JP2014233281A - Method for isolating esophageal epithelial stem cells - Google Patents

Abstract

The present invention provides a method for isolating esophageal epithelial stem cells from animal esophageal tissue, a screening method for anticancer substances effective for esophageal cancer, and a screening method for identifying carcinogenic factors that may cause esophageal cancer. To do. Esophageal epithelial cells are collected from esophageal tissue using proteolytic enzyme, and the esophageal epithelial cells are embedded in a gel matrix and cultured in a medium containing epidermal growth factor, thereby producing esophageal epithelial stem cells. Can be isolated. Further, screening of anticancer substances and carcinogens becomes possible using isolated esophageal epithelial stem cells. [Selection] Figure 2

Description

  The present invention relates to a method for isolating esophageal epithelial stem cells, a screening method for anticancer substances using the cells, and a screening method for carcinogenic factors.

  The esophagus is a long luminal organ that connects the oral cavity and stomach, and its wall consists of a mucosal layer composed of stratified squamous epithelium, a submucosal layer, a muscular layer, and an outer membrane.

  Esophageal cancer is a malignant tumor with a high incidence of morbidity in the 6th and 7th place by organ in terms of both incidence and mortality in Japan, but there are clear risk factors such as viruses and pathogens that cause the onset or smoking. This is a disease whose countermeasures are becoming clearer than those of other organs for which molecular targeted therapies are being developed.

  Esophageal cancer is an epithelial malignant tumor whose origin is the stratified squamous epithelium, but how the turnover of the stratified squamous epithelium of the esophagus is controlled has not been fully elucidated.

  Therefore, further elucidation of the cell proliferation mechanism of esophageal epithelial tissue is very important in establishing an effective treatment method for esophageal cancer.

  Patent Document 1 describes a method for culturing stem cells of intestinal mucosal epithelium, gastric mucosal epithelium and pancreatic ductal epithelium derived from mesoderm. However, examples of collecting and culturing tongue epithelial stem cells consisting of stratified squamous epithelium derived from ectoderm are not shown.

WO2010 / 090513

  As described above, a method for isolating esophageal epithelial stem cells has not yet been established. In order to analyze the homeostasis of esophageal epithelial tissue and elucidate the carcinogenic mechanism, it is urgent to establish a method for isolating esophageal epithelial stem cells.

  It is an object of the present invention to provide a method for isolating esophageal epithelial stem cells from animal esophageal tissue. Furthermore, by applying the isolation method, it is an object of the present invention to provide a screening method for an anticancer substance effective for esophageal cancer and a screening method for identifying a carcinogenic agent that is likely to cause esophageal cancer.

  As a result of repeated researches to solve the above problems, the present inventor collected esophageal epithelial cells from animal esophageal tissues using proteolytic enzymes, and embedded the esophageal epithelial cells in a gel matrix. It was found that esophageal epithelial stem cells can be selectively isolated by culturing using a medium containing epidermal growth factor.

  Furthermore, we found that esophageal epithelial stem cells isolated from animals exposed to carcinogenic factors changed to cells showing atypia that could progress to squamous cell carcinoma. It is thought that.

The present invention has been completed based on these findings and includes, for example, the subject matters described in the following sections:
Item 1. A method for isolating esophageal epithelial stem cells comprising the following steps (1) to (4):
(1) A process of recovering esophageal epithelial cells by treating esophageal tissue collected from an animal with a protease,
(2) a step of embedding the esophageal epithelial cells obtained in step (1) in a gel matrix;
(3) A step of contacting a cell culture medium containing an epidermal growth factor with a gel matrix containing esophageal epithelial cells to culture the esophageal epithelial cells, and (4) a step of collecting the cells grown in step (3).
Item 2. Item 2. The isolation method according to Item 1, further comprising a step of further treating the protease-treated esophageal epithelial cell with a chelating buffer between the step (1) and the step (2).
Item 3. Item 3. The isolation method according to Item 1 or 2, wherein the protease is dispase.
Item 4. Item 4. The isolation method according to any one of Items 1 to 3, wherein the animal is an animal exposed to a carcinogenic factor.
Item 5. A screening method for an anticancer substance comprising the following steps (a) to (d):
(A) treating esophageal tissue collected from an animal exposed to a carcinogenic factor with a protease to recover esophageal epithelial cells;
(B) a step of embedding the esophageal epithelial cells obtained in step (a) in a gel matrix;
(C) a step of culturing esophageal epithelial cells by contacting a cell culture medium containing epidermal growth factor with a gel matrix containing esophageal epithelial cells in the presence of a test substance;
(D) A step of determining that the test substance is an anticancer substance by using death or growth inhibition of esophageal epithelial cells as an index.
Item 6. Item 6. The method for screening an anticancer substance according to Item 5, comprising a step of further treating the protease-treated esophageal epithelial cell with a chelating buffer between the step (a) and the step (b).
Item 7. Item 7. The method for screening an anticancer substance according to Item 5 or 6, wherein the protease is dispase.
Item 8. Carcinogenic factor screening method comprising the following steps (i) to (iv):
(I) treating esophageal tissue collected from an animal exposed to the test factor with a protease to recover esophageal epithelial cells;
(Ii) a step of embedding the esophageal epithelial cells obtained in step (i) in a gel matrix;
(Iii) a step of contacting the cell culture medium containing epidermal growth factor with a gel matrix containing esophageal epithelial cells and culturing the cells; and (iv) indicating the degree of atypia of esophageal epithelial cells contained in the cultured tissue And determining the test substance as a carcinogenic factor.
Item 9. Item 9. The method for screening an anticancer substance according to Item 8, further comprising a step of treating the protease-treated tongue epithelial cell with a chelating buffer between the step (i) and the step (ii).
Item 10. Item 10. The method according to Item 8 or 9, wherein the protease is dispase.
Item 11. Esophageal epithelial stem cells expressing at least one selected from the group consisting of Bmil, type 5 keratin and type 14 keratin isolated from animal esophageal tissue.
Item 12. Item 12. The esophageal epithelial stem cell according to Item 11, wherein the animal is an animal exposed to a carcinogenic factor.
Item 13. Item 13. A cell mass obtained by culturing the esophageal epithelial stem cell according to Item 11 or 12.
Item 14. An esophageal epithelial stem cell that expresses at least one selected from the group consisting of Bmil, type 5 keratin, and type 14 keratin, obtained from the esophageal tissue of an animal, obtained by the step (1) according to item 1.
Item 15. An esophageal epithelial stem cell that expresses at least one selected from the group consisting of Bmil, type 5 keratin, and type 14 keratin, obtained from the esophageal tissue of an animal, obtained by the isolation method according to Item 1.
Item 16. Item 16. The esophageal epithelial stem cell according to Item 14 or 15, wherein the animal is an animal exposed to a carcinogenic factor.
Item 17. Item 17. A cell mass obtained by culturing the esophageal epithelial stem cell according to any one of Items 14 to 16.

  According to the isolation method of the present invention, esophageal epithelial stem cells can be isolated from animal esophageal tissue.

  In addition, the esophageal epithelial stem cells isolated by the isolation method of the present invention can be sterically proliferated in the gel matrix to form a spherical cell mass derived from one stem cell. Furthermore, since keratinization is observed in the cell mass formed from the esophageal epithelial stem cells isolated by the isolation method of the present invention, the esophageal epithelial stem cells isolated by the isolation method of the present invention are It is a differentiable cell as in vivo.

  Further, by applying the isolation method of the present invention, anticancer substances and carcinogenic factors can be screened. Anti-cancer effective for esophageal cancer by adding a test substance that seems to be an anti-cancer substance when culturing esophageal epithelial stem cells isolated from esophageal tissue of animals exposed to carcinogenic factors Substances can be screened.

  In addition, as an animal tissue used as a material, esophageal epithelial stem cells isolated from an animal exposed to a test factor that seems to be a carcinogenic factor are cultured, and the degree of atypia of the cell mass showing atypia appearing in the culture is used as an index. Thus, a carcinogenic factor capable of inducing esophageal cancer can be screened.

A. FIG. 5 shows changes in fluorescence color of esophageal tissue over time after tamoxifen was administered to Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mice. B. The graph which counted over time the positive rate of the cell from which the fluorescent color changed from green to another color is shown. A. A phase contrast microscopic image of esophageal epithelial cells collected from mouse esophageal epithelium by protease treatment is shown. B. The time-dependent change until the proliferation and epithelial tissue formation of the esophageal epithelial stem cell extract | collected by the isolation method of this invention is shown. A spherical cell mass is formed by three-dimensional culture. HE stained image of cell mass obtained by culture of esophageal epithelial stem cells (A) Immunostained image with type 5 cytokeratin antibody (B), Immunostained image with type 14 cytokeratin antibody (C), Ki-67 antibody An immunostaining image is shown. A. An HE-stained image of a cell mass obtained by culturing esophageal epithelial stem cells is shown. B. An electron micrograph of a cell mass obtained by culturing esophageal epithelial stem cells is shown. A. The esophageal epithelial stem cell extract ^| collected from the Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mouse | ^mouth which administered tamoxifen is cultured, The fluorescence image of the cell mass formed is shown. A short arrow indicates a cell expressing green fluorescence, and a long arrow indicates a cell whose fluorescent color is changed to blue or red. B. The image which culture | ^cultivated the esophageal epithelial stem cell extract ^| collected from the Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mouse | ^mouth , administered tamoxifen on the 4th day after culture ^| cultivation, and observed the fluorescence distribution of the cell mass over time is shown. The short arrow indicates the part where the fluorescent color changes to red, and the long arrow indicates the part where the fluorescent color changes to yellow. A. The schedule at the time of transplanting the cell mass obtained by culture | ^cultivating the tongue epithelial stem cell extract ^| collected from the Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mouse | ^mouth is shown. B. HE-stained image, fluorescent microscopic image and Ki-67, type 5 keratin and type 14 keratin immunostained image and HE-stained image after 2 weeks, type 5 keratin and type 14 keratin The immunostaining image of is shown. C. Fluorescence microscopic image of the transplanted tissue administered with tamoxifen after transplantation. A. The schedule when 4-nitroquinoline-1-oxide (4NQO), a carcinogen, is administered to mice is shown. B. Histological image of the esophagus of mice administered with 4NQO is shown. The left figure is a tissue image in which hyperplasia has occurred, and the right image is a tissue image that became tumor and became squamous cell carcinoma. Cytokine dependence was investigated in vitro when proliferating tongue epithelial stem cells. Add EGF, Noggin and R-Spondin1 to E + N + R group, add EGF and Noggin to E + N group, add EGF and R-Spondin1 to E + R group, add only EGF The data of the E alone group, the N + R group to which Noggin and R-Spondin1 were added, the N alone group to which only Noggin was added, and the R alone group to which only R-Spondin1 was added are shown. A. The schedule when 4-nitroquinoline-1-oxide (4NQO), a carcinogen, is administered to mice is shown. B. Histological image of the tongue of mice administered with 4NQO is shown. The left figure is a tissue image in which hyperplasia has occurred, and the right image is a tissue image that became tumor and became squamous cell carcinoma. After administration of 4NQO, tongue epithelial stem cells were isolated, and the cells were cultured to observe changes in cell mass morphology. A. A phase-contrast microscope image is shown. The arrow points to a cell mass that has an abnormal shape. B. HE staining image is shown. The arrow points to a cell mass that has an abnormal shape. An asterisk indicates a cell cluster exhibiting atypia. A strongly enlarged image of a cell cluster showing atypia is shown.

The isolation method of the present invention comprises the following four steps (1) to (4):
(1) Esophageal epithelial cell recovery process,
(2) a process of embedding esophageal epithelial cells in a gel matrix;
(3) an esophageal epithelial stem cell culture step, and (4) a cell collection step.

Hereinafter, each of these steps will be described.
(1) Recovery process of esophageal epithelial cells As animals collecting esophageal tissue, in addition to mammals such as primates, rodents, rabbits, cats, cloven-hoofs, and terrestrials, pheasants and ducks Includes the birds that belong to it. Preferred examples of mammals include humans, monkeys, dogs, cows, sheep, pigs, horses, rabbits, mice, rats, guinea pigs, and hamsters. More preferred are human, mouse, rat, pig and sheep, and most preferred are human and mouse.

  These animals may be healthy animals or animals suffering from neoplastic diseases such as cancer, or other epithelial diseases. Further, it may be a genetically modified animal such as a gene knockout animal, gene knockin animal, or transgenic animal, or an animal transplanted with a diseased tissue.

  A tissue is collected from the back of the animal's esophagus using scissors, a scalpel, or the like, and the tissue is minced into a tissue piece having a size of about 1 to 3 mm, for example.

  The minced tissue piece is washed, for example, about 1 to 5 times, more preferably about 3 to 5 times with a buffer or physiological saline to which 100 to 1000 μM, preferably 300 to 800 μM dithiothreitol (DTT) is added. The buffer to be used is not limited as long as it has a pH of about 6.8 to 7.5 and an osmotic pressure is isotonic with that in animal cells, but PBS is preferred. This washing step is preferably performed at about 0 to 4 ° C. and within about 5 to 40 minutes.

  Next, the collected tissue is treated with a proteolytic enzyme, and the esophageal epithelial layer is detached from the lamina propria.

  As the proteolytic enzyme, trypsin, collagenase, dispase and the like can be used. Dispase is preferable.

  In the case of proteolytic enzymes weighed by weight, the final concentration is 0.1 to 5% (w / v), preferably 0.2 to 1% (w / v) for 50 to 150 mg of tissue. About 1 ml of the enzyme solution is sufficient. In addition, when proteolytic enzyme is weighed by enzyme activity, about 1 ml of enzyme solution having a final concentration of 1 to 100 units / ml, preferably 30 to 80 units / ml, is applied to 50 to 150 mg of tissue. Use it.

  The protease solution used in the present invention is preferably PBS to which 1 to 100 units / ml of dispase is added.

  A tissue piece washed with a DTT addition buffer is placed in a proteolytic enzyme solution, and incubated at, for example, 28 to 37 ° C, preferably 34 to 37 ° C. The incubation time is, for example, about 15 to 120 minutes, more preferably about 45 to 90 minutes.

  After the incubation, the tissue piece is washed about 2-5 times with ice-cold PBS to stop digestion with proteolytic enzymes.

  Subsequently, the tissue piece can be treated with a chelate buffer as needed.

  “Chelating buffer” refers to a buffer containing a chelating agent having an action of chelating metal ions.

  The buffer component to be added to the chelating buffer is not limited as long as the pH is about 6.8 to 7.5 and the osmotic pressure is isotonic with animal cells, but disodium hydrogen phosphate, sodium chloride, potassium dihydrogen phosphate, It preferably contains potassium chloride or the like. For example, disodium hydrogen phosphate is in the range of 3 to 8.5 mM, sodium chloride is in the range of 90 to 150 mM, potassium dihydrogen phosphate is in the range of 1 to 10 mM, and potassium chloride is in the range of 1 to 5 mM, for example. The pH is preferably in the range of 7.2 to 7.5.

  Preferable chelating agents include citrate and ethylenediaminetetraacetate. More preferred is citrate.

  Examples of the citrate include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt, ammonium salt and the like, preferably sodium salt. The sodium salt is preferably disodium citrate or trisodium citrate, and more preferably trisodium citrate. These may be anhydrides or hydrates, but hydrates are preferred from the viewpoint of water solubility.

  Citrate can be added to the chelating buffer at a final concentration of 10 to 100 mM, more preferably 20 to 50 mM.

  The ethylenediaminetetraacetate is preferably a sodium salt or a potassium salt. More preferably, it is disodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate or a mixture thereof. These may be properly used depending on the pH of the buffer.

  The concentration of ethylenediaminetetraacetate is preferably about 100 to 5,000 μM.

  In addition to chelating agents, the chelating buffer can contain sugar alcohols, sugars, thiol group stabilizers, and the like.

  Examples of the sugar alcohol include isomaltitol, erythritol, xylitol, glycerol, sorbitol, palatinit, maltitol, maltotetritol, maltotriitol, mannitol, lactitol and the like. Glycerol, sorbitol or mannitol is preferable, and sorbitol is more preferable.

  The sugar constituting the sugar alcohol may be either D-form or L-form, but is preferably D-form.

  The sugar alcohol can be added to the chelate buffer at a final concentration of 10 to 100 mM, more preferably 30 to 60 mM.

  Examples of sugars include monosaccharides such as arabinose, galactose, xylose, glucose, sorbose, fructose, rhamnose, N-acetylglucosamine; isotrehalose, sucrose, trehalulose, trehalose, neotrehalose, palatinose, maltose, melibiose, lactulose, lactose, etc. The disaccharide can be exemplified. Preferred is sucrose, trehalose, palatinose, maltose or lactose, and more preferred is sucrose.

  The saccharide may be either D-form or L-form, but is preferably D-form.

  Saccharides can be added to the chelate buffer at a final concentration of 10 to 100 mM, more preferably 30 to 60 mM.

  As the thiol group stabilizer, β-mercaptoethanol or DTT can be used. The amount used is preferably about 0.1 to 2 mM.

  As a solvent for the chelating buffer, water such as ion-exchanged water or ultrapure water is preferable.

  The chelate buffer used in the present invention is 10 to 100 mM trisodium citrate, 10 to 100 mM D-sorbitol, 10 to 100 mM sucrose, 3 to 8.5 mM disodium hydrogen phosphate, 90 to 150. Most preferred is pH 7.2-7.5 containing mM sodium chloride, 1-10 mM potassium dihydrogen phosphate, 1-5 mM potassium chloride and 0.1-2 mM DTT.

  In the treatment with the chelate buffer, for example, 10 to 20 ml of the chelate buffer is added to 500 to 1000 mg of the tissue sample collected at the beginning, and it is about 5 to 20 minutes at 2 to 5 ° C., more preferably about 10 to 15 minutes. It can be carried out by incubating with gentle stirring with a stirrer at -100 rpm, more preferably 30-70 rpm.

  An esophageal epithelial cell containing an esophageal epithelial stem cell can be recovered by filtering the chelate buffer containing cells and tissue residue after completion of incubation through, for example, a sterilized 70 μm mesh and recovering the filtrate. Furthermore, put the residue on the mesh into a new tube, add ice-cold 10-20 ml chelate buffer, and mix vigorously by inversion about 20 times to suspend the cells containing esophageal epithelial stem cells remaining in the residue. be able to. This is filtered through a sterilized 70 μm mesh, and the filtrate is recovered, whereby esophageal epithelial cells including esophageal epithelial stem cells can be recovered from the residue.

  The collected cells can be concentrated as necessary. Concentration can be performed by centrifuging at about 800 to 1,200 r.p.m for about 5 to 15 minutes and removing the supernatant.

  The operation from the collection of esophageal tissue to the recovery of esophageal epithelial cells including esophageal epithelial stem cells is preferably performed within 1 to 3 hours, for example.

(2) Step of embedding esophageal epithelial cells in gel matrix Next, the esophageal epithelial cells collected in "1. (1) Step of collecting esophageal epithelial tissue" are embedded in a gel matrix and cultured. The collected cells may be used as they are or concentrated. Furthermore, Bim1 positive cells selected from the collected cells may be used. The method for selecting Bim1-positive cells will be described in the section “1. (4) Cell collection step” described later.

  Cultivation of the esophageal epithelial cells collected or concentrated or selected as necessary can be performed by two-dimensional or three-dimensional culture. In order to efficiently proliferate esophageal epithelial cells from one cell to a plurality of cells, three-dimensional culture is more preferable.

  In the three-dimensional culture method, esophageal epithelial cells collected from an esophageal tissue of an animal according to the method described in “1. (1)” above are embedded in a gel matrix, and the gel matrix containing the cells is contacted with a cell culture medium. Can be done.

  Examples of the base material used as the gel matrix include those containing polysaccharides, proteins, synthetic polymer hydrogels or peptide hydrogels. These may be used alone, but it is preferable to combine two or more.

  Preferred polysaccharides include dextran, alginic acid, hyaluronic acid, chondroitin sulfate proteoglycan, heparan sulfate proteoglycan and the like.

  When dextran is used as the base material of the gel matrix, for example, dextran can be modified with maleimide or the like and polymerized in accordance with a conventional method using polyethylene glycol, thiol crosslinker or the like.

  When alginic acid is used as the base material of the gel matrix, it can be polymerized according to a conventional method using a polymerization agent such as calcium chloride.

  When hyaluronic acid is used as the base material of the gel matrix, for example, a thiol group can be introduced into hyaluronic acid and polymerized in accordance with a conventional method using a thiol crosslinker together with heparan sulfate proteoglycan.

  Examples of the protein include those contained in the extracellular matrix of mammalian tissues such as collagen, elastin, entactin (nidogen), fibronectin, laminin and the like. Further, although it does not directly support the gel matrix, it may contain growth factors such as TGF-β, FGF, tissue plasminogen activator factor.

  As the collagen, type I, II, III or IV collagen can be used, more preferably type I, type III or type IV collagen, still more preferably type I or type IV collagen. A part of collagen may be enzymatically digested or hydrolyzed. When collagen is used as the base material of the gel matrix, it can be polymerized according to a conventional method using, for example, sodium hydroxide and / or sodium carbonate. Collagen may be used alone, but is more preferably used in combination with entactin, laminin and / or heparan sulfate proteoglycan.

  Examples of the synthetic polymer include polyethylene glycol, polyvinyl alcohol, or a copolymer thereof.

  When polyethylene glycol is used as the base material of the gel matrix, it can be polymerized in accordance with a conventional method such as chain polymerization or sequential polymerization using an acrylic group and / or methacrylic group at the end.

  When polyvinyl alcohol is used as the base material of the gel matrix, the polyvinyl alcohol can be modified with maleimide or the like and polymerized according to a conventional method using polyethylene glycol, thiol crosslinker or the like.

  Peptide hydrogel (Nippon Becton Decktonson) consists of about 1% purified artificial peptide and water, and can be polymerized by self-polymerization reaction.

A commercially available gel matrix can also be used. For example, 3-D Life Hydrogel series (CELLENDES) for 3D culture, Hyaluronic acid hydrogel (Glycosan Biosystems), Alginate 3D culture kit (PG Research), Collagen gel culture kit (Nitta Gelatin), BD Matrigel ^TM matrix (Nippon Becton Decktonson), BD ^TM Pure ^TM Matrix peptide hydrogel (Nippon Becton Decktonson), etc. are mentioned. In particular, the BD Matrigel ^™ matrix is preferred because it contains laminin, entactin, type IV collagen and heparan sulfate proteoglycan.

' ^TM ' indicates a registered trademark (hereinafter the same).

  Esophageal epithelial cells recovered from esophageal tissue by the method described in “1. (1)” above are embedded in a gel matrix within 12 hours, preferably within 6 hours, and more preferably within 3 hours from recovery. .

The cells for embedding should be about 0.1 to 5 × 10 ⁴ cells / μl, preferably about 0.5 to 1 × 10 ⁴ cells / μl, in a gel matrix solution to which a polymerization agent or a thiol crosslinker is added if necessary. For example, about 10 to 250 μl, more preferably about 100 to 200 μl is dispensed per well of a 24-well plate. What is necessary is just to adjust a dispensing amount suitably according to the magnitude | size of a well.

  Embedding is performed by polymerizing the gel matrix solution in the presence of cells.

  The polymerization of the gel matrix can be performed at 4 to 37 ° C, more preferably 25 to 37 ° C, and further preferably 35 to 37 ° C. The polymerization time can be 30 to 60 minutes, preferably 20 to 40 minutes.

(3) Esophageal epithelial cell culture process The basic culture medium is L-glutamine, epidermal growth factor, kinase inhibitor, transferrin, B-27 ^TM supplement (Life Technologies), N-acetyl cysteine, antibiotics, etc. Can be prepared.

  The basic medium is not particularly limited, and DMEM, DMEM / F12, advanced DMEM / F12, RPMI1640, advanced RPMI1640, and the like can be used. Preferably, it is DMEM / F12 or advanced DMEM / F12, and more preferably, it is devanted DMEM / F12. It is better not to add serum such as fetal calf serum to the basic medium.

When L-glutamine is added to the basic medium in advance, it is not necessary to add L-glutamine, but when it is not added, it is preferable to add L-glutamine. The addition amount can be added so as to be about 1 to 3 mM as L-glutamine. Further, instead of L-glutamine, a commercially available glutamine supplement such as GlutaMAX ^™ can be used. In this case, the supplement may be added so that the final concentration of the supplement is about 1 × of the manufacturer's recommended concentration.

  Epidermal growth factor (EGF) may be purified from mammals or may be obtained by gene recombination techniques. EGF is preferably derived from mammals such as humans, monkeys, mice, rats, cows, and more preferably humans or mice. The cell culture medium can contain EGF as a growth factor, preferably contains EGF as an essential component as a growth factor, and more preferably contains only EGF as a growth factor. The EGF concentration to be added is 1 to 300 ng / ml, preferably 10 to 200 ng / ml, more preferably 25 to 100 ng / ml as a final concentration with respect to the cell culture medium.

As the phosphorylase inhibitor, a ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitor can be added. As the ROCK inhibitor, (R)-(+)-trans-N- (4-pyridyl) -4- (1-aminoethyl) -cyclohexanecarboxamide · 2HCl · H ₂ O (Y-27632), 1- ( 5-isoquinolinylsulfonyl) homopiperazine dihydrochloride (fasudil or HA1077) and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl) sulfonyl] -hexahydro-1H- 1,4-diazepine dihydrochloride (H-1152) can be used, more preferably Y-27632. The concentration of the ROCK inhibitor added is 0.1 to 100 μM, preferably 1 to 50 μM, more preferably 5 to 20 μM as a final concentration with respect to the cell culture medium.

  As the transferrin, a holo type is preferable, and those supplied as N-2 supplements (for example, manufactured by Life Technologies) can be used. The N-2 supplement can be added at a final concentration with respect to the cell culture medium, for example, 0.5 × to 2 × which is the manufacturer recommended concentration. More preferably, the concentration is 1 ×.

The B-27 ^™ supplement can be added at a final concentration with respect to the cell culture medium, for example, 0.5 × to 2 × as recommended by the manufacturer. More preferably, it is about 1 × concentration.

  N-acetyl cysteine can be added to a final concentration of about 0.5 to 2 μM with respect to the cell culture medium.

  Antibiotics can be further added to the cell culture medium. As the antibiotic, penicillin, streptomycin or a mixture thereof can be added according to a conventional method.

  In the present invention, cytokines such as bone morphogenetic factor (BMP) antagonist, Wnt agonist, fibroblast growth factor (FGF) can be added to the cell culture medium as necessary.

  As the BMP antagonist, Noggin, DAN or DAN-like protein (Cerberus) or Gremlin can be used, more preferably Noggin. BMP antagonists may be purified from mammals or may be those obtained by gene recombination techniques. BMP antagonists are preferably derived from mammals such as humans, monkeys, mice, rats, cows, and more preferably humans or mice. The concentration of the BMP antagonist added is 1 to 500 ng / ml, preferably 10 to 300 ng / ml, more preferably 50 to 200 ng / ml as a final concentration relative to the cell culture medium.

  Wnt agonists include Wnt-1 / Int-1, Wnt-2 / Irp (Int-1-related protein), Wnt-2b / 13, Wnt-3 / Int-4, Wnt-3a, Wnt-4, Wnt- 5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a / 8d, Wnt-8b, Wnt-9a / 14, Wnt-9b / 14b / 15, Wnt-10a, Wnt-10b / 12, Wnt-11, Wnt-16, R-spondin 1, R-spondin 2, R-spondin 3 or R-spondin-4 can be used, preferably Wnt-3a, Wnt-7a or R-spondin 1, more preferably R-spondin 1 can be used.

  Wnt agonists are preferably derived from mammals such as humans, monkeys, mice, rats, cows, and more preferably humans or mice. The concentration of Wnt agonist added is 1 to 3000 ng / ml, preferably 100 to 2000 ng / ml, more preferably 500 to 1500 ng / ml in terms of the final concentration relative to the cell culture medium.

  As the FGF, any member of the bFGF family can be used, and those derived from mammals such as humans, monkeys, mice, rats and cows are preferable, and humans or mice are more preferable. The FGF concentration to be added is 1 to 3000 ng / ml, preferably 100 to 2000 ng / ml, more preferably 500 to 1500 ng / ml as a final concentration relative to the cell culture medium.

As a cell culture medium used in the present invention, advanced DMEM / F12 is used as a basic medium, 1 to 300 ng / ml of EGF is included as an essential component as a growth factor, and 1 × GlutaMAX ^™ , 0.1 to 100 μM Y is used. -27632, 0.5-2 × N-2 supplement, 0.5-2 × B-27 ^™ supplement and 0.5-2 μM N-acetyl cysteine are preferred.

  The cell culture medium and the gel matrix containing esophageal epithelial cells may be contacted by overlaying the cell culture medium on the gel matrix containing esophageal epithelial cells; cell culture of part or all of the gel matrix containing esophageal epithelial cells Examples include a method of immersing in a medium; a method of contacting a gel matrix containing esophageal epithelial cells and a cell culture medium through a cellulose filter or the like. A method of layering is preferable.

  When a cell culture medium is overlaid on a gel matrix containing esophageal epithelial cells, for example, when a gel matrix containing esophageal epithelial cells is polymerized using a 24-well plate, 300 to 1000 μl per well, preferably 500 ˜800 μl can be overlaid. The amount of the medium may be adjusted as appropriate according to the size of the well. The medium exchange can be performed by removing the part overlaid on the gel matrix and overlaying with a new cell culture medium. The medium can be exchanged once every 2 to 7 days, preferably once every 3 to 5 days.

Esophageal epithelial cells can be cultured in a commonly used CO ₂ incubator, with a temperature of around 37 ° C. and a CO ₂ concentration of around 5%.

  Esophageal epithelial cells cultured under the above conditions can start to grow on the second day after the start of culture and can be cultured for about 15 to 40 days after the start of culture.

(4) Cell collection process “Esophageal epithelial stem cells” combine the self-renewal capacity and multipotency derived from the esophageal epithelial tissue of animals, whether normal or not. It refers to the cell that has Multipotency refers to the ability to differentiate into one or more cells of the same cell lineage or different cell lineages.

  Esophageal epithelial stem cells have the ability to self-replicate and differentiate into mature squamous epithelial cells in vivo to form esophageal epithelium as well as to differentiate into taste bud cells. Differentiated cells lose their ability to self-replicate or proliferate.

  Therefore, the selection of whether or not the esophageal epithelial cells cultured in “1. (3) Esophageal epithelial cell culture process” is an esophageal epithelial stem cell is “1. (3) Esophageal epithelial cell culture process”. Can be carried out by culturing according to the method described in 1. above. Specifically, esophageal epithelial cells are cultured for about 10 to 60 days, more preferably about 12 to 30 days under the above-described culture conditions, and the cells having proliferation ability at that time are determined to be esophageal epithelial stem cells. . Among the esophageal epithelial cells collected in the process of “1. (1)” above, cells having no proliferative ability die in about 3 to 7 days. What is necessary is just to collect | recover the cell which has.

  The esophageal epithelial stem cells cultured in the above “1. (3) esophageal epithelial cell culture process” proliferate three-dimensionally in a state of being embedded in the gel matrix, and also differentiate, so that epithelial stem cells having proliferative ability And forming a cell mass containing differentiated esophageal epithelial cells.

  Therefore, esophageal epithelial stem cells can be selected by collecting the cell mass from the gel matrix using dispase or the like, loosening the cell mass, and then collecting cells having proliferation ability.

  Collection of cells having proliferation ability can be achieved by fraction collection using a cell sorter or the like using a fluorescent dye such as bisbenzimide such as Hoechst33342. Detect cells with a filter that can detect fluorescence at a wavelength of 405 nm and a filter that can detect fluorescence at a wavelength of 600 nm when excited with ultraviolet light, and fractionate in the S / G2 phase, and in the G0 / G1 phase A fraction of cells that emit fluorescence that is darker than the fraction or that does not emit fluorescence at all (side population fraction) may be collected.

  The “esophageal epithelial stem cell” can be said to be a cell that expresses at least one selected from the group consisting of Bmil, type 5 keratin, and type 14 keratin regardless of whether it is normal or not. Cells that express Bmi1 and type 5 keratin or type 14 keratin are preferred, and cells that express Bmi1, type 5 keratin and type 14 keratin are more preferred.

  As they mature, the expression of Bmi1, type 5 keratin and / or type 14 keratin is suppressed.

  Therefore, from the esophageal epithelial cells collected in the process of “1. (1)” above or from the cell mass cultured by the method described in “1. (3)” above, Bmil type 5 Cells expressing at least one selected from the group consisting of keratin and type 14 keratin can be identified as esophageal epithelial stem cells.

  Furthermore, cells expressing the above proteins can be selected and collected by conventional methods using, for example, magnetic beads, cell sorters, etc. using antibodies against these proteins.

  When the animal is, for example, a mouse, esophageal epithelial stem cells can be selected using a genetically modified mouse knocked in with a gene such as Cre recombinase as an animal from which esophageal tissue is collected.

  For example, transgenic mice can be obtained by the following method.

A knock-in mouse (Bmi1 ^{CreER / +,} etc.) in which a nucleic acid having a base sequence such as estrogen receptor binding Cre recombinase is incorporated into a locus corresponding to the 3 ′ untranslated region of Bmi1 mRNA is prepared, and a rainbow is separately added to the Rosa26 region. Mouse construct (having GFP cDNA sequence, CYP cDNA sequence, OFP cDNA sequence sandwiched between sequences such as loxp, loxN, lox2272 which are target sequences of Cre recombination downstream of CAG promoter, and RFP cDNA sequence downstream Rosa26 rainbow mice knocked in (see Rosa26 ^{rbw / +} mice, Red-Horse, K. et al., Nature 464, p549-553, 2010 and Rinkevich. Et al., Nature 476, p409-413, 2011) ). Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mouse, Bmi1 ^{CreER / CreER} / Rosa26 ^{rbw / +} mouse, Bmi1 ^{CreER / +} / Rosa26 ^{rbw / rbw} mouse Bmi1 ^{CreER / CreER} / Rosa26 ^{Get rbw / rbw} mouse etc. This mouse expresses GFP in Bmi1-expressing cells such as esophageal epithelial stem cells, but the administration of estrogen compounds such as tamoxifen deletes the sequence between loxp, loxN, lox2272, etc. inserted into Rosa26, and CFP (blue ), The fluorescence of either OFP (orange) or RFP (red) is expressed.

Therefore, Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mice, Bmi1 ^{CreER / CreER} / Rosa26 ^{rbw / +} mice, Bmi1 ^{CreER / +} / Rosa26 ^{rbw / rbw} mice Bmi1 ^{CreER / CreER} / Rosa26 ^{rbw / rbw} before recovery of esophageal tissue After administering an estrogen derivative such as tamoxifen to a mouse or the like, the cells are collected according to the method described in “1. (1)” above, and the collected cells are observed under a fluorescence microscope. Bim1-expressing cells change from green to blue, orange, or red fluorescent color by administration of estrogen derivative. Therefore, cells with a changed fluorescent color can be identified as esophageal epithelial stem cells, and a cell sorter is used. Thus, it is possible to collect cells in which these fluorescent colors have changed. In this case, the estrogen derivative such as tamoxifen may be administered at about 100 to 500 mg / kg (body weight) about 5 days before immediately before tissue collection.

Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mice, Bmi1 ^{CreER / CreER} / Rosa26 ^{rbw / +} mice, Bmi1 ^{CreER / +} / Rosa26 ^{rbw / rbw} mice Bmi1 ^{CreER / CreER} / Rosa26 ^{rbw / rbw} mice administered with estrogen derivatives From the above, the esophageal epithelial cells collected according to the process of “1. (1)” are cultured for about 2 to 4 days according to the processes of “1. (2)” and “1. (3)” and then collected. A cell sorter or the like can also be used to select cells whose fluorescence color has changed from green to another color as esophageal epithelial stem cells.

2. Esophageal epithelial stem cells isolated from animal esophageal tissue and cell mass obtained by culturing the cells (1) Esophageal epithelial stem cells isolated from animal esophageal tissue "Esophagus isolated from animal esophageal tissue" The term “epithelial stem cell” refers to an esophageal epithelial stem cell isolated from an esophageal tissue of an animal, and preferably expresses at least one selected from the group consisting of Bmi1, type 5 keratin and type 14 keratin, more preferably A cell that expresses Bmi1 and type 5 keratin or type 14 keratin, most preferably expresses Bmi1, type 5 keratin and type 14 keratin and has a proliferative ability.

  The method for isolating esophageal epithelial stem cells is not particularly limited, but preferably, the above-mentioned step “1. (1)” or the above “1. (1)”, “1. (2)” and “1. ) "Is preferred. Moreover, you may combine the process of said process "1. (4)" as needed.

(2) Cell mass obtained by culturing esophageal epithelial stem cells isolated from animal esophageal tissue "Cell mass obtained by culturing esophageal epithelial stem cells isolated from animal esophageal tissue" 2. A cell mass obtained by culturing esophageal epithelial stem cells as defined in (1). One cell mass is preferably proliferated from about 1, 2, or 3 esophageal epithelial stem cells.

  The cell mass is preferably a cell mass cultured in vitro for about 2 to 30 days, more preferably about 5 to 10 days, and even more preferably about 7 days.

  The method for culturing esophageal epithelial stem cells isolated from animal esophageal tissue is not particularly limited, but the cell mass cultured by the method described in the above “1. (2)” and “1. (3)” is preferable. .

The cell mass includes esophageal epithelial stem cells or cells differentiated from esophageal epithelial stem cells and esophageal epithelial stem cells into squamous epithelial cells and the like. The number of cells per cell mass is approximately 1 × 10 ⁴ to 1 × 10 ⁷ , preferably approximately 1 × 10 ^{5 to} 5 × 10 ⁶ .

3. Carcinogenic factor esophageal epithelial stem cells can also be isolated from animals exposed to the carcinogenic factor.

  “Carcinogenic factor” refers to a factor that damages DNA in a cell and causes the cell to acquire a proliferation ability that deviates from normal cell growth control, and further to acquire an invasion ability and / or metastasis ability. Carcinogenic factors include alkylating agents such as quinacrine mustard; calcineurin inhibitors such as cyclosporine; 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanone (NNK), dimethylnitrosamine, nitrosopyrrolidine, N ′ -N-nitroso compounds such as nitrosonornicotine; polycyclic aromatic hydrocarbons such as benzo [a] pyrene and benz [a] anthracene; heterocyclic amines such as PhIP; 2-naphthylamine, benzidine, aminobiphenyl, nitrobiphenyl Aromatic amines such as benzene; nitrogen oxides such as 4-nitroquinoline-1-oxide; metal particles such as cadmium, nickel, aluminum, polonium 210; hydrazine; acrylamide; acrylonitrile; urethane; coal tar; Aldehyde compounds such as cetaldehyde acrolein; Chemical carcinogenic factors such as mold toxins such as aflatoxin; Physical carcinogenic factors such as radiation, ultraviolet rays, heat, free radicals, friction, etc .; Biological factors such as EB virus and human papilloma virus, alcohol drinking Factors listed in the International Cancer Institute (IARC) Carcinogenic Risk List, such as behavior and smoking behavior.

  The carcinogenic factors in the present invention include substances whose carcinogenicity is not yet clear, in addition to factors that have already been confirmed to be carcinogenic.

  Carcinogen exposure includes environmental exposure and forced exposure. However, in the case of forced exposure, humans are excluded from the target animals.

  Forced exposure of a chemical carcinogen can be accomplished by exposing the chemical carcinogen orally or aerally.

  In the case of oral exposure, chemical factors can be added to drinking water at 0.1 to 500,000 ppm and ingested. Moreover, as another exposure mode, a chemical carcinogenic factor can be taken orally so that it may become 0.001-1,000 microgram / kg (body weight). Regardless of which method is employed, the animal can be orally ingested with a chemical carcinogenic factor continuously for about 1 to 1,000 days, more preferably for about 1 to 100 days, or intermittently.

  In the case of exposure via air, place the animal in an inhalation experiment device, etc., and make the chemical carcinogenic agent in particulate, dust, fume, mist, smoke, etc. aerosol, etc. It can be carried out by filling to about 1,000 ppm. Animal breeding in the apparatus can be performed continuously for 10 minutes to 24 hours a day, about 1 to 1,000 days, more preferably about 1 to 100 days, or intermittently.

  For example, as an example of forced exposure of carcinogenic factors in mice, when 4-nitroquinoline-1-oxide is used, it is mixed with drinking water so as to be 10 to 200 μg / ml and fed for 10 to 20 weeks. A method is mentioned.

The exposure of animals to physical carcinogenic factors is ionizing radiation, the cumulative exposure is about 0.001 to 10 Gy, and ultraviolet light with a wavelength of about 280 to 400 nm, the UV intensity is 0.35 to 1 mW / cm ² / It can be performed by irradiating the esophageal epithelium so as to be about sec.

  An animal may be exposed to a biological carcinogen by infecting the animal with a microorganism, such as a bacterium or virus, in contact with the animal at an amount of microorganisms that establishes the infection, orally or aerally. it can.

4). Screening method for anticancer substances Screening for anticancer substances effective for the treatment of esophageal cancer using esophageal epithelial cells obtained by treatment with protease from esophageal tissues collected from animals exposed to carcinogenic factors You can also

The method can be carried out by performing the following steps (a) to (d);
(A) a process of recovering esophageal epithelial cells from an animal exposed to a carcinogen;
(B) a step of embedding in a gel matrix;
(C) culturing esophageal epithelial cells in the presence of the test substance; and (d) determining the test substance as an anticancer substance.

Hereinafter, each of these steps will be described.
(A) Recovery process of esophageal epithelial cells from an animal exposed to a carcinogen Using an esophageal tissue of an animal exposed to the carcinogenic factor in the environment or an animal forcedly exposed to the carcinogenic factor described in “3.” above In the method described in “1.” above, the step of “1. (3)” is performed in the presence of a test substance that can be an anticancer substance, thereby screening an anticancer substance effective for esophageal cancer. be able to.

  Here, the test substance is an anti-tumor agent that has already been demonstrated to be effective against esophageal cancer, squamous cell carcinoma, or malignant tumors of other organs, as well as a substance whose anti-cancer activity has not yet been demonstrated. Is included.

  In addition, the collected esophageal tissues include tissues that already show hyperplasia, benign tumor, or cancer pathology.

(B) The process of embedding in a gel matrix It can carry out according to the method described in said "1. (2)".

(C) Step of culturing esophageal epithelial cells in the presence of the test substance The esophageal epithelial cells in the presence of the test substance may be cultured according to the method described in “1. (3)” above. By adding the substance to the medium, esophageal epithelial cells can be cultured in the presence of the test substance.

The amount of the test substance added to the cell culture medium can be set as appropriate using the IC ₅₀ of the test substance as an index, and for example, about 0.001 to 5,000 μM can be added. The test substance is preferably brought into contact with the esophageal epithelial cells at different concentrations in stages. Further, the contact between the test substance and the esophageal epithelial cells may be started from the day when the gel matrix containing the esophageal epithelial cells and the cell growth medium are contacted, but preferably the esophageal epithelial cells embedded in the gel matrix. It is preferable to start 2-5 days after the start of culture. The contact between the test substance and esophageal epithelial cells is preferably performed for 1 to 7 days, preferably 2 to 5 days.

  Further, as the control esophageal epithelial cells, esophageal epithelial cells cultured without contacting with the test substance for the same period as the esophageal epithelial cells contacted with the test substance can be used.

(D) The step of determining that the test substance is an anticancer substance The determination that the test substance is an anticancer substance is the time when contact culture of the test substance and esophageal epithelial cells is completed, or 1 to 5 days after the completion. Later, it can be determined by determining death or growth inhibition of esophageal epithelial cells as an index.

  The death of esophageal epithelial cells can be evaluated according to a conventional method, and can be evaluated by phase contrast microscopy using cell concentration or rupture as an index.

  Another method is to collect the cell mass of esophageal epithelial cells that have been contacted with the test substance, loosen the cell mass with dispase, etc. to obtain a cell suspension, and then perform propidinium iodide (PI) staining, etc. The dead cells may be detected with a flow cytometer or the like.

  As another method, the number of dead cells can be quantitatively evaluated by counting the number of cells in which apoptosis is induced in the cell mass of esophageal epithelial cells brought into contact with the test substance.

  Apoptosis-inducing cells can be evaluated by evaluation of nuclear enrichment by HE staining, TUNEL method, Annexin V binding, or the like. More preferably, detection is performed by TUNEL method or Annexin V binding. The TUNEL method may be performed on a tissue section using an optical microscope, or may be detected using a flow cytometer. Further, the detection with Annexin V can be evaluated using a flow cytometer or the like using the fluorescent label Annexin V.

  Whether or not the test substance is an anticancer substance can be determined by determining the dead cell rate and comparing the dead cell rate of the cell mass exposed to the test substance and the dead cell rate of the control esophageal epithelial cells. it can.

  When determining the dead cell rate using PI staining and a flow cytometer, the fraction of dead cells calculated by the analysis software attached to the flow cytometer can be used as the dead cell rate.

  When HE staining of tissue sections or TUNEL method is used, the dead cell rate is about 3 to 10 cell masses. The total number of cells and the number of dead cells per cell mass (in the case of HE staining) Count the number of cells enriched in nuclei, or the number of TUNEL-positive cells), and calculate the value obtained by dividing the number of dead cells per cell mass by the total number of cells per cell mass, and set the value to 100 It can be obtained by multiplying (the following formula 1). The cell death rate in the counted cell mass is averaged, and the cell death rate of the cell mass exposed to the test substance is compared with the cell death rate of the control esophageal epithelial cells.

When detecting cells fluorescently labeled with the TUNEL method or Annexin V using a flow cytometer, collect the cultured cell mass from the gel matrix and use TUNEL staining or fluorescently labeled Annexin V, etc. It can be fluorescently labeled. This fluorescence is detected by a flow cytometer, and the number of cells labeled with a fluorescent dye by TUNEL staining or Annexin V in, for example, 1 × 10 ⁶ cells can be counted to obtain the dead cell rate. .

  Even when evaluated by any of the above methods, the dead cell rate of the cell mass exposed to the test substance is about 10% or more, preferably about 20% or more than the dead cell rate of the control esophageal epithelial cells, More preferably, the test substance increased by about 30% or more can be determined to be an anticancer substance.

  In order to evaluate the growth inhibition of esophageal epithelial cells, according to a conventional method, collect the cell mass and loosen the cell mass, count the number of cells per well with a hemocytometer etc., or collect the cells in the gel matrix Thereafter, the number of cells can be compared with that of control esophageal epithelial cells by MTT assay or the like.

  Whether the cell growth is suppressed or not is evaluated by any of the above methods, the number of cells in the cell mass exposed to the test substance is preferably about 70% or less, preferably compared to the control esophageal epithelial cells. It can be determined that a test substance that is about 50% or less, more preferably about 30% or less, is an anticancer substance.

  In addition, evaluation of esophageal epithelial cell growth inhibition can also be performed using a flow cytometer. After collecting the cells in the gel matrix, nuclear staining is performed with PI staining or the like, and the ratio of cells in the S / G2 phase and G0 / G1 phase is measured with a philocytometer. Test substance in which the proportion of S / G2 phase of the cell mass exposed to the test substance was about 70% or less, preferably about 50% or less, more preferably about 30% or less, compared to the control esophageal epithelial cells Can be determined to be anti-cancer substances.

5. Screening Method for Carcinogenic Factors Using esophageal epithelial cells isolated from esophageal tissue collected from animals exposed to the test factor, it is possible to determine whether the test factor is a carcinogenic factor.

The method can be carried out by performing the following steps (i) to (iv);
(I) recovering esophageal epithelial cells from an animal exposed to the test factor;
(Ii) embedding esophageal epithelial cells in a gel matrix;
(Iii) culturing the cells; and (iv) determining that the test substance is a carcinogenic factor.

Hereinafter, each of these steps will be described.
(I) Step of recovering esophageal epithelial cells from the animal exposed to the test factor In addition to the known expressed substances described in “3.” above, the causal relationship with carcinogenesis is still clear for the test factor. Also included are chemical factors such as chemical substances that are not formed, physical factors such as electromagnetic waves, and biological factors such as microorganisms.

  Exposure includes environmental exposure and forced exposure. However, in the case of forced exposure, humans are excluded from the target animals.

  Forced exposure to a chemical test agent can be accomplished by exposing the chemical test agent orally or aerally.

  In the case of oral exposure, chemical factors can be added to drinking water at 0.1 to 500,000 ppm and ingested. Moreover, as another exposure mode, a chemical test factor can be taken orally so that it may become 0.001-1,000 microgram / kg (body weight). Regardless of which method is used, the animal is ingested by the chemical test factor orally continuously for about 1 to 1,000 days or intermittently.

  In the case of exposure via air, place the animal in an inhalation experimental device, etc., and set the chemical test factor in the form of aerosols such as particles, dust, fume, mist, smoke, etc. in the device. It can be carried out by filling to about 1,000 ppm. Animal breeding in the apparatus can be carried out continuously for 10 minutes to 24 hours a day, about 1 to 1,000 days, preferably about 1 to 100 days, or intermittently.

  Exposure to a physical test factor can be performed by irradiating an animal with electromagnetic waves such as radiation and light. The irradiation amount may be such that the electromagnetic wave is irradiated to such an extent that an acute symptom such as a tissue ulcer does not appear within about one month to one month after the irradiation of the electromagnetic wave. Further, the irradiation may be performed only once, or may be continuously performed about 1 to 1,000 times, preferably about 1 to 100 times.

  The exposure to the biological test factor may be performed by bringing the animal into contact with an amount of microorganisms sufficient to establish infection with the microorganism. As a contact method, a route suitable for infection with the microorganism may be selected.

  By the above method, collection of esophageal tissue from an animal exposed to the test factor and collection of esophageal epithelial cells can be performed according to the method described in “1. (1)” above.

(Ii) Step of embedding esophageal epithelial cells in gel matrix Recovery and culture of esophageal epithelial cells from animals exposed to the test factor is performed by gel of esophageal epithelial cells recovered in “5. (1)” above. The embedding in the matrix can be performed according to the method described in the above “1. (2)”. The culture period is preferably about 2 to 20 days, more preferably about 10 to 20 days. The cell mass obtained by this method is hereinafter referred to as a test factor-exposed cell mass.

  In this case, esophageal epithelial cells collected from a healthy animal that has not been exposed to the test factor as a control cell mass according to the method described in “1. (1)” above are collected from the animal that has been exposed to the test factor. A cell mass cultured by the method described in “1. (2)” above can be used for the same period as the cells.

(Iii) Step of culturing cells The esophageal epithelial cells embedded in the gel matrix in the step “5. (ii)” can be cultured according to the method described in “1. (3)” above. .

(Iv) A step of determining that the test substance is a carcinogenic factor For the test factor-exposed cell mass and the control cell mass, for example, by determining the degree of tissue atypia as an index, the test factor is determined to be a carcinogenic factor. Can do.

  The degree of atypia was determined by fixing the cell mass obtained in the above “5. (iii)” with 10% neutral buffered formalin and embedding in paraffin, followed by HE staining and the like under the optical microscope. It can be classified into the following grades I to IV from the tissue structure.

  Normal: No abnormality in the cell nucleus, cytoplasm, or tissue structure.

  Grade I: There are cells with enlarged cell nuclei than the cells present in the outermost cell layer of the control cell mass, but these cells are located in the center from the cells in the outermost layer of the spherically formed cell mass. When the thickness to the keratinized cell layer existing in 1 is 1, the cell layer is only about 1/3 from the outermost periphery.

  Grade II: Cells with enlarged cell nuclei are present in the outermost cell layer of the control cell mass, and the cells are located in the center from the outermost layer cell of the spherically formed cell mass. When the thickness to the existing keratinized cell layer is 1, the cell layer reaches about 2/3 from the outermost periphery.

  Grade III: Cells with enlarged cell nuclei exist than cells in the outermost cell layer of the control cell mass, and the cells reach the cornified layer in the center.

  Variability IV: Cell nuclei occupying enlarged cells, and chromatin in the nuclei appear to aggregate, so-called cancer cell-shaped cells appear.

  The above cancer cells can be identified using diagnostic criteria adopted for squamous cell carcinoma cells in histopathological diagnosis.

  Further, about 3 to 10 cell masses are observed for each of the control cell mass and test factor-exposed cell mass obtained in the culture of “5. (3)”, and are classified into normal or atypical grades 1 to 4. Count the number of cell clumps. Next, a variant index is obtained. The normal score is “1”, the score of variant 1 is “2”, the score of variant 2 is “3”, the score of variant 3 is “4”, and the score of variant 4 is “5”. Multiply the number of cell masses classified as atypical by the score value, add all the values to obtain the total, and divide the total value by the number of cell masses counted. , And a degree of atypical index (the following formula 2). A test factor that has a higher degree of atypicality index of the test factor-exposed cell mass than the control cell mass by about 10% or more, more preferably 30% or more can be determined as a carcinogenic factor.

  In addition, as another method for determining that the test factor is a carcinogenic factor, instead of observing under an optical microscope by HE staining, immunostaining the tumor marker SCC (Squamous cell carcinoma) antigen, cancer cells There is a method for evaluating the presence of the substance under a microscope.

  The SCC antigen includes neutral SCCA1 and acidic SCCA2, but it is more preferable to detect SCCA2.

  For the control cell mass and the test factor-exposed cell mass, prepare a paraffin-embedded section in the same manner as HE staining, then immunostain using anti-SCC antigen antibody as the primary antibody according to the conventional method, followed by nuclear staining After that, the labeling index of the anti-SCC antigen antibody is calculated.

  The labeling index of the anti-SCC antigen antibody is such that the total number of cells and the number of SCC antigen positive cells are counted for about 3 to 10 cell masses, and the number of SCC positive cells per cell mass is counted per cell mass. A value obtained by dividing the total number of cells can be obtained, and the value can be obtained by multiplying the value by 100 (Equation 3 below). The labeling index of the anti-SCC antigen antibody in the counted cell mass is averaged, and the value of the labeling index of the test agent-exposed cell mass is about 10% or more, more preferably about 30%, than the labeling index of the control cell mass. The test factor having a high value can be determined as a carcinogenic factor.

  When evaluating the carcinogenicity of a test factor, either an evaluation method using the degree of atypia or an evaluation method using SCC as an index may be adopted, but a cell showing atypia is not necessarily positive for SCC antigen. Therefore, it is more preferable to use the variant index as an index.

  When screening for an oncogenic factor that causes higher malignancy, it may be preferable to use the SCC antigen as an index. The grade of malignancy indicates a high ability to metastasize and infiltrate surrounding tissues.

6). Transplantation of esophageal epithelial stem cells isolated from animal esophageal tissue or cell mass obtained by culturing the cells into animals Esophageal epithelial stem cells isolated from animal esophageal tissue of the present invention or culturing the cells The resulting cell mass can be reimplanted into animal tissue or the like.

  Esophageal epithelial stem cells isolated from animal esophageal tissue can be transplanted as they are, but from the viewpoint of the survival rate of the cells after transplantation, it is preferable to transplant a cell mass obtained by culturing the cells.

The cell mass to be transplanted is a cell mass that has been cultured for about 2 to 10 days from the start of culture and has about 1 × 10 ⁶ cells per cell mass, for example, a stratified squamous epithelium of about 1 to 10 mm ^2. Each defect can be transplanted by floating in about 10 to 1,000 PBS or the like.

  The transplant site is preferably transplanted to a tissue below the epithelial base such as the lamina propria and the muscle layer.

Examples of the present invention are shown below, but the embodiments of the present invention are not construed as being limited to the examples.
1. Experimental example: Prior to the Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mouse production experiment, a Cre recombination cassette (Cre recombination and estrogen downstream of the IRES sequence) was placed in the 3 'untranslated region of the Bmi1 gene expressed in esophageal epithelial stem cells. Knock-in mice with the receptor fusion protein CreER sequence (see Bmi1 ^{CreER / +} mice, Sangiorgi, E. et al., Nat. Genet. 40 (7), p915-920, 2008) In the Rosa26 region, the rainbow mouse construct (with the GFP cDNA sequence, CYP cDNA sequence, and OFP cDNA sequence sandwiched between loxp, loxN, and lox2272 sequences, which are Cre recombinase target sequences, downstream of the CAG promoter, and further downstream Rosa26 rainbow mice (Rosa26 ^{rbw / +} mice, Red-Horse, K. et al., Nature 464, p549-553, 2010 and Rinkevich. Et al., Nature 476, p409) -413, are mated a reference 2011), Bmi1 ^{Cr eER / +} / Rosa26 ^{rbw / +} mice were obtained.

  This mouse expresses CreER in Bmi1-expressing cells according to the Bmi1 expression control, but the expressed CreER is present in the cytoplasm. By administering tamoxifen to this mouse, tamoxifen binds to the estrogen receptor sequence of CreER, and CreER translocates to the nucleus. Cre recombination of CreER transferred to the nucleus recognizes the loxp, loxN, and lox2272 sequences inserted in the Rosa26 region, and cuts out the DNA region sandwiched between the loxp, loxN, and lox2272 sequences.

  In cells before the DNA is cut out by Cre recombination, the cells express green fluorescence by GFP inserted in the Rosa26 region, but when the DNA is cut out by Cre recombination, the expression of GFP disappears and CFP (blue ), The fluorescence of either OFP (orange) or RFP (red) is expressed. Which fluorescence is expressed depends on how the DNA is cut.

  That is, the fact that the fluorescent color of a cell changes from green to another color by administration of tamoxifen means that it is an esophageal epithelial stem cell expressing Bmi1, or a cell derived from the esophageal epithelial stem cell.

2. Experimental Example 2: Identification of esophageal epithelial stem cells Adult Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mice (6 weeks old and later) were administered 8-10 mg of tamoxifen per 40 g of mouse weight after 3 days. Cells that changed from green to another color (red or blue) were observed immediately above (FIG. 1A). This cell is considered to be an esophageal epithelial stem cell expressing Bmil. When the state of cell proliferation was observed over time, it was observed that the number of red cells gradually increased and moved toward the upper part of the esophageal epithelium.

  Moreover, when the number of Bmi1-positive cells was counted, the number increased with time (FIG. 1B).

  This shows that cells expressing Bmil divide and proliferate, and are differentiated and mature. That is, it was shown that cells expressing Bmil are esophageal epithelial stem cells.

3. Example 1: Collection of mouse esophageal epithelial cells
After euthanizing Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mice, the esophagus of the mice was removed using dissecting scissors and cut into tissue pieces of approximately 2 mm using ophthalmic scissors.

  The obtained tissue piece was washed several times with PBS supplemented with 500 μM dithiothreitol (DTT), and then tissue was added to PBS supplemented with 50 units / ml dispase (Becton Dickinson Bioscience, Bedford, MA). The pieces were suspended and incubated at 37 ° C. for 60 minutes to peel off the epithelial layer and the lamina propria.

  Further, the tissue piece after dispase treatment was washed twice with ice-cold PBS, and then ice-cold 10 ml chelate buffer (27 mM trisodium citrate, 5 mM disodium hydrogen phosphate, 94 mM sodium chloride, 8 mM phosphorus) It was immersed in potassium dihydrogen acid, 1.5 mM potassium chloride, 0.5 mM DTT, 55 mM D-sorbitol, 44 mM sucrose, pH 7.3) and treated at 4 ° C. for 10 minutes while stirring slowly with a stirrer. The cells detached from the tissue piece were removed through a 70 μm mesh (70 μm mesh size, # REF352350; BD Falcon, Bedford, Mass.), And the cells that passed through the mesh were collected. The residue remaining on the mesh was transferred to a new 50 ml Corning tube, capped with 20 ml of ice-cold chelating buffer, and mixed vigorously by hand 20 times. The cells detached in the buffer were again passed through a 70 μm mesh, and the passed cells were collected again.

  The collected cells were collected in one conical tube.

A phase contrast microscopic image of the collected cells is shown in FIG. 2A.
4). Example 2: Culture of mouse esophageal epithelial cells and in vitro tissue formation Matrigel (registered trademark) (BD Becton Dickinson Co., Ltd.) at a cell density of 0.5-1 × 10 ⁴ cells / μl was obtained by the above method. And thinly spread on the bottom of the 24-well plate. After incubating at 37 ° C for 5 minutes to polymerize the gel, 750 μl of epithelial culture medium (Advanced DMEM / F-12, 1 × N-2, 1 × B-27, 1 μM N-acethyl cysteine (sigma), 1 × GlutaMAX (registered trademark) (Life Technologies), 50 ng / ml rmEGF (Peprotech), 100 ng / ml rmNoggin (Peprotech), 1000 ng / ml rhR-Spondin1-hFc, 10 μM Y-27632 (Sigma)). The epithelial culture medium was replaced with a new one every 4 days.

  Here, rhR-Spondin1-hFc is based on human R-Spondin1 cDNA donated by Tomizuka et al. (2005 Science, vol309, pp. 1256-1259, supplement), Ueno et al. (2003 Nat Immunol, vol4; pp. 457-463).

  Cells cultured by the above method were able to grow three-dimensionally. The state of growth over time was observed under a phase contrast microscope. As shown in FIG. 2B, cell proliferation was observed over time, and a spherical tissue was formed from one esophageal epithelial stem cell. Moreover, since the structure showed the pearl-like structure which exhibits a concentric circular layer shape, it was considered that it was a stratified squamous epithelial tissue.

  The cells collected in Example 1 also include differentiated epithelial cells, but the differentiated cells do not have proliferative capacity, and therefore the cells that have proliferated and formed cell masses in this experiment are esophageal epithelial stem cells. It was suggested that it originated.

5. Example 3: Histological evaluation of cell mass Furthermore, the tissue image of the formed cell mass was evaluated at the optical microscope level and the electron microscope level.
(1) Optical microscope level evaluation The cell mass was fixed with 4% paraformaldehyde, and a paraffin-embedded section was prepared, followed by HE staining or immunostaining.

  For immunostaining, use anti-type 5 keratin antibody (Covance, Denver, PA), anti-type 14 keratin antibody (Covance, Denver, PA) or anti-Ki-67 as a secondary antibody according to a conventional method. Used a peroxidase-labeled anti-rabbit immunoglobulin antibody and developed color using DAB as a substrate.

  As a result of HE staining, proliferating cells exist at the outermost periphery of the spherical tissue, the supplied keratin keratinized epithelial cell layer grows inward, and dead cells detached from the cell layer accumulate in the central part of the sphere. (FIG. 3A).

  Further, as a result of immunostaining, it was shown that type 5 keratin and type 14 keratin were strongly expressed in the outermost layer cells of the cell mass, and the expression decreased toward the inside (FIGS. 3B and 3C). . In addition, Ki-67, which is a marker for cells having proliferation ability, was positive in some of the cells present in the outermost layer of the cell mass (FIG. 3D). From this, the cells obtained by the method of Example 1 include esophageal epithelial stem cells that have proliferative ability and can mature into squamous epithelium, and these esophageal epithelial stem cells are present in the outermost layer of the cell mass. That was proved.

  In normal esophageal epithelial tissue, immature epithelial cells present at the base of the filiform papillary fistula strongly express keratin type 5 and type 14 keratin, but their expression as the cells migrate to the upper part of the filiform papillary as they mature Is known to decrease.

  Therefore, it was shown that the cell mass also has esophageal epithelial stem cells having proliferative ability on the outermost periphery of the spherical tissue, and the maturity of the cells increases as it goes to the center of the tissue.

  From these results, it was shown that the esophageal epithelial stem cells contained in the esophageal epithelial cells obtained in Example 1 not only proliferate, but mature and die in a certain direction.

  This proliferation was considered to reproduce the process of proliferation, differentiation and maturation of esophageal epithelial cells in vivo.

(2) Evaluation at electron microscope level The cell mass formed in Example 2 was fixed with 2.5% glutaraldehyde-added 0.1 M phosphate buffer (pH 7.4) and then fixed with 1% osmium tetroxide. . Thereafter, ultrathin sections were prepared by uranyl acetate staining and epoxy resin embedding according to a conventional method.

  FIG. 4 shows an HE-stained image and an electron microscope image of the cell mass obtained in Example 2. When the stratum corneum near the center of the cell mass was magnified and observed with an electron microscope, a concentric and ordered layered structure was observed (FIG. 4A right). In addition, when the vicinity of the boundary between the cell layer and the stratum corneum was magnified and observed with an electron microscope, a cell layer having an apoptotic and concentrated nucleus overlapped with a cell layer that has not yet undergone apoptosis, toward the center of the cell mass An image in which the cells gradually faded was observed (FIG. 4B right).

  This image shows that the keratinization process of normal esophageal epithelial tissue is similar, and that the keratin keratinized epithelial cell layer is also formed in the cell mass formed by culture.

  As described above in detail, from these experiments, cells were collected by the method of Example 1 and cultured by the method of Example 2, whereby the cells were separated into stratified squamous epithelial tissues having properties equivalent to those of normal esophageal epithelial cells. It has been shown that cell masses can be obtained.

6). Example 4: Examination of Monochromaticity of the Formed Tissue Next, it was examined whether the spherical cell mass obtained in Example 2 was formed from a single esophageal epithelial stem cell.
(1) Culture of esophageal epithelial cells isolated from Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mice treated with tamoxifen Once administered to give 8-10 mg tamoxifen (Sigma) per 40 g body weight of mice, 7 after tamoxifen administration Esophageal epithelial stem cells were collected from the esophagus of Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mice on the day in the same manner as in Example 1 and cultured in the same manner as in Example 2.

  As a result, as shown in FIG. 5A, the formed colonies each expressed single color fluorescence of green (short arrow), blue (long arrow), red (long arrow), or orange.

  This result indicates that each cell mass formed is formed from a single esophageal epithelial stem cell.

  The cell cluster emitting green fluorescence is considered to be cells derived from other than esophageal epithelial stem cells or cells to which tamoxifen has not been effective.

(2) Tamoxifen administration to esophageal epithelial stem cells collected from Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mice Next, esophageal epithelial cells were collected from the esophagus of Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mice as in Example 1. Then, the same culture as in Example 2 was started, and 4-hydroxytamoxifen was added to a final concentration of 40 ng / ml on the 4th day after the start of the culture, and the changes in Bmi1-positive cells were observed. did.

  As a result, from one day after tamoxifen administration, cells emitting orange (long arrow) or red (short arrow) fluorescence appeared, and an image in which the cells increased with time was observed (FIG. 5B). .

  This indicates that there are multiple esophageal epithelial stem cells expressing Bmil that have been divided from one esophageal epithelial stem cell on the 4th day after the start of culture, and that they continue to differentiate and mature without mixing afterwards. It was done.

  These results confirmed that the cell mass obtained by the culture method shown in Example 2 was derived from Bmi1-positive cells, that is, esophageal epithelial stem cells.

  The above results indicate that the cell mass obtained by the culture method of Example 2 is a cell mass formed by the proliferation of a single esophageal epithelial stem cell.

7). Example 5: Transplantation experiment of cell mass obtained by culture In order to prove that the cell mass cultured according to Example 2 can reconstruct squamous epithelial tissue in vivo, it was obtained by the same method as in Example 2. The obtained cell mass was collected from the gel matrix and transplanted to the tongue muscle layer of mice. The reason for selecting the tongue muscle for transplantation is that it is difficult to transplant a cell mass into the esophageal muscle layer because the esophagus of the mouse is thin.

About 10,000 esophageal epithelial cells are collected from the Bmi1 ^{CreER / +} / Rosa26 ^{rbw / +} mouse according to the method of Example 1 and cultured according to the method of Example 2 to obtain about 500 cell clusters. It was. The cell mass was injected into the tongue muscle of C57BL / 6J mice on the seventh day of culture. The experimental protocol is shown in FIG. 9A.

  One week after transplantation, the transplant site was collected and the tissue image was observed. As a result, a tissue mass showing a circular pearl-like tissue image appeared in the muscle layer (FIG. 9B). When this pearly tissue was observed with a fluorescence microscope, the expression of GFP was observed, indicating that the tissue mass was derived from the transplanted cell mass.

  Further, Ki-67 immunostaining showed that cells in the growth phase were present on the outermost periphery of cells expressing GFP. Furthermore, immunostaining of type 5 keratin and type 14 keratin showed that there was strong expression of type 5 keratin and type 14 keratin around the periphery of the GFP-positive cell mass.

  Furthermore, even when the tissue mass was immunostained 2 weeks after transplantation, expression of type 5 keratin and type 14 keratin was observed.

  These experiments showed that the cell mass obtained by the methods of Examples 1 and 2 has the ability to proliferate even in vivo, and can differentiate and mature into a stratified squamous epithelium.

  Furthermore, on the 7th day after transplantation, tamoxifen (Sigma) was administered once at 8-10 mg / 40 g of mouse body weight, and then fluorescence was observed on the 3rd day. As a result, cells whose color changed from green to orange were detected. (FIG. 9C). This also shows that esophageal epithelial stem cells can be isolated by the methods of Examples 1 and 2.

  In addition, since the transplanted cell mass can grow for at least one month in vivo, it was found that the transplanted cell mass can be further grown and survived using the in vivo niche environment.

  These results indicate that the cells or cell mass obtained by the methods of Examples 1 and 2 may be able to reconstruct normal squamous tissue in vivo.

8). Experimental example 3: Induction of esophageal epithelial cancer by 4-nitroquinoline-1-oxide (4NQO)
In order to confirm that administration of 4NQO can induce esophageal cancer, 4NQO at a concentration of 100 μg / ml was added to the drinking water of mice and administered for 16 weeks (FIG. 7A).

  Tissues were collected 24 weeks after the start of administration and observed for histology. As a result, squamous cell hyperplasia as shown in the middle of FIG. 7B and squamous cell carcinoma as shown in the right of FIG. 7B were observed.

  These data indicate that esophageal epithelial stem cells that may become cancer in the future can be isolated by exposing the animal to carcinogens before harvesting the esophageal epithelium.

9. Reference Example 1: Examination of Cytokine Dependence of Tongue Epithelial Stem Cells As a reference example, experimental results using tongue epithelial stem cells are shown.

  In Example 2, three types of cytokines, EGF, Noggin, and R-Spondin 1, were added to the medium for culturing tongue epithelial stem cells.

  Here, it was examined whether it is essential to combine these three types of cytokines.

  The examination was carried out by counting the formation rate of cell clusters from the tongue epithelial stem cells obtained according to the method of Example 1 using a medium supplemented with the following seven kinds of cytokines.

Specifically, the tongue epithelial cells collected according to Example 1 were seeded so that 0.5 to 1 × 10 ⁴ cells were embedded in 50 μl of Matrigel per well of a 24-well plate. The number of cell masses obtained was counted for 3 wells, and the cell mass formation ratio was calculated.

E + N + R group to which 3 types of EGF, Noggin and R-Spondin1 are added,
E + N group to which 2 kinds of EGF and Noggin are added
E + R group to which 2 kinds of EGF and R-Spondin1 are added
E alone group to which only EGF is added
N + R group to which two kinds of Noggin and R-Spondin1 are added
N alone group to which only Noggin is added
R alone group to which only R-Spondin1 is added The addition amount of each cytokine is 50 ng / ml for rmEGF, 100 ng / ml for rmNoggin, and 1000 ng / ml for rhR-Sondin1-hFc.

  As shown in FIG. 8, the cell mass formation rate tended to be slightly higher in the E + N + R group, but no significant difference was observed among the E + N + R group, the E + N group, the E + R group, and the E alone group.

  In contrast, in the N + R group, the N alone group, and the R alone group that did not contain EGF, the cell cluster formation rate was remarkably low.

  This indicates that EGF is essential for the proliferation and maturation of tongue epithelial stem cells, and that Noggin and R-Spondin1 are not required.

  This result is different from the result described in WO2010 / 090513 in which glandular epithelial cells are cultured.

Ten. Reference Example 2: Carcinogenicity experiment using tongue epithelial stem cell culture method In order to examine how carcinogens affect tongue epithelial stem cells, the carcinogens are administered to mice, and then tongue epithelial stem cells are collected. Then, the cells were cultured according to the method of Example 2 to observe changes in the growth form.

  Use 4-nitroquinoline-1-oxide (4NQO) (Wako) as a carcinogen, mix with drinking water in mice to a concentration of 100 μg / ml, administer for 16 weeks, and then rest for 8 weeks before tongue tissue Were collected (FIG. 9A). In accordance with the method of Example 1, tongue epithelial stem cells were collected and cultured in the same manner as in Example 2.

  FIG. 9B shows a tongue tissue image after 4NQO treatment. The left figure of FIG. 9B is a tissue image of a site showing hyperplasia, and the right figure is a tissue image of squamous cell carcinoma. It was shown that carcinogenesis was caused by 4NQO treatment.

  As shown in FIG. 10A, tongue epithelial stem cells collected from the tongue of mice administered with carcinogens were cultured and observed with a phase contrast microscope. In addition to the spherical cell mass shown in FIG. A cell cluster showing sex was observed (arrow). Further, when the cell mass was observed by HE staining, the cell mass obtained from the tongue epithelial stem cells administered with the carcinogen was denser in the outermost cells as shown in FIG. 2A, and moved toward the center of the cell mass. A cell cluster in which the number of cells decreased, that is, a so-called stratified squamous epithelial structure could not be formed was observed (FIG. 10B, *). In addition, cell clusters exhibiting atypicality or cell clusters (conformity degree III) in which the concentric structure collapsed were also observed (FIG. 10B, arrows).

  Further, FIG. 11 shows a strongly magnified image of the cell cluster showing atypia. It can be seen that some cells of the cell cluster proliferate irregularly and reach the center (grade III). If normal, the cells around the cell mass have the ability to proliferate, so the cell density at the outermost circumference of the cell mass is the highest, but in this cell mass, the cell density is lower than the cell density at the outermost circumference. The cell density is higher in the regular growth area. Therefore, it has shown that the cell which shows the proliferation form different from a normal tongue epithelial stem cell has appeared in culture | cultivation.

  From these results, it was shown that the tongue epithelial stem cell culture method of the present invention can evaluate the properties of cells exposed to a carcinogen in vivo, for example, the properties of cancer stem cells in vitro.

  Therefore, the method for isolating tongue epithelial stem cells of the present invention is expected to be applicable to elucidation of carcinogenic mechanisms and screening of anticancer substances.

Claims (13)

A method for isolating esophageal epithelial stem cells comprising the following steps (1) to (4):
(1) A process of recovering esophageal epithelial cells by treating esophageal tissue collected from an animal with a protease,
(2) a step of embedding the esophageal epithelial cells obtained in step (1) in a gel matrix;
(3) A step of contacting a cell culture medium containing an epidermal growth factor with a gel matrix containing esophageal epithelial cells to culture the esophageal epithelial cells, and (4) a step of collecting the cells grown in step (3). The isolation method according to claim 1, further comprising a step of treating the protease-treated esophageal epithelial cell with a chelating buffer between the step (1) and the step (2). The isolation method according to claim 1 or 2, wherein the protease is dispase. The isolation method according to any one of claims 1 to 3, wherein the animal is an animal exposed to a carcinogenic factor. A screening method for an anticancer substance comprising the following steps (a) to (d):
(A) treating esophageal tissue collected from an animal exposed to a carcinogenic factor with a protease to recover esophageal epithelial cells;
(B) a step of embedding the esophageal epithelial cells obtained in step (a) in a gel matrix;
(C) a step of culturing esophageal epithelial cells by contacting a cell culture medium containing epidermal growth factor with a gel matrix containing esophageal epithelial cells in the presence of a test substance;
(D) A step of determining that the test substance is an anticancer substance by using death or growth inhibition of esophageal epithelial cells as an index. 6. The method for screening an anticancer substance according to claim 5, further comprising a step of treating the protease-treated esophageal epithelial cells with a chelating buffer between the step (a) and the step (b). The method for screening an anticancer substance according to claim 5 or 6, wherein the protease is dispase. Carcinogenic factor screening method comprising the following steps (i) to (iv):
(I) treating esophageal tissue collected from an animal exposed to the test factor with a protease to recover esophageal epithelial cells;
(Ii) a step of embedding the esophageal epithelial cells obtained in step (i) in a gel matrix;
(Iii) a step of contacting the cell culture medium containing epidermal growth factor with a gel matrix containing esophageal epithelial cells and culturing the cells; and (iv) indicating the degree of atypia of esophageal epithelial cells contained in the cultured tissue And determining the test substance as a carcinogenic factor. The method for screening an anticancer substance according to claim 8, further comprising a step of further treating the protease-treated tongue epithelial cells with a chelating buffer between step (i) and step (ii). The method according to claim 8 or 9, wherein the protease is dispase. Esophageal epithelial stem cells expressing at least one selected from the group consisting of Bmil, type 5 keratin and type 14 keratin isolated from animal esophageal tissue. The esophageal epithelial stem cell according to claim 11, wherein the animal is an animal exposed to a carcinogenic factor. A cell mass obtained by culturing the esophageal epithelial stem cell according to claim 11 or 12.

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