JP2002034387A - Method for producing orthotopic grafted animal of colon cancer cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To readily cause colon cancer exposed to the interior of the alimentary canal cavity of an animal in a short period. SOLUTION: This method for producing an orthotopic grafted animal of colon cancer cells is characterized by carrying out a treatment for inducing inflammations in the large intestine of the animal and then grafting the colon cancer cells into the large intestine of the animal.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a colon cancer cell orthotopically transplantable animal capable of obtaining colon cancer exposed in the colon lumen in a short period of time.

[0002]

2. Description of the Related Art As colorectal cancer experimental animal models,
(1) a chemical carcinogenesis model using a carcinogen such as dimethylhydrazine, azoxymethane, and a nitroso compound; (2) a tumor suppressor gene knockout mouse such as APC and p53; and (3) a colon cancer cell transplantation model (an ectopic transplantation model ( Subcutaneous transplantation) or orthotopic transplantation model (transplantation under the mucosa from the colon scleral membrane) is common. Of these, in the chemical carcinogenesis model,
Although a tumor can be generated in the colonic mucosa, the experiment requires a long period of time, so that considerable labor and time must be spent to obtain the experimental result. In addition, the tumor suppressor gene knockout mouse is a model in which colorectal cancer develops because the gene for suppressing colorectal cancer does not function, and colorectal cancer exposed in the lumen in a relatively short time occurs. It was difficult to say that the model was easy to use because it was expensive and subject to various restrictions depending on the contract with the supplier. On the other hand, the conventional ectopic and orthotopic cell transplantation model has the advantage that the experimental results can be obtained in a short period of time, but since the transplantation site is different from the colonic mucosal surface, the tumor is exposed to the colonic mucosal surface It is heterogeneous in colorectal cancer models.

[0003]

Accordingly, an object of the present invention is to provide a method for producing a colon cancer cell transplanted animal in which a tumor is exposed on the mucosal surface of the colon lumen in a short period of time.

[0004]

Means for Solving the Problems In view of such circumstances, the present inventors have conducted intensive studies. As a result, after performing treatment for inducing inflammation in the large intestine, colon cancer cells were injected into the rectum and transplanted. The present inventors have found that colorectal cancer exposed in the lumen of the large intestine can be easily caused in a short period of time, and the present invention has been completed.

That is, the present invention provides a method for producing a colon cancer cell-transplanted animal, which comprises subjecting an animal to a treatment for inducing inflammation in the colon and then transplanting the colon cancer cell into the colon of the animal. It is intended to provide a colon cancer cell transplanted animal obtained by the method. Further, the present invention, when producing a colon cancer cell transplanted animal by the method, administering a test substance before or after performing a treatment to induce inflammation in the colon of the animal,
An object of the present invention is to provide a method for selecting an antitumor agent, which comprises comparing tumor formation between an animal to which the test substance has been administered and an animal to which the test substance has not been administered.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, animals
Mouse, rat, guinea pig, etc. can be used,
In particular, a mouse, which has many types of colorectal cancer cells to be transplanted and knowledge about them, is preferable. In the present invention, first, a treatment for inducing inflammation (inducing injury) is performed on the large intestine, particularly the colon, of the animal. Thereby, when the cancer cells are subsequently injected into the large intestine, the cancer cells can be established on the large intestinal mucosa. Specifically, it is preferable to administer to the animal a substance having an inflammation-inducing action such as dextran sulfate sodium (DSS), hydrochloric acid (HCl), acetic acid, and trinitrobenzenesulfonic acid (TNBS). For example, oral administration of DSS
Chemical treatment by rectal injection of HCl or acetic acid (eg, HC
and then neutralization with potassium hydroxide), and intrarectal injection of TNBS. Among them, it is particularly preferable to use DSS from the viewpoint of the appearance rate of the tumor and the like. The dose of these substances is not particularly limited as long as inflammation can be induced.
3.0 to 5.0% by weight for S, 0.1 to 5.0% for HCl
In the case of 0.5N and TNBS, the amount is preferably 1 to 2 mg.

After such treatment, induction of inflammation is confirmed by weight loss, diarrhea, bloody stool, etc., and then colon cancer cells are transplanted into the large intestine. The method of transplantation may be any of intrarectal injection, injection after incision, etc., and is not particularly limited, but intrarectal injection is preferred from the viewpoint of simplicity and cell fixation. As the colon cancer cells to be used, cancer cells syngeneic with animals are preferable. Examples of the combination of animal and colon cancer cells include BAL
B / c mice and Colon 26 cells, C57BL / 6 mice and CMT-93 cells, and the like. In addition, if an immunodeficient animal such as a nude mouse is used, it is possible to transplant a heterogeneous cell such as a human colon cancer cell. Transplantation may be performed by injecting colorectal cancer cells into the rectum, and is preferably fixed with a clip or the like so that the injected cells do not leak.

[0008] By processing as described above, 3-4
After a week, colon cancer exposed in the colon lumen can be caused, and a colon cancer transplanted animal can be easily produced. Since the method of the present invention involves transplanting cancer cells, it is unsuitable for analyzing the canceration process of cells during carcinogenesis.However, cancerous cells proliferate in mucous membranes to form tumors. Suitable for analyzing processes. Therefore,
Search and evaluation of factors (substances) that suppress the growth of colon cancer cells, including intestinal bacteria and intestinal bacterial metabolites,
It is also suitable for evaluating chemotherapeutic agents because it is a model for rectal cancer that is difficult to remove by surgery.

In order to select an antitumor agent using the colon cancer cell-transplanted animal obtained by the present invention, for example, when producing a colon cancer cell-transplanted animal as described above, a treatment for inducing inflammation in the colon of the animal. Before or after administering the test substance,
What is necessary is just to compare the tumor formation between the animal to which the test substance is administered and the animal to which the test substance is not administered. The administration method of the test substance may be any of oral, intravenous, intraperitoneal and subcutaneous administration.
The determination of tumor formation can be performed using the tumor appearance rate and tumor volume as indices. If tumor formation is suppressed in the animal to which the test substance has been administered, the test substance is expected to have an action as an antitumor agent.

[0010]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Animals: BALB / c female mice 7 weeks old were purchased,
MF feed (manufactured by Oriental Yeast Co., Ltd.) and water were freely available. (2) Tumor cells: Colon2 subcultured in vitro
Six cells (syngeneic tumor of BALB / c mouse) were used. Cells were cultured using fetal calf serum (10% FCS, Boeringer M
anheim) and kanamycin sulfate (100 μg / mL, SIGM)
The test was performed in RPMI1640 medium (GIBCO) supplemented with A). The cells grown in a monolayer on the bottom of the culture dish are peeled off using Cell Dissociation Solution (SIGMA) to form a single cell suspension, which is centrifugally washed and then RPMI1640 (F
(CS not added, kanamycin added). The number of cells in the cell suspension was calculated using a hemocytometer after dilution with Cell Dissociation Solution. 1 × 10 ⁷ cells / mL
The cell suspension adjusted to the above was used.

(3) Transplantation of Colon 26 into colonic mucosa: (a) HCl-KOH method; mice were fasted for 16 hours, anesthetized with Nembutal, and an intravenous indwelling catheter (Angioc)
0.1 mL of HCl (0.02, 0.05, or 0.1 N) was rectally injected using ath (24G-3 / 4 "), Becton Dikinson) to exfoliate the colorectal mucosa. 45 seconds later,
The same concentration of potassium hydroxide (0.
02, 0.05, or 0.1 N), and then washed by injecting 0.2 mL of Hanks buffer (HBSS). 6.5 hours after such operation, colon 26 cells were rectally injected into mice under Nembutal anesthesia (1 × 1).
0 ⁶ cells / mouse) and held 10 minutes with the anus so as not leak stopped in small clips (Natsume). (b) TNBS method; mice were fasted for 16 hours, anesthetized with Nembutal, and 0.1 mL using a venous indwelling catheter
(50% ethanol solution) of 2,4,6-TNBS (T
NBS (SIGMA), 1 mg / mouse) solution was injected rectally. 6.5 hours later, Colon 26 cells were injected in the same manner as in (a).

Four weeks after the transplantation operation of (3), the mice were exsanguinated and extirpated, and the colorectum was collected and cut along the long axis. When the tumor formation was observed with the naked eye and under a stereomicroscope, it was found that the mass was 0.
A tumor was observed in the rectum of two out of five mice in the 1N HCl-KOH treatment group and one in five mice in the TNBS treatment group.
These masses were injected intrarectally based on histopathological diagnosis
Colon 26 cells were confirmed to be colon tumors formed by colonization and proliferation.

Example 2 (1) Animals: 9-week-old BALB / c mice were purchased and fed food and water ad libitum, and 3 to 13 mice were used per group. (2) Tumor cells: Colon prepared as in Example 1
2.5 × 10 ⁷ or 5 × 10 ⁷ cell suspension with 26 cells
A suspension adjusted to s / mL was used.

(3) Inflammation induction on colonic mucosa (DSS)
Method): (a) Drinking water administration method using disposable type sterile water supply container; DS using disposable type sterile water supply container (AN pack and SE water supply nozzle, manufactured by Musashi)
S (molecular weight: about 25,000, manufactured by Tokyo Chemical Industry Co., Ltd.) was administered in drinking water. 5% by weight DSS solution prepared with sterile distilled water
Mice were allowed free access for 7 days. The sterile water supply was changed twice a week. (b) Mixed feed administration method using freeze-dried block feed: After adding 3, 4 or 5% by weight of DSS to MF feed (manufactured by Oriental Yeast Co., Ltd.), a block-shaped freeze-dried solid feed was prepared.
This diet was administered to 10 week old mice for 7 days.

(4) Transplantation of Colon 26 cells into colonic mucosa: After a mouse was fasted for 18 hours, Nembutal was anesthetized, and a Colon 26 cell suspension (40 μL) was injected into the rectum using an Eppendorf pipette (for 100 μL). . The anal part was fixed for 30 minutes using a small clip (jujube) to prevent the suspension from leaking.

Three to four weeks after the transplantation operation of (4), the mice are exsanguinated and extirpated, the colorectum is collected, and incised along the long axis.
After removing the contents, the mass formation was observed with the naked eye and under a stereoscopic microscope. The tumor appearance rate in each treatment was determined from the number of mice excluding mice that died of colitis due to DSS administration,
The results are shown in Table 1.

[0018]

[Table 1]

Example 3 In the same manner as in Example 2, after treating with a 5% by weight DSS solution,
A test substance was administered to a mouse transplanted with 2 × 10 ⁶ (cells / mouse) of lon26, and its antitumor effect was evaluated. Irinotecan hydrochloride (CPT-11,
Using Yakult Honsha Co., Ltd.), the amount shown in Table 2 was dissolved by heating in distilled water, then diluted and administered by forced stomach. CPT-11 administration was performed 7 days after tumor implantation, and 200 mg / kg administration was performed 5 times on days 7, 10, 13, 16, and 19 after implantation. On day 21 after the transplantation, tumor formation of each mouse was evaluated in the same manner as in Example 2. The tumor volume was determined by the following equation. Table 2 shows the results.

[0020] Tumor volume ^{= L × W 2/2 (} L; major axis of tumor (mm), W; minor axis of the tumor (mm))

[0021]

[Table 2]

[0022]

According to the present invention, colon cancer exposed in the colon cavity of an animal can be easily caused in a short period of time. The obtained colorectal cancer cell orthotopic transplant model animal can be suitably used for selection and evaluation of factors (substances) that suppress the growth of colorectal cancer cells, evaluation of chemotherapeutic agents, and the like.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Shoichi Kado 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (72) Inventor Minoru Ando 1-1-1-1 Higashi-Shimbashi, Minato-ku, Tokyo No. 19 Yakult Honsha (72) Inventor Kazumi Uchida 1-11-1 Higashi-Shimbashi, Minato-ku, Tokyo (72) Inventor Koji Nomoto 1-1-1, Higashi-Shimbashi, Minato-ku, Tokyo No. 19 Yakult Honsha (72) Inventor Masaki Morotomi 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo F-term (reference) 2G045 AA26 AA29 BA14 BB10 BB14 BB20 BB51 CB02 CB17 CB26 FA16 FA18 GC22

Claims (6)

[Claims] 1. A method for producing a colorectal cancer cell-transplanted animal, comprising subjecting an animal's colon to a treatment for inducing inflammation, and then transplanting colon cancer cells into the animal's colon. 2. The method according to claim 1, wherein the colorectal cancer cell-transplanted animal has colorectal cancer exposed in the lumen of the large intestine. 3. The method according to claim 1, wherein the colon cancer cells to be transplanted are cancer cells syngeneic with the animal. 4. The method according to claim 1, wherein the treatment for inducing inflammation in the large intestine comprises administering to the animal a substance selected from sodium dextran sulfate, hydrochloric acid and trinitrobenzene sulfonic acid. . A colorectal cancer cell-transplanted animal obtained by the method according to any one of claims 1 to 4. 6. When producing a colon cancer cell transplanted animal by the method according to any one of claims 1 to 4, a test substance is administered before or after performing a treatment for inducing inflammation in the colon of the animal, A method for selecting an antitumor agent, comprising comparing tumor formation between an animal to which the test substance has been administered and an animal to which the test substance has not been administered.