JP6851060B2 - Hematopoietic stem cell differentiation promoter - Google Patents

Description

The present invention relates to a hematopoietic stem cell differentiation promoter containing one or more amino acids selected from glutamic acid, leucine, valine, threonine and glycine as an active ingredient.

Blood is composed of blood cell cells and plasma, and has important functions for maintaining homeostasis of the living body such as oxygen transport, removal of waste products, and immune function. However, in the elderly, the number of blood cells in the blood decreases and the function declines due to aging, so the immunity to infectious diseases decreases, and various hematologic diseases such as anemia, neutrophil hypocytopenia, and thrombocytopenia It has been reported that it is easily affected and causes a decrease in QOL (Non-Patent Documents 1-4, etc.). Such a decrease in blood cell cells and a decrease in function are considered to be one of the causes of aging of hematopoietic stem cells that produce them (Non-Patent Document 5).

Hematopoietic stem cells are abundant in the bone marrow and are pluripotent and self-renewing cells that differentiate into all blood cells and continue to supply blood cells throughout the life of the individual. Therefore, by transplanting hematopoietic stem cells, it is possible to reconstruct whole blood cells from a state in which there are no blood cells. Therefore, as a means for treating blood diseases, immunodeficiency, etc., transplantation of self or the same type of hematopoietic stem cells is performed. However, it has been reported that the proliferative ability and differentiation ability of hematopoietic stem cells decrease with aging (Non-Patent Document 6), and even if hematopoietic stem cells are transplanted, the blood cell system cannot be reconstructed.

Therefore, it is considered important to restore the function of hematopoietic stem cells in order to prevent or improve the decrease in QOL caused by the failure of remodeling of the blood cell system with aging. So far, factors such as granulocyte macrophage colony stimulating factor (GM-CSF) and thrombopoietin (TPO) have been known as factors involved in promoting the differentiation of hematopoietic stem cells (Non-Patent Documents 7 and 8). However, there are various problems such as in vivo control and difficulty in continuous use on a daily basis, and it is desired to elucidate new factors to replace them. ing.

Disordered hematopoiesis and myelodysplasia in the elderly. J. Am. Geriatr Soc. 2003 Mar; 51 (3 Suppl): S22-6 Immunology: emerging challenges for an aging population. Immunology. 2007 Apr; 120 (4): 435-46. Epub 2007 Feb 15. Aging of the Innate Immune System: An Update. Curr Immunol. Rev. 2011 Feb 1; 7 (1): 104-115. Unexplained anemia in the elderly. Semin Hematol. 2008 Oct; 45 (4): 250-4. Doi: 10.1053 / j.seminhematol.2008.06.003. The ageing haematopoietic stem cell compartment. Nat. Rev. Immunol. 2013 May; 13 (5): 376-89. doi: 10.1038 / nri3433. Epub 2013 Apr 15. Age-associated characteristics of murine hematopoietic stem cells. J. Exp. Med. 2000 Nov 6; 192 (9): 1273-80. The molecular control of cell division, differentiation commitment and maturation in haemopoietic cells. Nature. 1989 May 4; 339 (6219): 27-30. The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells. Blood. 1996 Jun 15; 87 (12): 4998-5005.

An object of the present invention is to provide a novel factor that promotes the differentiation of hematopoietic stem cells in view of the above circumstances.

As a result of intensive studies to solve the above problems, the present inventors have found that one or more amino acids selected from glutamic acid, leucine, valine, threonine and glycine have an excellent effect of promoting differentiation of hematopoietic stem cells. The present invention has been completed.

That is, the present invention includes the following inventions.
(1) A hematopoietic stem cell differentiation promoter containing one or more amino acids selected from glutamic acid, leucine, valine, threonine and glycine as an active ingredient.
(2) A method for promoting the differentiation of hematopoietic stem cells, which comprises culturing the hematopoietic stem cells in the presence of the hematopoietic stem cell differentiation promoting agent according to (1).
(3) A method for producing blood cells, which comprises a step of culturing hematopoietic stem cells in the presence of the hematopoietic stem cell differentiation promoting agent according to (1).

Since the hematopoietic stem cell differentiation promoter of the present invention can promote the differentiation of hematopoietic stem cells and induce blood cells, it is possible to treat and improve blood and hematopoietic organ diseases caused by functional deterioration or deficiency of hematopoietic stem cells. And can be prevented. Further, since the hematopoietic stem cell differentiation promoter of the present invention contains an amino acid as an active ingredient, it has no side effects even when used on a daily basis and is highly safe. Therefore, it can be safely used for foods and drinks such as pharmaceuticals and health foods. Further, the blood cells produced by culturing the hematopoietic stem cells in the medium to which the differentiation promoter of the hematopoietic stem cells of the present invention is added can be used as a blood product.

The present invention will be described in detail below.
The hematopoietic stem cell differentiation promoter of the present invention contains one or more amino acids selected from glutamic acid, leucine, valine, threonine and glycine as active ingredients.

Glutamic acid, leucine, valine, threonine and glycine, which are the active components of the differentiation promoter of hematopoietic stem cells of the present invention, may be L-form, D-form or DL-form, respectively, but L-form. Is preferable. In addition, glutamic acid, leucine, valine, threonine and glycine may be in the form of free form or physiologically acceptable salt, respectively. The salt forms include organic acids (acetic acid, lactic acid, citric acid, tartaric acid, maleic acid, fumaric acid, etc.), inorganic acids (ammonia, hydrobromic acid, sulfuric acid, phosphoric acid, nitrate, perchloric acid, etc.), organic acids. Bases (ethylenediamine, propylenediamine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, triethanolamine, etc.) or inorganic bases (hydroxides or charcoal oxides of metals such as sodium, potassium, calcium, etc., or ammonia, etc.) ) And the salt formed. In addition, glutamic acid, leucine, valine, threonine and glycine are prepared by a chemical synthesis method, a fermentation method or a gene recombination method, extracted from animals and plants containing these amino acids, purified, or commercially available. You may.

As the active ingredients, glutamic acid, leucine, valine, threonine and glycine may be used alone, but it is preferable to use two or more in combination, and it is more preferable to use all five. When two or more kinds are used in combination, it is preferable to contain either one or both of glutamic acid and glycine.

In the present invention, the "hematopoietic stem cell" refers to myeloid cells (erythrocytes, platelets, granulocytes (eosinophils, eosinophils, neutrophils), monospheres, etc.) and lymphocytes via myeloid progenitor cells. A cell capable of differentiating into a lymphocytic cell (B cell, T cell, NK cell, etc.) via a lineage progenitor cell. Hematopoietic stem cells are characterized by being positive for the CD34 antigen and negative for the CD38 antigen (CD34 + CD38-).

In the present invention, "differentiation of hematopoietic stem cells" means that hematopoietic stem cells divide and proliferate into hematopoietic progenitor cells (myeloid progenitor cells, lymphoid progenitor cells) and from hematopoietic progenitor cells to mature blood cells. ..

In the present invention, "promotion of differentiation of hematopoietic stem cells" means that differentiation of hematopoietic stem cells is activated as compared with before administration or ingestion of the drug of the present invention. Whether or not the differentiation of hematopoietic stem cells has been activated can be determined in vitro by a method usually performed by those skilled in the art, for example, culturing in the absence of the differentiation promoting agent for hematopoietic stem cells of the present invention. It is possible to evaluate whether or not the expression level of the blood cell marker gene is significantly higher at the mRNA level or the protein level in the stem cells cultured in the presence of the differentiation promoter of the hematopoietic stem cells of the present invention as compared with the stem cells. it can. Examples of blood cell marker genes include Bmaj and Ter119 as red blood cell markers, Gr-1, Fcgr3 as granulocyte markers, and CD11b / CD18 (Mac-1), F4 / 80 and B cell markers as monocyte / macrophage cell markers. Examples include, but are not limited to, CD45R (Ptprc) and T cell markers such as Ly1 and CD2.

The hematopoietic stem cell differentiation promoter of the present invention can be used in the form of a drug or food to promote the differentiation of hematopoietic stem cells in vivo, as well as a cell culture additive for promoting the differentiation of hematopoietic stem cells, for research purposes. As a reagent, it can also be used to promote the differentiation of hematopoietic stem cells in vitro. Therefore, according to the present invention, a method for promoting differentiation of hematopoietic stem cells, which comprises culturing the hematopoietic stem cells in the presence of a differentiation promoting agent for hematopoietic stem cells containing the above-mentioned specific amino acids, and the above-mentioned specific amino acids. Provided is a method for producing a blood cell, which comprises a step of culturing the hematopoietic stem cell in the presence of a differentiation promoting agent for the hematopoietic stem cell containing.

Here, "blood cells" are all stages from hematopoietic stem cells to hematopoietic precursor cells (including pluripotent hematopoietic precursor cells and monophasic hematopoietic precursor cells) to finally functioning blood cells. Cells (excluding hematopoietic stem cells). Specifically, myelocyte progenitor cells, lymphoid progenitor cells, erythroblasts, myeloid blasts, promyelocytes, myelocytes, neutrophils, eosinophils, and eosinophils induced to differentiate from hematopoietic stem cells. , Erythrocytes, macronuclear cells, platelets, progenitor B cells, progenitor T cells, T cells, B cells, natural killer cells, monoblasts, monospheres, macrophages, dendritic cells and the like. In addition, the blood cells obtained by the method for producing blood cells of the present invention can be infused into a patient in the obtained state or by separating the target blood cells by a separation means such as centrifugation or a separation filter. It may be prepared into a formulation.

The medium used in the above method of the present invention can be prepared by using a medium generally used for culturing stem cells as a basal medium and adding the differentiation-promoting agent for hematopoietic stem cells of the present invention. For example, as the basal medium, a basal medium containing components (inorganic salts, carbohydrates, hormones, vitamins, fatty acids) necessary for the survival and proliferation of stem cells, specifically, Dulvecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basic Medium Eagle (BME), Dulvecco's Modern Eagle's Medium: Nutrient Mixture Medium (D-MEM / F-12) (Hank's balanced salt solution) and the like can be mentioned, but D-MEM / F-12 is preferable. In addition, basic fibroblast growth factor (bFGF) and / or leukocyte migration inhibitory factor (LIF) may be added to the medium as growth factors. In addition, if desired, the medium is epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferase, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27. -Supplement, N2-supplement, ITS-supplement, antibiotics, etc. may be contained.

The content of the differentiation-promoting agent for hematopoietic stem cells of the present invention in the medium can be appropriately changed depending on the type, number and combination of glutamic acid, leucine, valine, threonine and glycine used. For example, glutamic acid is preferably 7.5 mM or more. Is 15 mM or more, more preferably 30 mM or more, leucine is 1.5 mM or more, preferably 3 mM or more, more preferably 6 mM or more, valine is 1.5 mM or more, preferably 3 mM or more, more preferably 6 mM or more, and threonine is 1. The medium may be prepared so that the content is 5 mM or more, preferably 3.0 mM or more, more preferably 6.0 mM or more, and glycine is 0.9 mM or more, preferably 1.8 mM or more, more preferably 3.6 mM or more. The upper limit is not limited, but from the viewpoint of effectiveness and economy, all amino acids are 50 mM or less, preferably 35 mM or less.

The incubator used for culturing hematopoietic stem cells is not particularly limited as long as the stem cells can be cultured, and examples thereof include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. Be done.

The incubator may be cell non-adhesive or adhesive, and is appropriately selected depending on the intended purpose. As the cell adhesion incubator, one treated with a cell support substrate or the like using an extracellular matrix or the like may be used for the purpose of improving the adhesion to cells. Examples of the cell support substrate include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.

The culture conditions for hematopoietic stem cells may follow the usual conditions used for culturing hematopoietic stem cells, and no special control is required. For example, the culture temperature is not particularly limited, but is about 30 to 40 ° C, preferably 36 to 37 ° C. The CO ₂ gas concentration is, for example, about 1-10%, preferably about 2-5%. The culturing time is, for example, about 1 to 14 days, preferably 4 to 6 days. The medium is preferably changed once every 2 to 3 days, and more preferably every day. The culture conditions can be appropriately varied and set within a range in which hematopoietic stem cells can survive and proliferate and promote differentiation.

When the hematopoietic stem cell differentiation promoting agent of the present invention is administered in vivo, it can be administered as it is, but a composition such as a drug or a food or drink together with an appropriate additive as long as the effect of the present invention is not impaired. Can be blended with. The pharmaceutical product of the present invention also includes a drug used for animals, that is, a veterinary drug.

When the hematopoietic stem cell differentiation promoting agent of the present invention is provided as a pharmaceutical product, one or more amino acids selected from glutamic acid, leucine, valine, threonine and glycine (hereinafter referred to as "hematopoietic stem cell differentiation promoting amino acids"). ), Pharmaceutically acceptable and appropriately selected formulation substrates and carriers, excipients, diluents, binders, lubricants, coating agents, disintegrants or disintegrant aids, stabilization. Agents, preservatives, preservatives, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonic agents, pH adjusters, propellants, colorants, sweeteners, flavoring agents, fragrances Etc. may be appropriately added to prepare various formulations that can be orally or parenterally administered systemically or topically by various known methods. When the pharmaceutical product of the present invention is provided in each of the above forms, it can be produced by a manufacturing method usually used by those skilled in the art, for example, the manufacturing method shown in each article of the general formulation [2] formulation of the Japanese Pharmacopoeia.

The pharmaceutical product of the present invention can be administered orally or parenterally, but is preferably orally administered. When the drug of the present invention is orally administered, it is prepared in tablets (including sugar-coated tablets), capsules, granules, powders, troches, pills, liquid solutions for internal use, emulsions, syrups, suspensions, elixirs, etc. It may be a dry product that is converted or redissolved upon use. When the drug of the present invention is administered parenterally, it is formulated into an injection (for example, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), drip infusion, suppository, etc. and injected. In the case of pharmaceutical preparations, they are provided in the form of unit dose ampoules or multidose containers.

Formulations for oral administration include, for example, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; carboxymethyl cellulose, carboxymethyl cellulose calcium, etc. Disintegrants or disintegrants such as starch or hydroxypropyl cellulose; binders such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate, or talc. Coating agents such as hydroxypropylmethyl cellulose, sucrose, polyethylene glycol, or titanium oxide; bases such as vaseline, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat can be used. Not limited to these.

Solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, myoban water, and vegetable oil; isotonic agents such as glucose, sodium chloride, and D-mannitol; inorganic acids , Organic acids, inorganic bases, pH adjusters such as organic bases, and the like can be used, but the present invention is not limited thereto.

Further, in the formulation, one or more active ingredients other than the hematopoietic stem cell differentiation promoting amino acid, which is the active ingredient of the hematopoietic stem cell differentiation promoting agent of the present invention, may be used in combination. Examples of active ingredients suitable for combined use include inorganic acid salts of iron such as iron chloride, iron sulfate, and iron phosphate, organic acid salts of iron such as iron citrate, iron gluconate, and iron lactate, hem iron, and iron. Examples thereof include iron-protein conjugates such as ferritin, iron oxide and the like. The iron compound is preferably one that can be used in pharmaceuticals or foods, for example, iron citrate, sodium ferrous citrate, ferrous gluconate, iron lactate, ferrous fumarate, ferrous pyrophosphate, Examples thereof include ferric pyrophosphate and ferrous sulfate. As the iron compound, a commercially available product may be used, and one type or two or more types can be appropriately selected and used.

The content of the amino acid for promoting hematopoietic stem cell differentiation in the above preparation is not particularly limited, but is 0.1% by weight or more, preferably 1.0% by weight or more, and preferably 30% by weight or less, preferably 30% by weight or less, based on the total amount of the preparation. It is 10% by weight or less. Further, the method of adding the active ingredient in the formulation may be added in advance or may be added during the production, and may be appropriately selected in consideration of workability.

In the hematopoietic stem cell differentiation promoting agent of the present invention, since the hematopoietic stem cell differentiation promoting amino acid, which is an active ingredient, has a hematopoietic stem cell differentiation promoting action, a disease or pathological condition of blood and hematopoietic organs caused by functional deterioration or deficiency of hematopoietic stem cells. It is effective as a medicine for treating, improving, and preventing. Ineffective hematopoietic stem cell dysfunction or insufficiency is caused by, for example, insufficient differentiation of hematopoietic stem cells, which produces immature blood cells, but breaks down in the bone marrow without being sufficiently matured due to qualitative abnormalities. , And "dysplasia" in which the shape of the produced blood cell cells becomes abnormal. Therefore, diseases or pathological conditions of blood and hematopoietic organs caused by hypofunction or dysfunction of hematopoietic stem cells include, for example, aplastic anemia (panhemocytopenia), myelodystrophy syndrome (MDS), salacemia, and iron blast. Anemia, iron deficiency anemia, giant erythroblastic anemia, hemolytic anemia, erythroblastemia, congenital anemia (eg, sickle aplastic anemia), paroxysmal nocturnal pigmenturia, secondary anemia (infection, infection, Malignant tumor, chronic disease, renal disease, liver disease, anemia associated with endocrine disease, etc.), malignant lymphoma, multiple myeloma, myeloid proliferative disease (true polyemia, essential plateletemia, myeloid fibrosis, etc.) , Idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), thrombocytopenia, autoimmune hemolytic anemia, as well as general anemia (movement, shortness of breath, dizziness, dizziness) , Anemia, easy fatigue, malaise, loss of appetite, nausea, headache, pale face, skin stains and bears, ringing in the ears, stiff shoulders, keratitis, etc.). In addition, "hematopoietic stem cell dysfunction or deficiency" includes not only those caused by the above-mentioned diseases but also those caused by administration of anticancer agents and immunosuppressive agents, and radiotherapy for cancer.

The pharmaceutical product of the present invention functions as a preventive agent for suppressing the onset of the above-mentioned disease or pathological condition and / or as a therapeutic agent for improving the normal state.

Since the active ingredient of the pharmaceutical product of the present invention is extremely safe and has no side effects, when used as a therapeutic, ameliorating, or preventive drug for the above-mentioned diseases, mammals such as humans, mice, rats, rabbits, dogs, and cats can be fed. It can be administered orally or parenterally in a wide range of doses to animals.

The dose of the drug of the present invention can be appropriately determined according to the type of disease, the age, sex, body weight, degree of symptom, etc. of the administration target. For example, when orally administered to an adult (body weight 60 kg), the total amount of amino acids for promoting hematopoietic stem cell differentiation is about 10 mg to 50 g, preferably about 100 mg to 10 g per day.

In addition, the hematopoietic stem cell differentiation-promoting agent of the present invention can also be added to foods and drinks. In the present invention, foods and drinks include general foods and drinks, as well as foods other than pharmaceuticals that can be ingested for the purpose of maintaining or improving health, such as health foods, functional foods, health functional foods, or special purpose foods. It is used in the meaning of including. Health foods include foods provided under the names of dietary supplements, dietary supplements, supplements and the like. Health functional foods are defined by the Food Sanitation Law or the Food Promotion Law, and are based on specified health foods and nutritionally functional foods that can display specific health effects, functions of nutritional components, reduction of disease risk, etc., and scientific evidence. Includes foods with functional claims that can display the contents notified to the Commissioner of the Consumer Affairs Agency. In addition, special-purpose foods include foods for the sick, foods for the elderly, foods for babies, foods for pregnant women, etc. that indicate that they are suitable for a specific target person or a patient having a specific disease. The hematopoietic stem cell differentiation promoter of the present invention improves anemia and various symptoms associated with anemia, especially in elderly people, pregnant women, and diseases associated with menstruation and bleeding tendency (gastric ulcer, duodenal ulcer, gastrointestinal polyps and cancer, hemorrhoids, etc.). And when it is necessary to take it for a long period of time for prevention, it can be suitably used for the above-mentioned health foods and the like because it can be continuously taken on a daily basis. Here, the indications such as specific health effects and functions of nutritional components attached to foods and drinks are indications such as product containers, packaging, manuals, package inserts, product leaflets and pamphlets, newspapers and magazines, etc. It can be used as an advertisement for a product of.

Furthermore, when the food and drink of the present invention is used for mammals other than humans, it can be used in the sense of including pet food and feed.

The form of the food or drink may be any of edible forms such as solid, liquid, granular, granular, powdery, capsule-like, cream-like, and paste-like. In particular, in the case of the above-mentioned health foods, for example, tablets, rounds, capsules, powders, granules, fine granules, troches, and liquids (including syrup, milky, and suspension). Etc. are preferable.

The types of foods and drinks include breads, noodles, confectionery, dairy products, processed marine and livestock foods, fats and oils, processed fats and oils, seasonings, and various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks). , Milk beverages, etc.), concentrated stock solutions of the beverages, preparation powders, etc., but are not limited thereto.

The food and drink of the present invention may appropriately contain additives that are usually used depending on the type of food and drink. As the additive, any additive that is acceptable under the Food Sanitation Law can be used, and for example, sweeteners such as starch, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; citric acid, tartaric acid, etc. , Acidulants such as tartaric acid; Excipients such as dextrin and starch; Binding agents, diluents, fragrances, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspensions Examples include turbidants and preservatives.

When the food or drink of the present invention is a general food or drink, it can be produced by including a step of adding an amino acid for promoting hematopoietic stem cell differentiation in the normal manufacturing process of the food or drink. In the case of health foods, the above-mentioned method for producing pharmaceuticals may be followed. For example, in the case of tablet-shaped supplements, additives such as excipients are added to and mixed with amino acids for promoting hematopoietic stem cell differentiation, and tablets are tableted. It can be manufactured by applying pressure with a machine or the like. For capsule-shaped supplements, a liquid, suspension, paste, powder, or granular food composition containing an amino acid for promoting hematopoietic stem cell differentiation is filled in a capsule or encapsulated with a capsule base. Can be manufactured. Further, other materials as needed (e.g., iron, minerals such as potassium, vitamin C, vitamin B _2, vitamin B _6, vitamins such as vitamin B _12, folic acid, dietary fiber, etc.) may be added to it can.

The amount of the amino acid for promoting hematopoietic stem cell differentiation in the food or drink of the present invention may be an amount capable of exerting the differentiation promoting action of hematopoietic stem cells, but the general intake amount of the target food or drink, the form of the food or drink, the efficacy and efficacy. It may be appropriately set in consideration of the effect, taste, taste, cost and the like.

When the food or drink of the present invention is ingested for the purpose of preventing or ameliorating the above-mentioned disease or pathological condition, it varies depending on the condition of the subject to be ingested, the form of ingestion, the amount of food intake, etc. The total amount of amino acids for promoting hematopoietic stem cell differentiation per day is about 10 mg to 50 g, preferably about 100 mg to 10 g. The above amount may be ingested once, or may be ingested in several times (2 to 4 times). In the food and drink of the present invention, it is preferable that the amount of food and drink to be ingested at one time is packaged or filled in a container such as a bag or a bottle in order to use it as a guide for the amount of intake.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited thereto.

[Example 1] Measurement of hematopoietic stem cell differentiation promoting effect Differentiation of glutamic acid, leucine, valine, threonine, and glycine into hematopoietic stem cells using A-6 cells (mouse hematopoietic stem cells) (Physical and Chemical Research Institute) cultured in an undifferentiated state. An evaluation test of the promoting effect was conducted. The cell culture medium originally contains a certain amount of various amino acids for the growth and survival of cells. For example, DMEM / F12 (manufactured by Gibco) used as the basal medium for culturing A-6 cells in this test includes glutamic acid (0.05 mM), leucine (0.45 mM), valine (0.45 mM), and threonine (0.45 mM). ), Glycine (0.25 mM) is included. Therefore, in this test, as shown in Table 1, a medium (conditions 2 to 16) in which the concentration of one of the above five amino acids in the basal medium (condition 1) was changed, and the above five types. A medium (condition 17) in which all the amino acid concentrations of the above were increased at the same time was prepared, and the effect on the differentiation of hematopoietic stem cells was analyzed. In addition, the medium of each condition includes bovine fetal serum (20%; manufactured by Sigma), G-CSF (10 ng / mL; manufactured by Pepro Tech), IL-3 (10 ng / mL; manufactured by Pepro Tech), IL. -6 (100ng / mL; manufactured by Pepro Tech), SCF (100ng / mL; manufactured by Pepro Tech), EPO (25U / mL; manufactured by Pepro Tech), Insulin (10ng / mL; manufactured by SIGMA), Transferrin ( 10 ng / mL; manufactured by SIGMA) and 2-mercaptoethanol (100 μM; manufactured by Gibco) were added.

The A-6 cells were seeded by 1x10 ⁶ cells in 12-well plate with the addition of medium in each condition were cultured for 4 days. After 4 days of culturing, the cells were collected, washed twice with PBS (-), and RNA was extracted from the cells by Trizol Reagent (manufactured by Invitrogen). After reverse transcribing the extracted RNA to cDNA using the 2-STEP real-time PCR kit (manufactured by Applied Biosystems), real-time PCR (95 ° C: 15 seconds, 60 ° C: 30 seconds) by ABI7300 (manufactured by Applied Biosystems) , 40 cycles) of Ptprc (B cell marker: The decline in B lymphopoiesis in aged mice reflects loss of very early B-lineage precursors. J Immunol. 2003 Sep 1; 171 (5): 2326-30.) Gene expression was confirmed. Other operations were performed according to the prescribed method.

The primer set used for amplification of each gene is shown below.

[Primer set for Ptprc (B cell marker) gene]
Forward primer: 5'-ACCTGCTCGCACCACTGAA-3'(SEQ ID NO: 1)
Reverse primer: 5'-CCTGGATGATATGTGGTCTCTGAAG-3' (SEQ ID NO: 2)

[Primer set for internal standard glyceraldehyde 3-phosphate dehydrogenase (Gapdh) gene]
Forward primer: 5'-CCGTGTTCCTACCCCCAAT-3'(SEQ ID NO: 3)
Reverse primer: 5'-TGCCTGCTTCACCACCTTCT-3'(SEQ ID NO: 4)

The cell differentiation promoting effect is the gene-relative effect of the differentiation marker calculated as the ratio of the expression level of Ptprc (differentiation marker) in A-6 cells 4 days after culturing in the basal medium (condition 1) to the expression level of Gapdh mRNA, which is an internal standard. The value of the expression level (Ptprc gene expression level / Gapdh gene expression level) was set to 100%, whereas each differentiation in A-6 cells 4 days after culturing in a medium (conditions 2 to 17) in which the amino acid addition amount was changed. The value of the relative gene expression of the marker was calculated and evaluated. The results of these tests are shown in Table 1.

As shown in Table 1, the concentration of glutamic acid (condition 2-4), the concentration of leucine (condition 5-7), the concentration of valine (condition 8-10), and the concentration of threonine (condition 11-13) in the basal medium. , It was found that increasing the concentration of glycine (conditions 14-16) promotes the differentiation of hematopoietic stem cells. Furthermore, when the concentrations of all five amino acids were increased at the same time (Condition 17), the differentiation promoting effect became even more remarkable. From the above, it was confirmed that glutamic acid, leucine, valine, threonine, and glycine have a remarkable effect of promoting the differentiation of hematopoietic stem cells. In addition to the cells used in this experimental example, a similar test was performed on commercially available human hematopoietic stem cells, and a similarly remarkable effect of promoting differentiation of hematopoietic stem cells was observed.

The hematopoietic stem cell differentiation-promoting agent of the present invention can efficiently induce the differentiation of hematopoietic stem cells into blood cells. Therefore, the hematopoietic stem cell differentiation-promoting agent of the present invention can be used in the manufacturing field of pharmaceutical products for treating, improving, and preventing diseases or pathological conditions of blood and hematopoietic organs caused by functional deterioration or deficiency of hematopoietic stem cells.

Claims (3)

A hematopoietic stem cell differentiation promoter containing an amino acid mixture consisting of glutamic acid, leucine, valine, threonine and glycine (excluding methionine) as an active ingredient. A method for promoting the differentiation of hematopoietic stem cells, which comprises culturing the hematopoietic stem cells in the presence of the hematopoietic stem cell differentiation promoting agent according to claim 1. A method for producing blood cells, which comprises a step of culturing hematopoietic stem cells in the presence of the hematopoietic stem cell differentiation promoter according to claim 1.