JPWO2005093083A1 - Disease prediction method and use method thereof - Google Patents

Abstract

By differentiation-inducing cells including tissue stem cells derived from adipose tissue into specific cells, and measuring the characteristics of the specific cells, it is possible to apply to pharmaceutical screening methods, food screening methods, and the like.

Description

  The present invention relates to a disease prediction method using cultured cells in fields such as biology and medicine, and a method for measuring characteristics of cultured cells.

  Hyperlipidemia, diabetes, and hypertension, which are called lifestyle-related diseases in Japan, are so common that they may accidentally be merged into the same individual, but rather than each person developing separately, It has been revealed that this is a group of diseases that often develop with obesity. Therefore, WHO has proposed such a series of syndromes as “metabolic syndrome (metabolic syndrome)”, and has come to be diagnosed and treated worldwide (Kazuhisa Maeda: Endocrinology / Diabetic Medicine Mar. 2004 (Vol18 No. 3)). p234-240).

  In recent years, obesity has increased due to changes in environmental factors such as overnutrition and lack of exercise. The increase in obese males is particularly remarkable in Japan as well as in Western countries. The first 10 since the birth of Heisei in the 60s, obese men (BMI> 25), considered the most prone to metabolic syndrome and also a target for the complications of atherosclerotic disease. In just a year, it has almost doubled.

  Globally, the frequency of diabetes continues to increase with increasing obesity, and as in other Asian countries, it can be recognized as an important risk factor in Japan. It is rather expensive than European countries. However, it should be noted that the frequency of obesity exceeding the so-called BMI 30 (11%) is very low compared to other countries. It can be said that it is a large folklore feature of the metabolic syndrome that the Japanese have that it is easy to have diabetes even if there is no excessive weight gain. In general, the frequency (32%) of obese people with a BMI of 25 or more is equivalent to the BMI of 30 or more in other countries (Kazuhisa Maeda, Nakamura: Japan Medical Association Journal, Vol. 130, No. 1 (July 1, 2003) p31 -P36).

  Therefore, it can be said that the prediction of metabolic syndrome associated with obesity such as diabetes is extremely difficult for Japanese compared to Western countries with many obese people. In other words, the significance of so-called tailor-made treatment or tailor-made treatment that can predict an individual's disease or the effect of a therapeutic drug in advance is considered to be very large particularly in Japan.

  Under such circumstances, one of the most promising aspects of post-genomic research is the elucidation of genetic factors of multifactorial genetic diseases, mainly lifestyle-related diseases. The body is said to be an individual difference in the genome called a single nucleotide polymorphism (SNP) rather than a genetic abnormality. As a research method, the classical candidate gene approach has been the main method so far, but recently, a broader genre of candidate genes has been tried, such as the sibling pairing method and candidate region approach, and the whole genome correlation analysis. Such an analysis method is taken (Kazuhisa Maeda, Toru Funabashi, Koji Matsuzawa: Cell Vol. 35 No. 7, 2003 p258-261).

  There have been a lot of snippets that have been reported to have some correlation with type 2 diabetes-related pathologies, but the results have not always reproduced the results, and the effects are often weak. It is far from explaining the whole picture of heredity. Among them, snipps that are particularly noted are β3 adrenergic receptor (β3AR), PPARγ, and adiponectin.

  β3AR promotes heat production by uncommon protein (UCP1) in brown adipose tissue, and promotes lipolysis in white adipose tissue. Information that can be reduced to the clinical setting includes β3AR polymorphism, β3AR agonist as an anti-obesity drug, which is easily fattened (disease susceptibility) at the same level of diet and exercise as individuals with low basal metabolism and no polymorphism It is possible that the expected effects of the drug are different (drug reactivity).

  PPARγ, which is a master gene for the differentiation and proliferation of adipose tissue, is also a target molecule for an insulin sensitizer. With regard to the activity-reduced Pro12Ala polymorphism of γ2 isoform (specific for adipose tissue), results of being less obese and less likely to become diabetic have been reported including meta-analysis. Decreased activity type Pro12Ala is thought to be less likely to cause obesity and insulin resistance, especially under high fat diets. In fact, even Japanese populations who have immigrated to the United States have a more pronounced effect of making Ala type diabetes less likely It has been.

  Adiponectin secreted from adipose tissue discovered by the inventor in the human genome project (Maeda K, et al .: Biochem Biophys Res Commun 221 (2): 286-9, 1996) has an anti-arteriosclerotic action and a glucose tolerance improving action. Recently, there was a correlation between intron snippets, blood adiponectin levels, and abnormal glucose metabolism. In the future when adiponectin and related drugs will be clinically applied, it is expected that this sniptyping will be used for patient selection and evaluation of prognosis (Kazuhisa Maeda: Toru Funabashi: Endocrine, Metabolism p15- 19).

  In addition to the analysis of multifactorial genetic diseases that have been promoted centering on the conventional candidate gene approach, an international collaborative database creation of haplotypes, which are a combination of multiple snippets, and a candidate area approach are being carried out. In Japan, an important national strategy has started a large-scale snippet-related project in collaboration with ministries and agencies, which is collectively called the Millennium Genome Project. In particular, in the multi-center collaborative research started in 2002, whole genome correlation analysis has been started, and certain results have been achieved in myocardial infarction and rheumatoid arthritis.

  However, the results currently being obtained even in such a national approach are only that the disease-related genes of myocardial infarction and rheumatoid arthritis were identified and reported (Kazumoto Yasuda: Annual Review 2004 Endocrine, Metabolism p49-58). ). In addition, there is no system that actually screens and administers drugs that should be tailor-made to the most deadly, that is, to determine the effect before administration, and innovative technology is strongly desired. It was.

  The present invention has been made with the intention of solving the problems of the prior art as described above. That is, an object of the present invention is to provide a novel disease prediction method based on a completely different idea from the prior art. Moreover, an object of this invention is to provide the utilization method. Furthermore, an object of the present invention is to provide a novel method for measuring characteristics of cultured cells.

  In order to solve the above-mentioned problems, the present inventors have studied and developed from various angles. As a result, it has been surprisingly found that a disease can be predicted by inducing differentiation of a cell containing a tissue stem cell isolated from adipose tissue into a specific cell and measuring the characteristics of the specific cell. The present invention has been completed based on such findings.

  That is, the present invention provides a disease predicting method characterized by differentiation-inducing cells including tissue stem cells isolated from adipose tissue into specific cells and measuring the characteristics of the specific cells.

  The present invention also provides a disease prediction system using the disease prediction method.

  In addition, the present invention provides a novel method for measuring the characteristics of cultured cells.

  Furthermore, the present invention provides a pharmaceutical screening method, a food screening method, and a therapeutic method using the disease prediction system.

  The method of inducing differentiation of cells containing tissue stem cells isolated from adipose tissue described in the present invention into specific cells and measuring the characteristics of the specific cells can predict diseases efficiently. .

FIG. 1 shows that tissue stem cells collected from human adipose tissue in Example 2 can differentiate into mature adipocytes. FIG. 2 is a diagram showing the results of comparing the lipid droplet storage capacity of mouse A and mouse B in Example 3. FIG. 3 is a diagram showing the results of comparing the sugar uptake capacities of mouse A and mouse B in Example 3. FIG. 4 is a diagram showing the results of comparing the free fatty acid resolution of mouse A and mouse B in Example 3. FIG. 5 is a diagram showing the results of comparing the anti-atherosclerotic hormone secretion ability of mouse A and mouse B in Example 3. FIG. 6 is a graph showing the results of comparison of body weights before and after loading of mouse A and mouse B in Example 4. FIG. 7 is a graph showing the results of comparing the glucose metabolism capacity before and after loading of mouse A and mouse B in Example 4. FIG. 8 is a graph showing the results of comparison of lipid metabolism before and after loading of mouse A and mouse B in Example 4. FIG. 9 is a diagram showing the results of comparing the degree of progression of arteriosclerosis after loading of mouse A and mouse B in Example 4. FIG. 10 is a diagram showing the results of comparing the sugar uptake capacities when mice were administered drugs with and without insulin load in Example 5 in Example 5. FIG. 11 shows the results of comparison of glucose metabolism under drug administration in mouse A and mouse B in Example 5. FIG. 12 is a diagram showing how cells containing tissue stem cells differentiate into cells other than fat in Example 7.

The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
(Definition of terms)
The present invention will be described below by enumerating definitions of terms particularly used in the present specification.

  As used herein, “adipocyte” is a general term for fat storage tissues. Among the loose connective tissues, there are particularly fatty ones and subcutaneous fat tissues. Each fat cell is surrounded by lattice fibers, and capillaries are densely distributed between the cells to form a fat body. As used herein, any adipose tissue can be a source. A fat body is an almost constant mass or tufted adipose tissue independent of other tissues, and in vertebrates, it exists in the abdominal cavity in contact with the kidneys and gonads. Often white, yellow or orange.

  Each organ can differentiate from organ-derived progenitor cells or somatic stem cells, but cells containing tissue stem cells are one of the most readily available cells in the body. In addition, it can contribute to the prediction of various cell functions and the development of tailor-made therapeutics, in addition to the ability to differentiate into multiple strains.

  As used herein, a “cell” is defined in the same way as the broadest meaning used in the art, and is a structural unit of a tissue of a multicellular organism that is enclosed in a membrane structure that isolates the outside world, Refers to a living organism having self-regenerative ability and having genetic information and its expression mechanism. Any cell can be targeted in the method of the present invention. The number of “cells” used in the present invention can be counted through an optical microscope. When counting through an optical microscope, counting is performed by counting the number of nuclei. The tissue is used as a tissue slice, and hematoxylin-eosin (HE) staining is performed, whereby the extracellular matrix (for example, elastin or collagen) and the nucleus derived from the cells are dyed with a dye. This tissue piece can be examined with an optical microscope, and the number of nuclei per specific area (for example, 200 μm × 200 μm) can be estimated and counted. The cells used herein may be naturally occurring cells or artificially modified cells (eg, fusion cells, genetically modified cells). The source of cells can be, for example, a single cell culture, or such as cells from a normally grown transgenic animal embryo, blood, or body tissue, or a normally grown cell line Examples include but are not limited to cell mixtures. Moreover, such a supply source can also be used as a cell as it is.

  The cells used in the present invention may be cells derived from any organism (for example, eelfishes, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.) as long as there are adipocytes or their counterparts. Can be used. Preferably, such cells are mammalian (eg, monoporous, marsupial, rodent, winged, winged, carnivorous, carnivorous, long-nosed, odd-hoofed, cloven-hoofed , Rodents, scales, sea cattle, cetaceans, primates, rodents, rabbits, etc.). In one embodiment, cells derived from primates (eg, chimpanzees, Japanese monkeys, humans), particularly cells derived from humans, are used, but are not limited thereto.

  As used herein, “stem cell” refers to a cell having a self-renewal ability and having pluripotency (ie, “pluripotency”). Stem cells are usually able to regenerate the tissue when the tissue is damaged. As used herein, stem cells can be embryonic stem (ES) cells or tissue stem cells (also referred to as tissue stem cells, tissue-specific stem cells or somatic stem cells), but are not limited thereto. In addition, as long as it has the above-mentioned ability, an artificially produced cell (for example, a fusion cell described herein, a reprogrammed cell, etc.) can also be a stem cell. Embryonic stem cells refer to pluripotent stem cells derived from early embryos. Embryonic stem cells were established for the first time in 1981 and have been applied since 1989 to the production of knockout mice. In 1998, human embryonic stem cells were established and are being used in regenerative medicine. Unlike embryonic stem cells, tissue stem cells are cells in which the direction of differentiation is limited, are present at specific positions in the tissue, and have an undifferentiated intracellular structure. Therefore, tissue stem cells have a low level of pluripotency. Tissue stem cells have a high nucleus / cytoplasm ratio and poor intracellular organelles. Tissue stem cells generally have pluripotency, have a slow cell cycle, and maintain proliferative ability over the life of the individual. As used herein, stem cells can be tissue stem cells, preferably mesenchymal stem cells, although embryonic stem cells can also be used depending on the situation.

  In the present specification, the term “stem cell” refers to a tissue aggregate containing at least a certain amount of stem cells. Therefore, in the present invention, for example, stem cells collected from adipocytes after collagenase treatment (such as stem cells used in the Examples) can be used as the stem cells, but are not limited thereto.

  When classified according to the site of origin, tissue stem cells are classified into, for example, the skin system, digestive system, myeloid system, and nervous system. Examples of skin tissue stem cells include epidermal stem cells and hair follicle stem cells. Examples of digestive tissue stem cells include pancreatic (common) stem cells and liver stem cells. Examples of myeloid tissue stem cells include hematopoietic stem cells and mesenchymal stem cells. Examples of nervous system tissue stem cells include neural stem cells and retinal stem cells.

  As used herein, “adipose stem cell” refers to a stem cell derived from an adipose tissue. Some methods for separating such stem cells are known, for example, WO 00/53795; WO 03/022988; WO 01/62901; Zuk, P. et al. A. , Et al. Tissue Engineering, Vol. 7, 211-228, 2001; Zuk, P .; A. , Et al. , Molecular Biology of the Cell Vol. , 13, 4279-4295, 2002, etc. or a modification thereof. Specifically, for example, (1) The lipoaspirate is sufficiently washed with physiological saline using a separatory funnel; (2) It is confirmed that the lipoaspirate is sufficiently separated in the upper layer and the physiological saline is sufficiently separated in the lower layer. And discard the lower layer. Repeat this until the physiological saline is almost transparent to the naked eye; (3) Add 0.075% collagenase / PBS in the same amount as the lipoaspirate and incubate for 30 minutes at 37 ° C. with good agitation; 4) Add an equal volume of 10% serum-added DMEM to the above sample; (5) Centrifuge the above sample at 1200 g for 10 minutes; (6) Add PBS to the pellet and suspend it at room temperature as appropriate (eg, 10-7 minutes); (7) The above sample can be filtered by suction using a 100 μm caliber mesh; and (8) The filtrate can be centrifugated at 1200 g for 5 minutes. Here, it is within the technical scope of those skilled in the art to scale up or scale down the protocol according to the adjustment amount.

  As cell markers for these cells, for example, selected from the group consisting of CD4, CD13, CD34, CD36, CD49d, CD71, CD90, CD105, CD117, CD151; or CD105, CD73, CD29, CD44 and Sca-1. Cell surface markers include but are not limited to them. Alternatively, the surface antigens of mesenchymal stem cells are considered to be CD105 (+), CD73 (+), CD29 (+), CD44 (+), CD14 (−), CD34 (−), CD45 (−). It is understood that cells that exhibit at least any one of these, preferably two or more, more preferably all of these properties are preferred as the cells used in the present invention.

  As used herein, “fibroblast” refers to a cell that supplies the fiber component of the supporting tissue and forms an important component of the fibrous connective tissue. Tissue sections have a flat, long outline and often show irregular protrusions. The cytoplasm contains mitochondria, Golgi apparatus, centrosome, small fat globules, etc., but does not show special differentiation. The nucleus is elliptical and often exists in close proximity to the collagen fibers. It is said that fibroblasts isolated from adipose tissue often contain stem cells.

  As used herein, “differentiated cells” refers to cells with specialized functions and morphology (eg, muscle cells, nerve cells, etc.), and unlike stem cells, there is no or almost no pluripotency. . Examples of differentiated cells include epidermal cells, pancreatic parenchymal cells, pancreatic duct cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, osteoblasts, skeletal myoblasts, neurons, vascular endothelial cells, pigment cells, Examples include smooth muscle cells, fat cells, bone cells, chondrocytes. As used in the present invention, the differentiated cells may be in the form of a population or tissue.

  Any culture solution can be used for the cells of the present invention as long as the cells are maintained or differentiated into desired differentiated cells. Examples of such a culture solution include, but are not limited to, DMEM, P199, MEM, HBSS, Ham's F12, BME, RPMI 1640, MCDB104, MCDB153 (KGM), and mixtures thereof. Such cultures include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate-2-phosphate, ascorbic acid and its derivatives, glycerophosphate, estrogen and its derivatives, Progesterone and its derivatives, Androgen and its derivatives, Growth factors such as aFGF, bFGF, EGF, IGF, TGFβ, ECGF, BMP, PDGF, pituitary extract, pineal gland extract, retinoic acid, vitamin D, thyroid hormone, fetal bovine Demethylation of serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate (sodium selenite, etc.), linolenic acid, 3-isobutyl-1-methylxanthine, 5-azanthidine Agent, Histone deacetylating agents such as lycostatin, cytokines such as activin, LIF and IL-2 / IL-6, hexamethylenebisacetamide (HMBA), dimethylacetamide (DMA), dibutyl cAMP (dbcAMP), dimethyl sulfoxide (DMSO), Including one or a combination of iododeoxyuridine (IdU), hydroxyurea (HU), cytosine arabinoside (AraC), mitomycin C (MMC), sodium butyrate (NaBu), polybrene, selenium, cholera toxin, etc. It may be left.

  As used herein, “in vivo” or “in vivo” refers to the inside of a living body. In a particular context, “in vivo” refers to the location where the target tissue or organ is to be placed.

  As used herein, “in vitro” refers to a state in which a part of a living body is removed or released “in vitro” (eg, in a test tube) for various research purposes. A term that contrasts with in vivo.

  In this specification, “ex vivo” refers to a series of operations when a target cell for gene transfer is extracted from a subject, a therapeutic gene is introduced in vitro, and then returned to the same subject again. It is called ex vivo.

In one embodiment, the conditions for culturing in the regenerative treatment method of the present invention, and the conditions for providing a culture of a desired organ, tissue or cell part used in the present invention or a stem cell capable of differentiating into the cells are the cells. Any of those usually used for culturing tissues or organs can be used. As an example of such a culture condition, for example, 37 ° C. and 5% CO ₂ can usually be used as the culture condition. Any medium can be used as the medium to be used. For example, a medium containing DMEM / Ham12 (1: 1), 10% FCS, insulin / colotoxin and the like can be used. Further, if necessary, a culture solution containing differentiation factors (for example, EGF (10 ng / ml) in advance) may be used, but is not limited thereto.

In one preferred embodiment, differentiation factors can be added for culturing in the methods of the invention. Such differentiation factors include, for example, DNA demethylating agents (such as 5-azacytidine), histone deacetylating agents (such as trichostatin), nuclear receptor ligands (such as retinoic acid (ATRA), vitamin D ₃ , T3 etc.), cell growth factor (activin, IGF-1, FGF, PDGF, TGF-β, BMP2 / 4 etc.), cytokine (LIF, IL-2, IL-6 etc.) hexamethylenebisacetamide, dimethylacetamide, dibutyl Examples include, but are not limited to, cAMP, dimethylthiol sulfoxide, iododeoxyuridine, hydroxylurea, cytosine arabonoside, mitomycin C, sodium butyrate, aphidicolin, fluorodeoxyuridine, polybrene, sesin and the like. For differentiation into the cornea, a differentiation factor called EGF can be added. Any other differentiation factor described herein can also be used for other differentiated cells.

  The present invention is a part of a multifaceted approach to analyze organically related issues in order to overcome the problems of the prior art, especially for lifestyle related diseases such as obesity, diabetes, hyperlipidemia, arteriosclerosis, etc. As an intensive tailor-made treatment, we propose a function evaluation method for lifestyle-related diseases using adipose tissue-derived stem cells.

  Although we have succeeded in predicting disease onset and tailor-made treatment using mice by this method, first of all, the β3 adrenergic receptor (β3AR), PPARγ, adiponectin, which have been reported as obesity and diabetes-related genes. Note that all of these are proteins expressed in the adipose tissue itself. In other words, it is possible to indirectly prove the validity of the present invention in which such a group of gene proteins can be observed comprehensively.

  The present inventor has found that cells containing tissue stem cells in adipose tissue can be predicted to become obese in the future or to be associated with lifestyle-related diseases without being influenced by environmental factors such as snippets. Mature adipocytes occupying 99.9% or more of adipose tissue are hypertrophied cells in obese humans, and have the characteristics of thin cells in lean humans, and are already affected by the environment. On the other hand, tissue progenitor cells, which occupy very few tissues, have recently been proven to differentiate into tissues other than fat, and maintain the genetic background inherent in the individual who is not affected by the environment at all. It can be said that it is an intact cell.

  The present inventor succeeded in easily measuring metabolic functions after separating and culturing and differentiation of the cells. This metabolic function is a metabolic function inherent in an adipose tissue inherent in an individual. Therefore, if this system is used, 1) it is possible to quantify that an individual is likely to become obese in the future or a lifestyle-related disease such as diabetes. 2) The effects of drugs under development or already developed drugs. The effect can be determined without administration to a living body. In other words, tailor-made treatment aimed at snipes becomes possible. The present inventor was able to prove this experimentally using adipose tissue-derived stem cells of a pediatric mouse that has already been found to cause diabetes when it becomes an adult mouse.

  In addition, by using the present invention to create a database of multiple samples, the effects of anti-obesity drugs, health foods, etc. can be examined in vitro using human adipose tissue, so that it can be easily and accurately determined from many candidates. Become.

  Examples of the adipose tissue used in the present invention include, but are not particularly limited to, subcutaneous fat and abdominal visceral fat. The animal species is not particularly limited, and examples thereof include humans, monkeys, dogs, pigs, guinea pigs, mice and rats. According to the present invention, when the adipose tissue of these animals is used, it is possible to know a disease that is likely to occur when the animal matures. Here, although it differs with animal species, it is good within 12 months after birth, Preferably within 3 months, More preferably within 1 month. In the case of humans, those within 20 years after birth, preferably within 10 years, more preferably within 5 years are good. For the purpose of human treatment, it is most effective to use the adipose tissue of young human beings. According to the present invention, if the adipose tissue for a young human is used, a disease that is likely to occur when the person becomes an adult can be understood, treatment is started before the onset of disease, and the onset is suppressed in advance. It becomes possible. Furthermore, even if the disease occurs, the patient can be optimally treated.

  In the present invention, it is necessary to isolate cells containing tissue stem cells from adipose tissue. The method is not particularly problematic as long as it follows a conventional method, and examples thereof include a method of centrifuging using an enzymatic method and a method of separating using a kit. In terms of operation, the latter method is an efficient and simple method.

  In the present invention, it is necessary to induce differentiation of cells including tissue stem cells isolated from adipose tissue into specific cells, and to measure the characteristics of the specific cells. The specific cell is not particularly limited, and examples thereof include mature adipocytes, bone-related cells, nerve-related cells, muscle-related cells, and vascular endothelial cells. When mature adipocytes are selected as cells here, the characteristics include one or more of lipid droplet storage volume, lipid droplet resolution, sugar metabolism capacity, specific protein secretion amount, etc. There are no particular restrictions. In this case, the specific protein includes one consisting of leptin, adiponectin, TNF-α, VEGF, HGF, or two or more, but is not particularly limited. When predicting a disease, specific cells of an individual to be predicted based on the characteristics of the specific cells obtained from an individual who has not suffered from lifestyle-related diseases, obesity, hyperlipidemia, diabetes, and arteriosclerosis These characteristics should be compared. Here, with respect to the amount of lipid droplets stored and the amount of TNF-α secretion, the characteristics of the specific cells obtained from individuals who have not suffered from lifestyle-related diseases, obesity, hyperlipidemia, diabetes, and arteriosclerosis are used as standards. As an increase of at least 10% or more, preferably 20% or more, more preferably 30% or more, the individual to be predicted suffers from diseases such as lifestyle-related diseases, obesity, hyperlipidemia, diabetes, and arteriosclerosis. It becomes clear that it is easy. Individuals who are not suffering from lifestyle-related diseases, obesity, hyperlipidemia, diabetes, or arteriosclerosis with respect to lipid droplet resolution, sugar metabolism capacity, leptin secretion, adiponectin secretion, VEGF secretion, HGF secretion The individual desired to be predicted to be reduced to at least 10% or less, preferably 20% or less, more preferably 30% or less on the basis of the characteristics of the specific cells obtained from the above are lifestyle-related diseases, obesity, hyperlipidemia It becomes clear that the disease is susceptible to diseases such as diabetes, diabetes, and arteriosclerosis. Furthermore, if the cells containing tissue stem cells isolated from young adipose tissue before being affected by the environment are induced to differentiate into mature adipocytes, the future metabolic capacity of the individual from whom the adipose tissue was collected, That is, it becomes possible to predict the likelihood of becoming one or more of lifestyle diseases, obesity, hyperlipidemia, diabetes, and arteriosclerosis. Vascular endothelial cells can measure cytokines such as VEGF and HGF with a kit, and serve as an index of angiogenic ability, that is, arteriosclerosis. Bone cells can be predicted to be susceptible to osteoporosis by measuring bone metabolism. Nerve and muscle cells can also be used to predict organ function.

  When the specific cell is a bone-related cell, a nerve-related cell, or a muscle-related cell, the characteristics include one of bone metabolic function, nerve and muscle cell function, or two or more, There is no particular restriction. In particular, if the cells containing tissue stem cells isolated from adipose tissue before childhood affected by the environment are induced to differentiate into bone-related cells, the disease of bone metabolism of the individual from whom the adipose tissue was collected is predicted. become able to.

  According to the present invention, if a tissue of an animal individual can be used, it becomes possible to recognize a disease that is likely to occur when it becomes an adult. A disease prediction system by combining the mature animal individuals by measuring the characteristics of the specific cells by inducing differentiation of cells including tissue stem cells isolated from adipose tissue at a young age into specific cells. It becomes. That is, if this predictive system is used, drug screening can be performed by a method of selecting an animal that does not become diseased by continuing to administer some drug to an animal animal that is efficiently diseased. In the case of the present invention, the drug that can be developed is not particularly limited, and examples thereof include anti-obesity drugs, anti-diabetic drugs, and anti-arteriosclerotic drugs. In the same way, the functional food can be screened by the prediction system shown in the present invention.

  Furthermore, if the individual animal is a human, the adipose tissue of the child's childhood is collected in advance and differentiated into a specific cell. When the function of the specific cell is measured, the person becomes an adult. Thus, it is possible to understand a disease that is likely to occur, and it is possible to start treatment before the onset and to suppress the onset in advance. Furthermore, even if the disease occurs, the patient can be optimally treated. In the treatment, identification of the individual under treatment is based on the characteristics of the specific cells obtained from the individual who has already suffered from lifestyle-related diseases, obesity, hyperlipidemia, diabetes, and arteriosclerosis. It may be carried out while comparing and examining the characteristics of the cells. Here, with respect to the amount of accumulated lipid droplets and the amount of TNF-α secreted, at least 10% or less, preferably 20% or less, more preferably 30%, based on the characteristics of the specific cells obtained from the individual before treatment. If decreased below, it becomes clear that the individual has improved from diseases such as lifestyle-related diseases, obesity, hyperlipidemia, diabetes, and arteriosclerosis. Further, the resolution of lipid droplets, the ability to metabolize sugar, the amount of leptin secretion, the amount of adiponectin secretion, the amount of VEGF secretion, and the amount of HGF secretion are at least 10% or more based on the characteristics of the specific cells obtained from the individual before treatment. If it is increased to preferably 20% or more, more preferably 30% or more, it is clear that the individual is improved from diseases such as lifestyle-related diseases, obesity, hyperlipidemia, diabetes, and arteriosclerosis. It becomes.

It can be said that the present invention is one of the advantages that a supply source from adipose tissue can be provided. Extraction from adipose tissue has the advantage that it can be collected without stress, and it can be extracted at the outpatient level and can be repeated. It should be noted that there are advantages such as being able to obtain in large quantities.
[Example]
Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.

  The reagents used in the following examples were obtained from Wako Pure Chemicals and Sigma unless otherwise specified. Animal breeding, National Society for Medical Researchg created "Principles of Laboratory Animal Care" and the Institute of Laboratory Animal Resource is created, National Institute of Health has published "Guide for the Care and Use of Laboratory Animals" (HIN Publication, No. 86-23, 1985, revised), in accordance with the standards stipulated in Osaka University School of Medicine, and in accordance with the spirit of animal welfare. When human subjects were used, experiments were conducted after obtaining prior consent in accordance with the standards of the Ministry of Health, Labor and Welfare.

(Adjustment of cells including tissue stem cells)
In this example, cells were first prepared from adipose tissue from humans who gave consent to this experiment. The lipoaspirate was thoroughly washed with physiological saline. After confirming that the liposuction material was sufficiently separated in the upper layer and the physiological saline was sufficiently separated in the lower layer, the lower layer was discarded, and this was repeated until the physiological saline became almost transparent with the naked eye. In this experimental example, it was performed seven times. Adipose tissue was added with 40 ml of 0.075% collagenase / PBS (Gibco) in the same amount as 40 ml, and incubated at 37 ° C. with good agitation for 1 hour. To this sample, the same amount of 10% serum-added DMEM was added and centrifuged at 1200 × g for 10 minutes.

To the pellet obtained by centrifugation, 0.16M HN ₄ Cl / PBS (Gibco) was added and suspended, and incubated at 25 ° C. for 20 minutes. The sample was subjected to suction filtration using a mesh (Whatman) having a diameter of 100 μm. The filtrate was centrifuged at 1200 × g for 5 minutes. This cell was a fibroblast-like cell.

(Differentiation of cells including tissue stem cells)
Next, the cells in the cells prepared in Example 1 were primarily cultured, subcultured, and then differentiated into mature adipocytes. The number of subcultures is preferably up to about 4 generations. Specifically, cells containing tissue stem cells grown to confluence were treated with an adipocyte differentiation medium every two days. On the first and second days of differentiation, the cells were treated with 10% CCS-DMEM medium containing 0.5 mM IBMX, 1 μM dexamethasone, 1 μM triglitazone, 5 μg / ml insulin. On the 4th day, the cells were treated with 10% CCS-DMEM medium containing triglitazone and insulin, and after the 6th day, they were treated with 10% CCS-DMEM medium containing 500 ng / ml insulin. The obtained results are shown in FIG. It was found that tissue stem cells collected from adipose tissue were induced to differentiate into mature adipocytes by the above method.

(Functional evaluation using post-differentiation adipocytes derived from mice)
In this Example, 5 each of 6-week-old apo E knockout mice (hereinafter referred to as mouse A) and arteriosclerosis-onset double knockout mice (hereinafter referred to as mouse B) that have not yet been observed to have metabolic abnormalities, and adipose tissue according to the above method The derived stem cells were collected and differentiated into mature adipocytes, and the following measurements were performed.
(Fat droplet storage capacity)
Differentiated cells were stained with intracellular fat globules by the Oil Red O method. Furthermore, the amount of fat was quantified by extracting with an organic solvent and comparing with cells containing tissue stem cells. For Oil Red O, a Wpit Pure Chemicals Lipit Assay Kit was used. Alternatively, as a simpler method, differentiated adipocytes at the time of staining were photographed under a weak microscope and the stained area occupying the visual field was used as the differentiation ability. The obtained results are shown in FIG. FIG. 2 shows that mature adipocytes obtained from arteriosclerosis-type double knockout mice are more abundant than mature adipocytes obtained from 6-week-old apoE knockout mice that have not yet had metabolic abnormalities. It can be seen that the amount of drops stored is high.
(Sugar uptake ability)
^The sugar uptake ability was measured using ³ H-2-deoxyglucose as a substrate. Cytochalasin B (30 μM) was used as a negative control as an inhibitor of Glut4, a transporter of sugar uptake in adipocytes. Insulin 100 nM was defined as a positive control based on the sugar uptake when no insulin was added. The obtained results are shown in FIG. FIG. 3 shows that mature adipocytes obtained from arteriosclerosis-type double knockout mice are more sucrose than mature adipocytes obtained from 6-week-old apoE knockout mice that have not yet had metabolic abnormalities. It turns out that the amount of uptake is low.
(Fatty acid resolution)
Isoproterenol (1 μM), which is a β3 receptor agonist, was added to quantitate fatty acids released by neutral lipolysis. The obtained results are shown in FIG. FIG. 4 shows that mature adipocytes obtained from arteriosclerosis-type double knockout mice are more fatty acids than mature adipocytes obtained from 6-week-old apoE knockout mice that have not yet undergone metabolic abnormalities. It can be seen that the resolution is high.
(Anti-sclerosis hormone secretion)
Next, the anti-arteriosclerotic effect of the prepared cells was examined. About adiponectin, ELISA was performed using a cell secretion as a sample using an anti-adiponectin antibody. The ELISA was performed using an ELISA kit available from Otska. The obtained results are shown in FIG. FIG. 5 shows that adiponectin is obtained from mature adipocytes obtained from arteriosclerosis-type double knockout mice, compared to mature adipocytes obtained from 6-week-old apoE knockout mice that have not yet undergone metabolic abnormalities. It turns out that the amount of secretion is low.

  After applying a load to the mouse, changes in the living body were examined. A 6-week-old apo E knockout mouse (hereinafter referred to as mouse A) and an arteriosclerosis-onset double knockout mouse (hereinafter referred to as mouse B) used in the previous examples, each having 5 Western-type diet (21% crude) Various kinetics were measured after feeding (oriental yeast containing fat, 0.15% cholesterol). Body weight (FIG. 6), glucose metabolic capacity (FIG. 7), and lipid metabolism (FIG. 8) were fasted from the previous day and examined at the stage where the influence of meal time was removed. For metabolic capacity, fasting blood glucose level and fasting triglyceride level were measured using a kit.

  Atherosclerosis (FIG. 9) was dissected after anesthesia of the mouse, and three aorta were examined histopathologically. The aorta specimens were observed at three sites: proximal, middle, and distal. Staining was performed by HE dyeing and oil red O dyeing. A characteristic change was a localized plaque composed of cells with a large cytoplasm in the form of fine foam in HE staining. In oil red O staining, lipid droplets of various sizes were observed in the cytoplasm of the cells (lipoid plaques). These plaques swelled on the luminal side of the aorta, often showing mild connective tissue growth near the surface.

  When viewed by aortic site, the formation of lipoid plaques was found in all cases proximally, and the changes were clearly higher than in other sites. The proximal aortic wall with high lipoid plaques was accompanied by medial thickening with medial myocardial misalignment. These changes were apparent in the media located under the lipoid plaque, and fine lipid droplets were also observed in media media muscle cells with oil red O staining.

  Intensity of lesion: 0; No change 1; Very mild 2; Mild 3; Moderate 4; Severe (Lipoid plaque strength: Very mild, mild; less than 1/3 of the luminal wall, moderate Degree: 1/3 of the vessel wall facing the lumen but relatively thin (cells are about 3 layers), altitude: 1/3 or more of the vessel wall facing the lumen and thick spots) Turned into.

(Tailor-made treatment for mouse adipocytes before loading)
The sugar uptake ability in functional evaluation using the mouse-derived post-differentiation adipocytes used in Example 3 was measured. When stimulated with troglitazone, a PPARγ agonist, 24 hours ago, both cells had the same level of sugar uptake. That is, it was considered that troglitazone is a cell that can improve the sugar uptake ability in this mouse (FIG. 10).

(Studies on glucose metabolism after administration of food and drugs to mice)
The mouse load group used in Example 4 was fed with a diet so that troglitazone was 0.1%. As expected in Example 5, troglitazone improved the metabolic kinetics of mice. That is, in Examples 5 and 6, it was proved that the method for establishing tailor-made treatment according to the present invention was correct (FIG. 11).

(Differentiation of cells including tissue stem cells into cells other than fat)
The cell group used in Example 1 was confirmed to differentiate into cells other than fat cells. For differentiation into neurons, 1 mM beta-mercaptoethanol was added to DMEM medium supplemented with 20% serum and cultured for 24 hours. The next day, the cells were washed twice with PBS, and then 5 mM beta-mercaptoethanol was added to serum-free DMEM medium. (Woodbury et al., J Neurosci Res 61; 364-370, 2000). The obtained result is shown in FIG.

The method described in the present invention makes it possible to easily and accurately quantify the likelihood of an individual having a lifestyle-related disease and other diseases. By using this system, it is possible to examine the effects of a certain kind of medicine on each person, and tailor-made treatment becomes possible. Furthermore, for example, if this method is converted into a large-scale database using a kit that can easily collect fat, application to development screening of drugs and functional foods is strongly expected. Therefore, the present invention is extremely useful in the fields of medicine, biology and the like.

Claims (29)

  A method for predicting a disease, characterized in that a cell containing tissue stem cells isolated from adipose tissue is induced to differentiate into a specific cell, and the characteristics of the specific cell are measured.   The disease prediction method according to claim 1, wherein the adipose tissue is a childhood.   The disease prediction method according to any one of claims 1 and 2, wherein the specific cells include mature adipocytes.   The disease according to claim 3, wherein the mature adipocyte has one or more of lipid droplet retention, lipid droplet resolution, sugar metabolic capacity, and specific protein secretion. Prediction method.   The disease prediction method according to claim 4, wherein the specific protein comprises one or more of leptin, adiponectin, TNF-α, VEGF, and HGF.   4. The disease prediction method according to claim 3, wherein mature adipocytes are characterized by comprehensively analyzing changes in expression profiles of β3 adrenergic receptor, PPARγ, and adiponectin adipose tissue expression genes.   The disease prediction method according to any one of claims 1 to 6, wherein the disease to be predicted consists of one or more of lifestyle-related diseases, obesity, hyperlipidemia, diabetes, and arteriosclerosis. .   The disease prediction method according to any one of claims 1 and 2, wherein the specific cells are bone cells, chondrocytes, nerve cells, muscle cells, and vascular endothelial cells.   The disease prediction method according to claim 8, wherein the characteristic of the specific cell is one of bone metabolism, cartilage function, nerve function, and muscle function, or two or more.   10. The disease according to any one of claims 1, 8, and 9, wherein the disease to be predicted is one of bone disease, cartilage disease, neurological disease, muscle disease, and diabetic complications, or two or more. Disease prediction method.   The disease prediction method according to any one of claims 1 to 10, wherein the adipose tissue is derived from a human.   The disease prediction method according to claim 1, wherein the adipose tissue is of a subject himself / herself who is predicted to have the disease.   The disease prediction method according to claim 1, wherein the isolation is by enzyme treatment.   The disease prediction system according to the disease prediction method of Claims 1-13.   A disease prediction system using a database of results obtained from many individuals according to the disease prediction method of claims 1 to 13.   A pharmaceutical screening method using a prediction system comprising either one or both of claims 14 and 15.   The method of screening a pharmaceutical product according to claim 16, wherein the pharmaceutical product is any one of an anti-obesity drug, an anti-diabetic drug, an anti-arteriosclerotic drug, an osteoporosis therapeutic drug, and a diabetic complication therapeutic drug.   A food and a food ingredient screening method using the prediction system according to any one of claims 14 and 15.   The treatment method using the prediction system described in any one of Claims 14 and 15.   The method of treatment according to claim 19, wherein the method of treatment is tailored to the respective disease of the patient.   A method for measuring cell characteristics, characterized in that cells containing adipose tissue-derived tissue stem cells are induced to differentiate into specific cells and the characteristics of the specific cells are measured.   The measurement method according to claim 21, wherein the adipose tissue is a childhood.   The measurement method according to any one of claims 21 and 22, wherein the specific cell comprises a mature adipocyte.   24. The measurement according to claim 23, wherein the characteristics of mature adipocytes are any one of, or two or more of, lipid droplet storage volume, lipid droplet resolution, sugar metabolic capacity, and specific protein secretion amount. Method.   The measurement method according to claim 24, wherein the specific protein comprises one or more of leptin, adiponectin, TNF-α, VEGF, and HGF.   The measurement method according to claim 23, wherein the characteristics of mature adipocytes are comprehensive analysis of expression profile changes of β3 adrenergic receptor, PPARγ, and adiponectin adipose tissue expression genes.   The measurement method according to any one of claims 21 and 22, wherein the specific cells are bone cells, chondrocytes, nerve cells, muscle cells, and vascular endothelial cells.   28. The measuring method according to claim 27, wherein the characteristic of the specific cell is any one or two or more of bone metabolism, cartilage function, nerve function, and muscle function. The measurement method according to any one of claims 21 to 28, wherein the adipose tissue is derived from a human.