WO2007004635A1 - Method for differentiation culture of preadipocyte into adipocyte by coculture with macrophage, and screening of substance capable of affecting the differentiation process of preadipocyte into adipocyte - Google Patents

Abstract

A culture method for inducing the differentiation of a preadipocyte into an adipocyte, the method comprising co-cultivating the preadipocyte with a macrophage in a culture medium free from PPAR-Ϝ agonist and IBMX; an adipocyte produced by the culture method; a screening method for a substance capable of affecting the differentiation process of a preadipocyte into an adipocyte, characterized by using the culture method; a substance capable of affecting the differentiation process of a preadipocyte into an adipocyte, the substance being given by the screening method; a kit for the screening; and others.

Description

 Specification

 Differentiation culture method of preadipocytes into adipocytes by co-culture with macrophages, and screening of substances that affect the differentiation process

 Technical field

 [0001] The present invention relates to a culture method for differentiating preadipocytes into adipocytes, a screening method for substances that affect the differentiation process, and the like.

 Background art

 [0002] In recent years, the prevalence of arteriosclerotic diseases such as coronary artery disease and cerebrovascular disorder secondary to metabolic syndrome has increased. This is associated with almost all arteriosclerotic conditions, including metabolic syndrome is obesity, impaired glucose tolerance associated with insulin resistance, dyslipidemia, hypertension, and thrombus formation and inflammation with elevated PAI-1 and CRP, respectively. Because. Therefore, in order to prevent and treat these diseases, there is an urgent need for elucidation of the onset physiology of metabolic syndrome and drugs to prevent and treat them.

[0003] The involvement of adipose tissue, particularly visceral adipose tissue, has been shown in the onset of metabolic syndrome. Adipocytes, which are the main constituent cells of adipose tissue, are derived from pre-adipocytes! /, A fibroblast-like mesenchymal cell, and prematurely mature into preadipocytes and then adipocytes. In this process, adipocytes produce many secretory factors (adipocyte power-in), and these secretory factors are involved in the development of metabolic syndrome. Therefore, by using a culture system that differentiates preadipocytes into adipocytes, we elucidate the pathogenesis of metabolic syndrome, screen for drugs that control the amount of metabolic processes, and use them for metabolism. Attempts have been made to control the onset of the syndrome.

[0004] However, the conventional differentiation culture method of human preadipocytes into adipocytes uses a medium supplemented with PPAR-γagost and isobutylmethylxanthine (ΙΒΜΧ) that are not present in the living body as essential components. (See Non-Patent Document 1), PPAR-γagoist and PPAR-γagogo are particularly useful when screening drugs using a forceful system that interacts with candidate drugs. -Candidate drugs due to the strong action of strikes Identification becomes difficult. Therefore, it is appropriate to perform drug screening using the conventional conventional culture method.

 [0005] In addition, there are blood-derived mononuclear cells in adipose tissue, and recently, it has been reported that at least a part of adipocytes is secreted by macrophages infiltrating the adipose tissue. (See Non-Patent Document 2). However, the interaction between adipose precursor cells and macrophages has been studied in a differentiation culture system for preadipocytes, or a screening system for substances using the same.

 Non-patent literature l: Hemmrich K et al., Differentiation 73: 28-35 (2005)

 Non-Patent Document 2: Bruun JM et al, J Clin. Endocrinol Metab. 90: 2282-2289 (2005) Disclosure of the Invention

 Problems to be solved by the invention

[0006] The problem to be solved by the present invention is that it is closer to the situation in the living body without using PPAR-γagost and sputum! It is to provide a method for screening substances that affect the sorting process.

 Means for solving the problem

[0007] As a result of intensive studies in view of the above circumstances, the present inventors obtained adipocytes without using PPAR-yagost and IBMX by co-culturing adipose precursor cells with macrophages. As a result, the present invention has been completed.

That is, the present invention provides:

 (1) a culture method for differentiating adipose precursor cells into adipocytes, characterized by co-culturing adipose precursor cells with macrophages in a medium that does not contain PPAR—yagost and IBMX;

 (2) The method according to (1), characterized in that adipose precursor cells and macrophages are isolated and co-cultured with a permeable material,

 (3) The cell is derived from a human, (1) or (2),

 (4) A fat cell obtainable by the method according to any one of (1) to (3),

(5) Using the culture method described in (1) or (2), a candidate substance is added to the medium, and the fat precursor A method for screening a substance that affects the differentiation process from preadipocytes to adipocytes, characterized by examining the differentiation of cells into adipocytes,

 (6) The method according to (5), wherein the cell is derived from human.

 (7) A substance that can be obtained by the method according to (5) or (6) and that affects the differentiation process from preadipocytes to adipocytes,

 (8) From a preadipocyte to an adipocyte, characterized by using the culture method according to (1) or (2) and adding a candidate substance to the medium to examine differentiation from a preadipocyte into an adipocyte A kit for screening substances that affect the separation process of

 (9) The kit according to (8), wherein the cell is derived from human.

 Is to provide.

 The invention's effect

 [0009] According to the present invention, there is provided a culture method for differentiating adipose precursor cells into adipocytes under conditions closer to those in the living body, without using the artificial substances PPAR-yagost and IBMX. Adipocytes that can be obtained by the method, a method for effectively screening for substances that influence the process of dividing fat precursor cells into fat cells using the culture method, and fat precursor cells that can be obtained thereby Substances that influence the process of differentiation into adipocytes, and kits for powerful screening are provided.

 Brief Description of Drawings

 [0010] FIG. 1 is a microscopic image of oil red O-stained adipocytes obtained by co-culturing adipose precursor cells with macrophages for 7 days. The scale bar is 49.9 μm.

 FIG. 2 is a graph showing the lipid content of adipocytes obtained by co-culturing adipose precursor cells with macrophages.

 FIG. 3 is a graph showing the effect of ECGC on the differentiation process from preadipocytes to adipocytes by oil red O staining.

[Fig. 4] Fig. 4 shows the effects of ECGC on the differentiation process from preadipocytes to adipocytes, adiponectin, AP-2, glucose transporter, IRS-1, leptin, LPL, PAI-1, TGF-β, It is a graph shown by expression of TNF-a and resistin. White is without EC GC, black is co-cultured with adipocytes in the presence of ECGC Is.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0011] In one embodiment, the present invention is characterized in that adipose precursor cells are co-cultured with macrophages in a medium that does not contain PPAR-yagost and IBMX. The present invention relates to a culture method. Since PPAR-γ agonist and I ΒΜΧ are artificial substances that are not produced in vivo, the culture method of the present invention using a medium that does not include both PPAR-y agonist and IB MX uses these substances. Conventional differentiation culture methods (for example, PPAR-yagost and IBMX are considered essential in the differentiation culture method of human adipose precursor cells into adipocytes. Compared to IBMX, which is essential for differentiation culture methods), it is very superior in that the culture is performed under conditions closer to those in the living body. In this way, for example, the mechanism of human adipose precursor cell force can also be investigated more accurately and in detail.

 [0012] The preadipocytes used in the present invention can be obtained by means and methods known to those skilled in the art. For example, it is induced from a stem cell such as a mesenchymal stem cell or an ES cell that is isolated from an animal, in particular, a visceral adipose tissue or a subcutaneous adipose tissue force. However, it is not limited to these.

 [0013] The animal species from which the preadipocytes are derived is not particularly limited, and is preferably a mammal including, for example, a mouse, a rat, a rabbit, a dog, a cat, a rabbit, a horse, a monkey, and the like, more preferably a human It comes from. For example, by using human preadipocytes, it becomes possible to study the pathogenesis of metabolic syndrome and the like.

 [0014] The animal species from which the macrophages co-cultured with the preadipocytes in the method of the present invention are not particularly limited, and may be the same or different from the animal species from which the preadipocytes are derived. Good. Preferably, cells are derived from the same animal species, more preferably from the same individual. For example, by using adipose precursor cells and macrophages derived from humans, preferably from the same person, adipose cells that have been proliferated in vitro according to the present invention in reconstruction after mastectomy of breast cancer patients are used. It can also be used.

[0015] In this specification, co-culture means that preadipocytes and macrophages can interact with each other. Thus, culturing these cells. For example, fat precursor cells and macrophages may be mixed and cultured in the same container. Alternatively, fat precursor cells and macrophages may be isolated and cultivated with a permeable material. Cells and macrophages can be placed in separate containers, and these containers can be connected by permeable means and cultured. Here, permeability means a property that allows cells to pass through substances such as medium force-in produced and secreted from medium components and cells. In the present invention, the preferred co-culture mode is economical, or the convenience such as subsequent assembly and operation is considered.

1, the preadipocytes and macrophages are isolated from each other by a permeable material. A strong culture form can be realized by using an incubator such as Coaster / Corning or Millipore. Isolation means that the preadipocytes are not in direct contact with the macrophage. Those skilled in the art can select and use various incubators and isolation means according to the purpose and desired conditions.

[0016] As the medium used in the present invention, a medium well known in the art can be used, and can be appropriately selected and used according to the cells to be used, the culture conditions, and the purpose. For example, DMEMZF-12 medium containing 10% FCS, 66 nM insulin, 0.033 mM piotin, 0.017 mM pantothenic acid may be used. In addition, the adipose precursor cells may adhere to the base material in a strong medium, or may be suspended in a low-adhesion culture dish.

 [0017] It will be apparent to those skilled in the art that the object of the present invention can also be achieved by adding the culture supernatant of macrophages to the medium of preadipocytes. Therefore, such a culture form is also included in the co-culture of the present invention.

 [0018] In another aspect, the present invention provides a fat cell obtainable by the above-described method for culturing fertilizer. Powerful adipocytes, which do not contain artificial substances and are cultured under conditions similar to those in vivo, have properties similar to those in vivo. Therefore, it becomes possible to conduct in vitro experiments under conditions close to in vivo using the fat cells obtained by the present invention.

[0019] In a further aspect, the present invention is characterized in that a fat cell obtainable by a culture method for differentiating the aforementioned preadipocytes into a fat cell is administered to a subject. The present invention relates to a method for treating or preventing diseases such as ataxia, leprosy, and lipodystrophy. For example, treatment can be performed by performing the above method using adipose precursor cells derived from a subject with lipodystrophy and transplanting the obtained adipocytes into the subject.

 [0020] In a still further aspect, the present invention uses an adipocyte obtainable by the above-described culture method for differentiating the preadipocyte into an adipocyte. It relates to a treatment method. This method can be applied to, for example, breast reconstruction and various cosmetic operations. Preferably, problems such as side effects can be avoided by using adipocytes differentiated from autologous adipose precursor cells.

 [0021] In yet another embodiment, the present invention relates to a method for screening for a substance that affects the differentiation process from preadipocytes to adipocytes using the culture method described above. Since the differentiation culture method of the present invention is a method for separating adipose precursor cells under conditions close to those in a living body without containing an artificial substance, a screening method performed using this method was obtained. This has the advantage of increasing the possibility that the substance will show the same effect in vivo.

 [0022] To influence the separation process means to suppress, stop or promote the differentiation of preadipocytes into adipocytes. Specifically, reducing the differentiation rate from preadipocytes to adipocytes, delaying the differentiation, incompletely or completely stopping the differentiation, reducing the number of adipocytes, or On the contrary, increasing the differentiation rate, increasing the number of adipocytes, and further allowing the differentiated adipocytes to become younger.

 [0023] The screening method of the present invention is carried out by adding a candidate substance to the differentiation culture system and examining differentiation from adipose precursor cells to adipocytes. There are various candidate substances, for example, ECGC (epigalocatechin gallate) analogs, derivatives or mutants, or macrophages, which are known to suppress the differentiation of preadipocytes into adipocytes Substances known to act to suppress or promote activity

Or a force including a mutant thereof, and the like.

[0024] As a means for examining the separation, for example, the number of fat cells is calculated by microscopic observation. Observe the size, number, fat content, etc. of lipid droplets by staining with oil red o Substances whose expression decreases or increases with differentiation from adipocyte precursor cells to adipocytes, such as a P2, CD36, adiponectin , Resistin, GLUT4, TNF-a, PAI-1 etc. are measured by quantitative PCR or immunoplot. In the screening method of the present invention, as a control, differentiation from adipose precursor cells to adipocytes in the above differentiation culture system when no candidate substance is added is also examined, and compared with differentiation when a candidate substance is added. If the differentiation is suppressed in the candidate substance-added culture system, the candidate substance is considered to be a substance that suppresses the differentiation, and conversely, if the differentiation is promoted! It is considered to be a substance that promotes the differentiation.

[0025] If, for example, a substance that suppresses differentiation into adipocytes using a human-derived adipose precursor cell is found by the screening method of the present invention, it may be obesity, diabetes, hypertension. It is considered to be a substance useful for the treatment or prevention of diseases such as hyperlipidemia, metabolic syndrome, arteriosclerotic disease, and ischemic heart disease. In addition, if a substance that promotes differentiation is found, it is considered to be a substance useful for the treatment or prevention of diseases such as malnutrition, itchiness, and lipodystrophy. Since the screening method of the present invention is carried out in the absence of PPA-γ agonist and ΙΒΜΧ, it is possible to find useful substances that are close to in vivo conditions and that can eliminate the effects of these substances and that are useful. It can be said to be an easy, efficient and economical method. Furthermore, by using the patient-derived cells and the screening method of the present invention, it becomes possible to create drugs and cells for tailor-made treatment. In addition, for example, it is possible to screen for substances that are useful for producing meat that meets the needs of consumers by using fat precursor cells derived from sea bream and inducing adipocytes efficiently at desired sites. .

 [0026] Therefore, in a further aspect, the present invention relates to a substance that can be obtained by the above-described screening method and that influences the process of dividing preadipocytes into adipocytes. For example, a substance that promotes differentiation from preadipocytes into adipocytes may be used in the culture method of the present invention to increase the number of adipocytes obtained.

[0027] In yet another embodiment, the present invention screens a substance that affects the differentiation process from preadipocytes to adipocytes, characterized by using the culture method described above. Providing a kit for The kit of the present invention may include a container, a device, a culture medium and the like for performing the co-culture of the present invention. Usually, an instruction manual is attached to the kit.

 [0028] In the screening method or kit of the present invention, means for facilitating screening and promoting efficiency, such as an oil red O staining solution for confirming differentiation into adipocytes, quantitative PCR. Use primers and Z or probes for ELISA, ELISA, antibodies for immunoplots, etc.

 [0029] Hereinafter, the present invention will be described in more detail and specifically with reference to Examples, but the Examples are not intended to limit the present invention.

 Example 1

 [0030] A. Preparation of human adipose precursor cells

 Human adipose tissue-derived adipose precursor cells were suspended in a basic medium (DMEMZF-12 medium containing 10% FCS) to a concentration of 100,000 ZmL, and 1.5 mL of this suspension was seeded in a 6-well culture dish. . The basal medium was changed the next day, and every 3 days thereafter, and adipose precursor cells were grown until confluent.

 [0031] B. Preparation of human monocyte-derived macrophages

 Palin was collected from the periphery to 30 mL. After separating lymphocytes at a specific gravity of 1.066, 15 mL of the separated solution was transferred to a 50 mL tube. Next, 30 mL of blood was layered and centrifuged at 500 g, 16 ° C. for 40 minutes, and separated from the bottom into approximately three layers of red blood cells, lymphocyte separation liquid (colorless and transparent), and plasma (yellow and transparent). A white cell layer located between the lymphocyte separation solution and plasma was collected and suspended in a medium. After washing by centrifugation (3 times), the resulting cell pellet was suspended in DMEM / F-12 medium containing 10% human serum. A culture of 1.5 mL of this suspension in the upper chamber of 6-well Transwell (Coaster / Corning) (10% human serum-containing DMEMZF—12 medium 2. 7 mL pre-added to the bottom chamber) Seeded on insert. The medium was changed after 1 hour and the next day and every 3 days thereafter.

 [0032] C. Induction of preadipocytes into adipocytes by co-culture with macrophages

The medium of the 6-well culture dish obtained in A was replaced with 2.7 mL of DMEM / F12 medium containing 10% human serum, 66 nM insulin, 0.033 mM piotin, 0.017 mM pantothenic acid. Next, replace the culture insert medium of B Transwell with the above medium. 6 It was inserted into a well culture dish and co-culture of adipose precursor cells and macrophages was started. Change the medium every 3 days, and oil red O stains the cells at 0, 3, 5, 7, 9, and 14 days after the start of co-culture.

The lipid content was measured. As a control, one cultured with preadipocytes alone was used. When the macrophage and adipose precursor cells were co-cultured, the lipid content of the cells was confirmed to be differentiated into adipocytes, which was larger than the lipid content of the cells when cultured alone with the adipose precursor cells (Fig. 1). And Figure 2).

 Example 2

 [0033] Inhibition of preadipocyte differentiation into adipocytes by ECGC

 A. Confirmation of mist suppression by oil red O staining

 According to the method described in Example 1, co-culture of preadipocytes and macrophages (MF) was started. The medium was supplemented with ECGC at concentrations of 1, 10, and 100 M, respectively. Cells on day 7 after the start of co-culture were stained with oil red O, and the lipid content was measured. As controls, those cultured in the absence of ECGC and co-cultured with macrophages and those cultured with fat precursor cells alone were used. It was confirmed that ECGC was suppressed in a dose-dependent manner by dividing preadipocytes into adipocytes in co-culture with macrophages (see Fig. 3).

 [0034] B. Confirmation of Suppression by Quantitative PCR

According to the method described in Example 1, adipose precursor cells were co-cultured with macrophages in a medium containing 100 M ECGC. On the 7th day after the start of culture, RNA was extracted from the adipocytes induced to differentiate. Using a TaqMan (registered trademark) probe (Applied Bio Systems), PCR (internal standard: GAPDH) is performed, and adiponectin, AP-2, glucose transporter, IRS-1, leptin, LPL, PAI-1, TGF-β, TNF-a, and resistin were quantified. The expression of adiponectin, glucose transporter, leptin, and TGF-β, which are known to decrease during adipocyte differentiation, is greater in the presence of ECGC compared to those cultured in the absence of ECGC. It was confirmed that the number of cells was increased compared to the cultured cells (see Fig. 4). In addition, the expression of AP-2, IRS-1, LPL, PAI-1, TNF-α, and resistin, which are known to increase with adipocyte differentiation, was cultured in the absence of ECGC. It was confirmed that it was reduced in cells cultured in the presence of ECGC compared to those (see Fig. 4). From these results, the co-culture of the present invention In the system, it was found that substances that affect the differentiation of preadipocytes into adipocytes can be screened.

Industrial applicability

 The present invention provides a culture method for differentiating adipocytes from preadipocytes without using PPAR-yagost and IBMX that do not exist in the body, and a screening method for substances that affect such a differentiation process. Therefore, in the field of pharmaceuticals, for example, in the field of development, manufacturing, health food, etc. of preventive, therapeutic or diagnostic agents for diseases such as metastatic syndrome, research on adipocytes and related cells It can be used in fields.

Claims

The scope of the claims  [1] A culture method for differentiating adipose precursor cells into adipocytes, characterized by co-culturing adipose precursor cells with macrophages in a medium that does not contain PPAR-yagonist and IBMX.  [2] The method according to [1], wherein the preadipocytes and macrophages are isolated and co-cultured with a permeable material. [3] The method according to claim 1 or 2, wherein the cell is derived from a human. [4] An adipocyte obtainable by the method according to any one of claims 1 to 3. [5] Differentiation from preadipocytes to adipocytes, characterized by using the culture method according to claim 1 or 2 and adding a candidate substance to the medium to examine differentiation from preadipocytes to adipocytes A method of screening for substances that affect the process. 6. The method according to claim 5, wherein the cell is derived from a human.  [7] A substance that can be obtained by the method according to claim 5 or 6 and that affects the differentiation process from preadipocytes to adipocytes.  [8] The differentiation from preadipocytes to adipocytes, characterized by using the culture method according to claim 1 or 2 and adding a candidate substance to the medium to examine differentiation from preadipocytes to adipocytes. A kit for screening substances that affect the process.  [9] The kit according to claim 8, wherein the cell is derived from a human.