims

The scope of the claims

[I] A non-human vertebrate model having transplanted cells under the kidney capsule,

An animal model, wherein the transplanted cells can be distinguished from the vertebrate cells by non-invasive observation of the vertebrate using an observation marker.

[2] The animal model according to claim 1, wherein the position and Z or proliferation of the transplanted cells can be determined in real time by noninvasive observation of the vertebrate.

[3] The animal model according to claim 1 or 2, wherein the transplanted cells are dispersed cells.

[4] The animal model according to any one of claims 1 to 3, wherein the transplanted cells have the observation marker capable of noninvasive observation.

[5] The animal model according to any one of claims 1 to 4, wherein the observation marker is a fluorescent marker or a luminescent marker.

[6] The animal model according to any one of [1] to [5], wherein the vertebrate is an immunodeficient mouse.

[7] The animal monot according to claim 6, wherein the immunodeficient mouse is a NOG mouse.

[8] The method according to any one of claims 1 to 7, wherein the transplanted cells are derived from a human endometrium! The animal model described in any one of the above.

[9] The transplanted cell is derived from a diseased lesion of a human disease,

~ 7 !, Animal model as described in somewhere.

10. The animal model according to claim 9, wherein the disease is eclampsia.

[II] A screening method for a therapeutic agent for the disease, comprising an administration step of administering a candidate substance for the therapeutic agent for the disease to the animal model according to claim 9.

[12] A screening method for a therapeutic agent for endometriosis, comprising an administration step of administering a candidate substance for the therapeutic agent for endometriosis to the animal model according to claim 8 or 10.

[13] A method for producing an animal model, comprising a transplantation step of transplanting cells under the kidney capsule of a non-human vertebrate, After the cells are transplanted under the kidney capsule, the vertebrate cell lines can be distinguished by non-invasive observation of the vertebrates.

[14] The production according to claim 13, wherein the location and Z or proliferation of the cells can be determined in real time by noninvasive observation of the vertebrates after transplantation under the kidney capsule. Method.

15. The production method according to claim 13 or 14, wherein the cells are dispersed when transplanted.

[16] The production method according to any one of [13] to [15], further comprising an introduction step of introducing the observation marker capable of noninvasive observation into the cell.

[17] The production method according to claim 16, wherein the observation marker is a fluorescent marker or a luminescent marker.

[18] The production method according to claim 16 or 17, wherein the observation marker is introduced into the transplanted cell by introducing an expression vector having a gene encoding the observation marker. .

19. The production method according to claim 18, wherein the expression vector is a viral vector.

[20] The production method according to any one of claims 16 to 19, further comprising a selection step of selecting cells into which the observation marker is introduced using the observation marker as an index before the transplantation. .

[21] a second introduction step of introducing a selection marker into the cells before the transplantation;

20. The production method according to any one of claims 16 to 19, further comprising a selection step of selecting a cell into which the observation marker is introduced using the selection marker as an index.

[22] The production method according to any one of [13] to [21], wherein the cell is derived from a human eclampsia lining.

[23] The production method according to any one of [13] to [21], wherein the cells are derived from a lesion of a human disease.

[24] A non-human vertebrate model, An animal model of endometriosis having cells isolated from the human endometrium under the kidney capsule.

[25] The animal motenore according to claim 24, wherein the vertebrate is an immunodeficient mouse.

26. The animal moire according to claim 25, wherein the immunodeficient mouse is a NOG mouse.

[27] A screening screen for an endometriosis drug, comprising an administration step of administering a candidate drug for the treatment of eclampsia to the animal model according to any one of claims 24 to 26. Jung way.

[28] Reproducing a human endometrial environment characterized by including an administration step of administering an exogenous steroid hormone to the animal model according to any one of claims 8, 10, and 24-26. how to.