JP2015140334A - Inducer of differentiation to tissue repairing macrophage and method of producing tissue repairing macrophage - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inducer of differentiation to a tissue repairing macrophage.SOLUTION: This invention provides an inducer of differentiation to a tissue repairing macrophage comprising a shell membrane or a hydrolysate thereof and a method of producing a tissue repairing macrophage using the same.

Description

  The present invention relates to a differentiation-inducing agent for tissue-repairable macrophages and a method for producing tissue-repairable macrophages.

  Monocytes in peripheral blood pass through the blood vessel wall and invade tissues, and then differentiate into macrophages under the influence of various environmental factors and inducers in each tissue. Macrophage phenotypes include inflammatory macrophages induced by stimulation with Th1 cytokines such as interferon (IFN) -γ, tumor necrosis factor (TNF) -α, and lipopolysaccharide (LPS) (hereinafter referred to as “inflammatory MΦ”). Or “M1MΦ”) and tissue-repairing macrophages induced to differentiate by Th2 cytokines such as interleukin (IL) -4 and IL-13 (hereinafter referred to as “tissue-repairing MΦ” or “M2MΦ”) It is known that there are at least two types. Furthermore, it is known that macrophages include resting macrophages in addition to the active forms represented by these.

  Inflammatory MΦ has a high expression level of inflammatory cytokines such as TNF-α and IL-1, and induces oxidative stress and neutrophil infiltration through their secretion, decomposing necrotic tissue, foreign matter and bacteria It is thought that they play a part in their phagocytosis. On the other hand, tissue-repairing MΦ highly expresses IL-10, transforming growth factor (TGF) -β, etc., and decreases inflammatory cytokine secretion from inflammatory MΦ through the secretion of IL-10. It is considered that it is also involved in tissue repair through secretion of TGF-β, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and the like.

  In recent years, the role of macrophages in the wound healing process has attracted attention. The wound healing process, observed over time, "bleeding / coagulation phase" where platelets aggregate, "inflammatory phase" where inflammatory cells such as neutrophils migrate and remove foreign bodies, angiogenesis, granulation and re-epithelialization It consists of a four-stage process of “growth phase” in which morphogenesis is performed and “reconstruction phase” in which granulation is absorbed and healing is completed. In an actual wound, a series of vital reactions occur in which these processes overlap and lead to healing. In cases of wounds with underlying diseases such as diabetes (DM) and peripheral arterial injury (PAD), it is thought that the wound's ordered healing cascade is impaired, so that the wound is refractory. In the transition from the proliferative phase to the proliferative phase, the balance of inflammatory MΦ and tissue repair MΦ is led by M2, that is, the conversion from inflammatory macrophages or resting macrophages to tissue repair macrophages plays an important role. It is thought that

  Therefore, an object of the present invention is to provide a differentiation inducer for tissue repairable macrophages and a method for producing tissue repairable macrophages.

As a result of intensive studies to solve the above problems, the present inventors have found that eggshell membranes or hydrolysates thereof induce differentiation of inflammatory macrophages into tissue repairable macrophages, thereby completing the present invention. It came.
That is, the present invention provides the following (1) to (19).

(1) An agent for inducing differentiation into a tissue-repairing macrophage, comprising an eggshell membrane or a hydrolyzate thereof.
(2) The differentiation-inducing agent according to (1), which has a concentration-inducing action on tissue-repairable macrophages.
(3) The differentiation inducing agent according to (1) or (2), which has a concentration-dependent macrophage growth promoting action.
(4) The differentiation inducer according to any one of (1) to (3), wherein the eggshell membrane or a hydrolyzate thereof is an eggshell membrane hydrolyzate obtained by alkaline hydrolysis of the eggshell membrane.
(5) The differentiation inducer according to any one of (1) to (4), which induces differentiation from inflammatory macrophages to tissue repairable macrophages.
(6) The differentiation inducer according to (5), wherein at least one tissue repairable macrophage marker is expressed while at least one type of inflammatory macrophage marker is expressed.
(7) The differentiation inducer according to any one of (1) to (4), which induces differentiation from monocytes to tissue repairable macrophages.
(8) The differentiation inducer according to any one of (1) to (7), wherein the administration route is on the skin, transdermally, intradermally or subcutaneously.
(9) A method for inducing differentiation into a tissue repairable macrophage, comprising a step of contacting the differentiation inducing agent according to any one of (1) to (8) with an inflammatory macrophage or monocyte in vitro.
(10) A method for producing a tissue repairable macrophage, comprising a step of contacting the differentiation inducer according to any one of (1) to (8) with an inflammatory macrophage or monocyte in vitro.

(11) The differentiation inducer according to (7), wherein differentiation is induced from monocytes to tissue repairable macrophages via resting macrophages.
(12) The differentiation inducer according to (5), wherein the inflammatory macrophages are inflammatory macrophages derived from humans or mice.
(13) The differentiation inducer according to (7), wherein the monocytes are monocytes derived from humans or mice.
(14) The differentiation induction method according to (9), comprising the following steps:
(A) adding the differentiation-inducing agent according to any one of (1) to (8) to a culture solution containing inflammatory macrophages or monocytes, and (b) using the culture solution of (a). Culturing inflammatory macrophages or monocytes.
(15) The production method according to (10), comprising the following steps:
(A) adding the differentiation-inducing agent according to any one of (1) to (8) to a culture solution containing inflammatory macrophages or monocytes, and (b) using the culture solution of (a). Culturing inflammatory macrophages or monocytes.
(16) A method for producing a macrophage composition comprising the following steps:
(A) a step of recovering the tissue repairable macrophage produced by the production method according to (10) or (15), and (b) a step of dispersing the recovered tissue repairable macrophage in physiological saline, serum or plasma.
(17) A method for treating an intractable wound comprising the following steps:
(A) collecting inflammatory macrophages or monocytes from a patient having an intractable wound;
(B) The inflammatory macrophages or monocytes collected from the patient are contacted with the differentiation inducer according to any one of (1) to (8) in vitro, and the inflammatory macrophages or monocytes are tissue-repairing macrophages. The process of differentiating into
(C) recovering the tissue repairable macrophages differentiated in (b),
(D) Dispersing the tissue repairable macrophages recovered in (c) in physiological saline, serum or plasma to produce a tissue repairable macrophage composition; and (e) injecting the tissue repairable macrophage composition thus produced. Administering to said patient.
(18) The refractory wound is at least selected from the group consisting of diabetic (neurogenic) ulcers, arterial (ischemic) ulcers, venous stasis ulcers, ulcers due to Buerger's disease and ulcers associated with collagen diseases The treatment method according to (17), which is one intractable wound.
(19) The treatment method according to (17), wherein the patient has at least one basic disease selected from the group consisting of diabetes, peripheral arterial injury, varicose veins, Buerger's disease and collagen disease.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the differentiation inducer to a tissue repairable macrophage and a tissue repair macrophage can be provided.

  The differentiation inducer of the present invention does not affect inflammatory macrophages and can increase the conversion to tissue repairable macrophages in a concentration-dependent manner.

FIG. 1 shows the differentiation rate of Comparative Examples (Comp. Ex.) 1 and 2 and Examples (Ex.) 1 to M1MΦ (inflammatory macrophages, F4 / 80 (+) CD11b (+) cells) ( %). FIG. 2 shows the differentiation rate of Comparative Examples (Comp. Ex.) 1 and 2 and Examples (Ex.) 1 to M2MΦ (tissue repairable macrophages, F4 / 80 (+) CD206 (+) cells). It is a graph showing (%). FIG. 3 is a graph showing a cell proliferation index of Comparative Example (Comp. Ex.) 2 and Examples (Ex.) 1 to 3, with Comparative Example (Comp. Ex.) 1 as 100.

[Induction agent for tissue repairing macrophages]
The differentiation-inducing agent for tissue repairable macrophages of the present invention contains eggshell membranes or hydrolysates thereof as active ingredients.

<Egg shell membrane or its hydrolyzate>
The eggshell membrane is a thin film inside the avian egg shell. The main component is protein, and the outer side is rough and the inner side is a two-layered network structure, which is rich in flexibility and toughness.

  The adhesion to the tissue by the mesh structure, moderate moisture retention and breathability is suitable as a wound dressing material, so in the old days when wrestlers were injured, eggshell membranes were applied to the wounds and the wounds were cured quickly. For example, eggshell membranes are known to be effective as wound dressings (dressing materials).

  The eggshell membrane is not particularly limited as long as it is an eggshell membrane derived from a bird's egg, and any eggshell membrane of any bird's egg can be used as it is or as a powder or a hydrolyzate. As eggs for birds, for example, eggs of chickens, pheasants, turkeys, ducks, ducks, aigamo, quail, pigeons, partridges, grouse, swordfish, porcupines, ostriches, crows, sparrows, swallows, guns, swans, etc. can be used. .

  The eggshell membrane is preferably used as eggshell membrane powder or eggshell membrane hydrolysate from the viewpoint of dispersibility and solubility in water or an aqueous solvent.

(Egg shell membrane powder)
As a method for producing eggshell membrane powder, there is a method in which eggshell membranes are separated from avian eggs such as chicken eggs, dried, pulverized and sieved. The particle size of the powder can be controlled by the degree of pulverization and the size of sieving.
Examples of eggshell membrane powder include EM powder (300) (manufactured by Kewpie).

(Egg shell membrane hydrolyzate)
The eggshell membrane hydrolyzate can be produced by dissolving bird eggs such as chicken eggshell membrane with alkali such as sodium hydroxide, neutralizing the alkali, filtering, desalting, microfiltration, Examples thereof include a method for producing a hydrolyzate in a powder form by freeze-drying and sieving, a method for preparing a hydrolyzate in a solution state after desalting, adjusting components and performing microfiltration. Further, it can also be produced by the eggshell membrane hydrolyzate production method described in, for example, JP-A-1-275512, JP-A-5-97897, JP-A-2009-132661, and the like.
Examples of the eggshell membrane hydrolyzate include EM protein-P (produced by Kewpie, powder), EM protein-L (produced by Kewpie, liquid), and the like.

《Dosage form》
The differentiation inducer of the present invention is preferably used in vitro against inflammatory macrophages or monocytes collected from a living body, but may be used directly on a living body. Since the differentiation-inducing agent of the present invention contacts inflammatory macrophages or monocytes in vivo and promotes differentiation induction into tissue-repairing macrophages, the step of collecting inflammatory macrophages or monocytes is not necessary. On the other hand, there is an advantage that it is minimally invasive.

  The dosage form when the differentiation-inducing agent of the present invention is used for a living body is not particularly limited, and includes, for example, tablets including orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, dissolving tablets, and the like; hard capsules, Capsules including soft capsules, etc .; granules including foamed granules; powders; oral liquids including elixirs, suspensions, emulsions, limonades, etc .; syrups including syrups, etc .; oral jellys; Oral tablets including lozenges, sublingual tablets, buccal tablets, adhesive tablets, gums, etc .; oral sprays; oral semisolids; mouthwashes; infusions, implants, sustained injections, etc. Injections; inhalants including inhaled powders, inhalants, aerosols, etc .; eye drops; ophthalmic ointments; ear drops; nasal powders, nasal drops; suppositories; semisolid for rectal use; enema Vaginal tablets; vaginal suppositories; external solid preparations including powders for external use; liniment Liquid preparations for external use, including aerosols, lotions, etc .; summary sprays, including aerosols, pump sprays, etc .; ointments; creams; gels; and tapes, patches, etc. . Among these, a dosage form that can be administered locally is preferable because it can directly affect the wound site.

《Pharmaceutically acceptable additive》
In addition, the differentiation-inducing agent of the present invention may use pharmaceutically acceptable additives in the production thereof, and examples of such additives include starch, gelatin, glucose, lactose, and fructose. , Maltose, magnesium carbonate, talc, magnesium stearate, methylcellulose, carboxymethylcellulose or a salt thereof, gum arabic, polyethylene glycol, p-hydroxybenzoic acid alkyl ester, syrup, water, ethanol, propylene glycol, petrolatum, carbowax, glycerin, Examples include sodium chloride, sodium sulfite, sodium phosphate, citric acid, collagen, sodium alginate, sucrose and the like.

<Macrophages and monocytes>
The differentiation inducer of the present invention induces differentiation of inflammatory macrophages or monocytes into tissue repairable macrophages.

《Macrophage》
In the present invention, macrophages are human cells that are positive for human macrophage marker CD14 (hereinafter sometimes referred to as “CD14 (+)”) or mouse macrophage marker F4 / 80 is positive ( Hereinafter referred to as "F4 / 80 (+)"). Furthermore, macrophages can be classified into active macrophages, including inflammatory macrophages and tissue repair macrophages, and resting macrophages, depending on their phenotype.

  Hereinafter, active macrophages, inflammatory macrophages, tissue repair macrophages and resting macrophages will be described.

(Active macrophages)
In the present invention, the activated macrophage is CD14 (+), and the human inflammatory macrophage marker CD80 is also positive (hereinafter sometimes referred to as “CD80 (+)”), F4 / 80. (+), Mouse cells that are positive for mouse inflammatory macrophage marker CD11b (hereinafter sometimes referred to as “CD11b (+)”), human tissue repair macrophage marker CD206 is positive (hereinafter referred to as “CD11b (+)”) "CD206 (+)" in some cases) or mouse cells that are positive for CD206, which is a marker for mouse tissue repair macrophages (hereinafter sometimes referred to as "CD206 (+)"). Say. That is, activated macrophages include inflammatory macrophages and tissue repair macrophages.

(Inflammatory macrophages)
In the present invention, inflammatory macrophages mean human cells that are CD14 (+) and CD80 (+), or mouse cells that are F4 / 80 (+) and CD11b (+). Inflammatory macrophages, also known as classical activated macrophages, enhance immunity and induce and promote inflammation.

(Tissue repairing macrophages)
In the present invention, tissue-repairing macrophages means human cells that are CD14 (+) and CD206 (+), or mouse cells that are F4 / 80 (+) and CD206 (+). Tissue repairing macrophages, also known as wound healing macrophages, anti-inflammatory macrophages, suppress immunity and direct inflammation to termination.

(Resting macrophages)
In the present invention, resting macrophages are human cells that are CD14 (+) and are negative for both CD80 and CD206 (may be referred to as “CD80 (−)” and “CD206 (−)”, respectively). Or F4 / 80 (+) means a mouse cell in which both CD11b and CD206 are negative (may be described as “CD11b (−)” and “CD206 (−)”, respectively). Resting macrophages are in an inactive state (resting state), although monocytes infiltrated into the tissue from the blood are differentiated into macrophages.

《Monocyte》
Monocytes are immature phagocytes that circulate in the blood, function as antigen-presenting immune cells, and have the function of taking in and degrading pathogens and foreign substances by phagocytosis.
In the present invention, monocytes derived from humans or mice can be used as monocytes. Among these, human-derived monocytes are preferable, and human peripheral blood-derived monocytes (human peripheral blood monocytes) are more preferable.

The monocytes may be commercially available products such as monocytes derived from human or mouse peripheral blood, or may be monocytes collected by apheresis from whole peripheral blood collected from a donor. The apheresis method is not particularly limited as long as it can separate monocytes from whole blood, but it is preferable to use a blood component separation device. Examples of the blood component separation device include COBE ^(R) Spectra (manufactured by Terumo BCT), COM. Examples include TEC (manufactured by Fresenius Kirby). Further, monocytes can be separated and collected from whole blood by density gradient centrifugation without using a blood component separation device. When blood is collected from a donor, granulocyte colony stimulating factor (G-CSF) may be administered to the donor several days before blood collection.

[Method of inducing differentiation of tissue repairing macrophages]
The differentiation inducer of the present invention can induce differentiation from inflammatory macrophages or monocytes into tissue repairable macrophages.

  The method of inducing differentiation from inflammatory macrophages or monocytes into tissue repairable macrophages with the differentiation-inducing agent of the present invention (hereinafter sometimes simply referred to as “the differentiation-inducing method of the present invention”) is inflammatory macrophages or monocytes. It includes a step of contacting the sphere with the differentiation inducer of the present invention in vitro.

  The method of bringing the inflammatory macrophage or monocyte into contact with the differentiation inducer of the present invention is not particularly limited, and examples thereof include a method of culturing the inflammatory macrophage or monocyte in the presence of the differentiation inducer of the present invention. For example, it is preferable to add the differentiation inducer of the present invention to a medium containing inflammatory macrophages or monocytes. In addition, any method can be used as long as it exhibits the same effect as the method of adding the differentiation inducer of the present invention to the medium.

Specifically, for example, the differentiation induction method of the present invention includes the following steps:
(A) adding the differentiation-inducing agent of the present invention to a culture solution containing inflammatory macrophages or monocytes, and (b) culturing inflammatory macrophages or monocytes using the culture solution of (a).

As a medium for inflammatory macrophages or monocytes, a conventionally known medium can be used as long as the object of the present invention is not impaired. For example, Gibco ^(R) RPMI 1640 medium (Life Technologies), HL-1 known composition, serum-free medium (Lonza), monocyte adhesion medium (Promocell), mononuclear cell medium (Promocell), etc. Can be used.

The culture condition is not particularly limited as long as it is a method suitable for culturing inflammatory macrophages or monocytes. For example, inflammatory macrophages or monocytes are preferably used as a medium in a medium of 1 × 10 ⁰ to 1 × 10 ⁷ cells / mL. More preferably, after suspending to a density of 1 × 10 ² to 1 × 10 ⁶ cells / mL and seeding on a culture plate, the temperature is preferably 30 to 39 ° C., more preferably 35 to 39 ° C. A method of culturing at 37 ° C., preferably for 1 to 10 days, more preferably 1 to 7 days, and preferably under CO ₂ condition through about 5% carbon dioxide can be mentioned. .

  The concentration of the differentiation-inducing agent of the present invention is not particularly limited, but is preferably 0.01 to 1 mg / L as the solid content concentration of the eggshell membrane powder or eggshell membrane hydrolyzate as the active ingredient. Within this range, differentiation can be induced more efficiently from inflammatory macrophages or monocytes to tissue repairing macrophages.

Before culturing using the medium to which the differentiation inducer of the present invention is added, inflammatory macrophages or monocytes may be precultured. In the pre-culture, the temperature is preferably 30 to 39 ° C., more preferably 35 to 39 ° C., still more preferably 37 ° C., and the time is preferably 4 to 48 hours, more preferably 8 to 36 hours, preferably A method of incubating under conditions of CO ₂ through 5% carbon dioxide can be mentioned.

  The tissue-repairing macrophages obtained by inducing differentiation of inflammatory macrophages or monocytes in the culture solution are subsequently purified using high-purity tissue-repairing macrophages by using cell recovery, separation, and purification methods by known methods. Can be obtained efficiently and in large quantities.

[Method for producing tissue repairing macrophages]
By using the differentiation inducing method of the present invention described above, tissue repairable macrophages can be produced from inflammatory macrophages or monocytes (the tissue repairable macrophages of the present invention are hereinafter referred to as “tissue repairable macrophages of the present invention”). ").

Although the use of the tissue repairable macrophage of the present invention is not particularly limited, for example, it can be used for the treatment of intractable wounds described later.
When the tissue repairable macrophage of the present invention is used in this application, the tissue repairable macrophage should be used as a tissue repairable macrophage composition by a method for producing a tissue repairable macrophage composition described later from the viewpoint of ease of handling. Is preferred.

[Method for producing tissue repairing macrophage composition]
The tissue repairable macrophages produced by the above-described method for producing tissue repairable macrophages are collected by a known method such as centrifugation or membrane separation, purified, washed, etc. as desired, and then dispersed in physiological saline, serum or plasma. By doing so, a tissue repairable macrophage composition can be produced (the tissue repairable macrophage composition produced may be hereinafter referred to as “the tissue repairable macrophage composition of the present invention”). The tissue repairable macrophage composition of the present invention may be further used by mixing with a conventionally known additive that is pharmacologically acceptable.

  Here, serum refers to blood (whole blood) coagulated and platelets and coagulation factors are removed, and plasma refers to cellular components (red blood cells, Leukocytes and platelets are excluded.

  The use of the tissue repairable macrophage composition of the present invention is not particularly limited. For example, the tissue repairable macrophage composition can be used for the treatment of intractable wounds described later.

[Tissue repairable macrophage composition and treatment of refractory wound using the same]
The tissue repairable macrophage composition of the present invention is suitable for use in the treatment of intractable wounds. In patients with refractory wounds (mammals, preferably humans), it is considered that the wound is refractory due to impaired ordered healing of the wound, but the macrophage or macrophage composition of the present invention is This is because administration to a patient can be expected to promote wound healing by exerting an action in accordance with the healing cascade stage at the wound site.

  Here, the intractable wound refers to a chronic, incurable wound, and is not particularly limited. For example, diabetic (neurogenic) ulcer, arterial (ischemic) ulcer, venous stasis ulcer, Buerger disease And ulcers associated with collagen disease. Examples of basic diseases of intractable wounds include diabetes, peripheral arterial injury, varicose veins, deep vein thrombosis, Buerger's disease, collagen disease and the like.

  As a method for administering the tissue repairing macrophage composition to a patient, injection is preferable, and examples thereof include intramuscular injection, intravenous injection, intradermal injection, and subcutaneous injection.

Also, the patient receiving the tissue repair macrophage composition and the inflammatory macrophage or monocyte donor used to produce the tissue repair macrophage composition may be allogeneic (human or mouse). Preferably, they are syngeneic humans or mice, more preferably humans or mice of the same individual. Syngeneic refers to, for example, the relationship between identical twins and pure animals. If the patient and donor are syngeneic or the same individual, they are genetically identical and therefore do not have serious side effects such as rejection.
In addition, when serum or plasma is used in the production of a tissue repairable macrophage composition, a sample collected from the patient can be used so that the patient to whom the tissue repair macrophage composition has been administered becomes a virus or other pathogen. The risk of infection can be reduced.

  Examples of the present invention will be described below, but the present invention is not limited thereto.

[Comparative Example 1]
(Pre-culture)
Metabolic syndrome model mice (C57BL / 6J mice, manufactured by Nippon SLC Co., Ltd.) were injected intraperitoneally with an inflammation inducer (thioglycolate medium), and intraperitoneal macrophages were collected 72 hours later. Since thioglycolate medium causes inflammation, macrophages (hereinafter sometimes simply referred to as “MΦ”) exist as M1MΦ.
The collected MΦ is adjusted to about 1 × 10 ⁶ cells / mL with a macrophage colony stimulating factor (M-CSF) -containing RPMI-1640 medium and seeded on a multiplate (6 wells) for adherent cell culture (manufactured by Nippon Becton). And pre-cultured for 24 hours in an environment of 37 ° C. and 5% CO ₂ .
(Main culture, cell count)
After exchanging the RPMI-1640 medium, the cells were further cultured for 7 days under the same conditions as the preculture, and then the cells were collected from each well. The collected cells were labeled with mouse macrophage marker F4 / 80, mouse inflammatory macrophage marker CD11b, mouse tissue repair macrophage marker CD206, and flow cytometry (Cytomics FC500, Beckman Coulter). Was used to measure the total number of cells per mL of culture, the number of F4 / 80 (+) CD11b (+) cells, and the number of F4 / 80 (+) CD206 (+) cells.
(Calculation of differentiation rate)
From the measured data, the differentiation rate into inflammatory macrophages (M1MΦ) was determined as the ratio of F4 / 80 (+) CD11b (+) cells to total cells = {(F4 / 80 (+) CD11b (+) cells) / The total number of cells} × 100 (%) was calculated. Similarly, the ratio of F4 / 80 (+) CD206 (+) cells in the total cells = {(F4 / 80 (+) CD206 (+) cells) / total The number of cells} × 100 (%) was calculated. The results are shown in Table 1, FIG. 1 and FIG.

[Comparative Example 2]
(Pre-culture)
The preculture of Comparative Example 1 was used.
(Main culture, cell count)
After changing the RPMI-1640 medium, Th2 cytokines (IL-4, IL-10 and IL-13) were added to each well so that the final concentration of each was 10 ng / mL.
Then, after further culturing for 7 days under the same conditions as the preculture, cells were collected from each well. The collected cells were labeled with mouse macrophage marker F4 / 80, mouse inflammatory macrophage marker CD11b, mouse tissue repair macrophage marker CD206, and flow cytometry (Cytomics FC500, Beckman Coulter). Was used to measure the total number of cells per mL of culture, the number of F4 / 80 (+) CD11b (+) cells, and the number of F4 / 80 (+) CD206 (+) cells.
(Calculation of differentiation rate and cell proliferation index)
From the measured data, the differentiation rate into inflammatory macrophages (M1MΦ) was determined as the ratio of F4 / 80 (+) CD11b (+) cells to total cells = {(F4 / 80 (+) CD11b (+) cells) / The total number of cells} × 100 (%) was calculated. Similarly, the ratio of F4 / 80 (+) CD206 (+) cells in the total cells = {(F4 / 80 (+) CD206 (+) cells) / total The number of cells} × 100 (%) was calculated. The results are shown in Table 1, FIG. 1 and FIG.
Further, from the measurement data, the ratio of the cell proliferation index for Comparative Example 1 to the total cell number of Comparative Example 1 relative to the total cell number of Comparative Example 2 = (total number of cells of Comparative Example 2 / total number of cells of Comparative Example 1) × Calculated as 100. The results are shown in Table 1 and FIG.

[Examples 1 to 3]
(Pre-culture)
The preculture of Comparative Example 1 was used.
(Main culture, cell count)
After replacing the RPMI-1640 medium, the final concentration of EM protein-P is 0.01 mg / mL (Example 1), 0.1 mg / mL (Example 2), or 1 mg / mL (Example 3) To each well.
Then, after further culturing for 7 days under the same conditions as the preculture, cells were collected from each well. The collected cells were labeled with mouse macrophage marker F4 / 80, mouse inflammatory macrophage marker CD11b, mouse tissue repair macrophage marker CD206, and flow cytometry (Cytomics FC500, Beckman Coulter). Was used to measure the total number of cells per mL of culture, the number of F4 / 80 (+) CD11b (+) cells, and the number of F4 / 80 (+) CD206 (+) cells.
(Calculation of differentiation rate and cell proliferation index)
From the measured data, the differentiation rate into inflammatory macrophages (M1MΦ) was determined as the ratio of F4 / 80 (+) CD11b (+) cells to total cells = {(F4 / 80 (+) CD11b (+) cells) / The total number of cells} × 100 (%) was calculated. Similarly, the ratio of F4 / 80 (+) CD206 (+) cells in the total cells = {(F4 / 80 (+) CD206 (+) cells) / total The number of cells} × 100 (%) was calculated. The results are shown in Table 1, FIG. 1 and FIG.
Further, from the measurement data, the ratio of the cell proliferation index with respect to Comparative Example 1 to the total number of cells in Comparative Example 1 with respect to the total number of cells in each of Examples 1 to 3 = (the total number of cells in each of Examples 1 to 3) The total number of cells in Example 1) × 100 was calculated. The respective results are shown in Table 1 and FIG.

It can be seen that there is almost no difference in the differentiation rate into M1MΦ (inflammatory macrophages) between Examples 1 to 3 and Comparative Example 1 (FIG. 1).
On the other hand, it can be seen that there is a large difference in the differentiation rate into M2MΦ (tissue repairable macrophages) between Examples 1 to 3 and Comparative Example 1 (FIG. 2). Moreover, the differentiation rate to M2MΦ increases depending on the concentration of the differentiation-inducing agent (eggshell membrane hydrolyzate) of the present invention and reaches 98% at 1 mg / mL (FIG. 2).
From the above, it can be seen that the differentiation inducer of the present invention does not affect M1MΦ (inflammatory macrophages) and increases differentiation into M2MΦ (tissue repairing macrophages) in a concentration-dependent manner.
Moreover, in Examples 1-3, it turns out that a cell proliferation parameter | index also increases depending on the density | concentration of the differentiation-inducing agent of this invention (FIG. 3).

Claims (9)

  An agent for inducing differentiation into a tissue-repairing macrophage, comprising an eggshell membrane or a hydrolyzate thereof.   The differentiation-inducing agent according to claim 1, which has a concentration-inducing action on tissue-repairable macrophages in a concentration-dependent manner.   The differentiation-inducing agent according to claim 1, which has a concentration-dependent macrophage growth promoting action.   The differentiation inducer according to any one of claims 1 to 3, wherein the eggshell membrane or a hydrolyzate thereof is an eggshell membrane hydrolyzate obtained by alkaline hydrolysis of the eggshell membrane.   The differentiation inducing agent according to any one of claims 1 to 4, which induces differentiation from an inflammatory macrophage to a tissue repairing macrophage.   The differentiation-inducing agent according to claim 5, wherein at least one tissue repairing macrophage marker is expressed while at least one inflammatory macrophage marker is expressed.   The differentiation inducer according to any one of claims 1 to 4, which induces differentiation from monocytes to tissue repairable macrophages.   The differentiation inducer according to any one of claims 1 to 7, wherein the administration route is dermal administration, transdermal administration, intradermal administration, or subcutaneous administration.   A method for producing a tissue repairing macrophage from inflammatory macrophages or monocytes, comprising a step of contacting the differentiation inducing agent according to any one of claims 1 to 8 with inflammatory macrophages or monocytes in vitro.