WO2007004469A1

WO2007004469A1 - Method for differentiation culture of preadipocyte into mature adipocyte and screening of substance capable of affecting the differentiation process of preadipocyte into mature adipocyte

Info

Publication number: WO2007004469A1
Application number: PCT/JP2006/312793
Authority: WO
Inventors: Akifumi Matsuyama; Yoshiki Sawa; Yuzuru Kanakura
Original assignee: Osaka University
Priority date: 2005-07-04
Filing date: 2006-06-27
Publication date: 2007-01-11

Abstract

A culture method for inducing the differentiation of a preadipocyte into a mature adipocyte, the method comprising cultivating the preadipocyte in a floating state; a mature adipocyte produced by the method; a screening method for a substance capable of affecting the differentiation process of a preadipocyte into a mature adipocyte, characterized by using the culture method; a substance capable of affecting the differentiation process of a preadipocyte into a mature adipocyte, which is given by the screening method; a kit for the screening; and others.

Description

Specification

Methods for differentiation and culture of preadipocytes into mature adipocytes, and screening for substances that affect the differentiation process

[0001] The present invention relates to a culture method for differentiating preadipocytes into mature adipocytes, characterized by culturing preadipocytes in a suspended state, and mature adipocytes obtainable thereby, A screening method for substances that affect the differentiation process from preadipocytes to mature adipocytes, characterized by using a culture method, and the differentiation process from preadipocytes to mature adipocytes that can be obtained thereby Material, as well as kits for such screening. 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 for treating it.

[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 adipose precursor cells, a fibroblast-like mesenchymal cell, and preadipocytes that encapsulate many lipid droplets, and then become unicellular Lipid droplets differentiate and mature into spherical mature adipocytes occupying most of the cells. During this process, adipocytes produce many secretory factors (adipocytes), and it is known that these secretory factors are involved in the development of progeny Hi syndrome. Preadipocytes produce secretory factors that suppress the development of metabolic syndrome such as adiponectin, but more mature mature adipocytes reduce the production of adiponectin and promote the development of metabolic syndrome such as TNF It is known to produce secretory factors. Therefore, by studying the differentiation process from preadipocytes to mature adipocytes and inhibiting the differentiation, Although it is believed that the disease group can be prevented, a culture system that differentiates preadipocytes into mature fat cells has not been established so far, and substances having such inhibitory action are screened in vitro. It is impossible.

[0004] Conventionally used cells obtained by the method of maintaining differentiation of preadipocytes enclosing a large number of fat droplets on a culture dish or scaffold are scaffolds of visceral tissue and subcutaneous fat tissue. It differs greatly from adipocytes not only in protein expression pattern but also in morphology (see Non-Patent Documents 1 and 2). Further, most of the conventional culture systems for differentiation of preadipocytes into adipocytes use mouse-derived preadipocytes. Therefore, the establishment of a differentiation culture system from animal-derived, particularly human-derived preadipocytes to mature adipocytes, has not been successful, and remains a major issue. In addition, adherent cells generally have a biological activity that is significantly reduced when exposed to suspension, or die if maintained for a long time in suspension. The cells are not cultured in a suspended state.

Non-Patent Document 1: Tchknoia T et al., Am J Physiol Endocrinol Metab 288: E267-E277 (2 005)

Non-Patent Document 2: Hemmrich K et al., Differentiation 73: 28-35 (2005)

Disclosure of the invention

Problems to be solved by the invention

[0005] The problem to be solved by the present invention affects the culture method of differentiating preadipocytes into spherical mature adipocytes encapsulating monocytic lipid droplets, and the differentiation process into mature adipocytes, such as preadipocyte force It is to provide a screening method for substances.

Means for solving the problem

[0006] As a result of intensive studies in view of the above circumstances, the present inventors have surprisingly found that the three-dimensional environment, that is, the two-dimensional environment such as on the culture dish, has not been obtained in a suspended state. It has been found that by culturing fat cells, mature adipocytes that are morphologically and physiologically very similar to fat cells in the animal body can be obtained, and the present invention has been completed.

That is, the present invention provides:

(1) A preadipocyte is characterized by culturing in a suspended state. A culture method for differentiation into mature fat cells,

(2) The method according to (1), wherein the culture in a suspended state is performed in a siliconized culture vessel or a low adhesion culture vessel,

(3) The method according to (1), wherein the culture in a suspended state is performed using a hanging 'drop culture method,

(4) The cell is derived from a human, (1) to (3),

(5) A mature adipocyte obtainable by the method according to (1) to (4),

(6) A method for screening a substance that affects the differentiation process from preadipocytes to mature adipocytes, characterized by using the culture method according to (1) or (2),

(7) The cell is derived from a human, (6) The method according to,

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

(9) A kit for screening a substance that affects the differentiation process from preadipocytes to mature adipocytes, characterized by using the culture method according to (1) or (2),

(10) The cell is derived from a human, (9) the kit according to

Is to provide.

The invention's effect

[0008] According to the present invention, a method for culturing mature adipocytes, and a method and kit for screening mature adipocytes obtainable thereby, and substances that affect the differentiation process from preadipocytes to mature adipocytes. Etc. are provided. The present invention is excellent in that mature adipocytes can be obtained using human-derived preadipocytes, and that the obtained mature adipocytes are very similar to adipocytes present in vivo. Have

Brief Description of Drawings

[0009] FIG. 1 is a micrograph of cells after culturing for 2 days in a state where preadipocytes are suspended.

[FIG. 2] FIG. 2 is a micrograph of mature adipocytes induced by culturing for 4 days in a state in which preadipocytes are suspended. FIG. 3 is a micrograph of cells after preadipocytes have been cultured for 4 days under adhesion conditions.

FIG. 4 is a graph showing the influence of ECGC on the differentiation process from preadipocytes to mature adipocytes by the expression of adiponectin. The white color indicates adiponectin expression in preadipocytes before the start of suspension culture, the black color indicates adiponectin expression after culturing the preadipocytes in the presence of ECGC for 4 days in the presence of ECGC.

FIG. 5 is a graph showing the effect of ECGC on the differentiation process from preadipocytes to mature adipocytes by the expression of PAI-1. The white color indicates PAI-1 expression in preadipocytes before the start of suspension culture, the black color indicates PAI-1 expression in the absence of ECGC, and the hatched line indicates PAI-1 expression after 4 days in the presence of ECGC.

BEST MODE FOR CARRYING OUT THE INVENTION

[0010] The present invention relates to a culture method for differentiating preadipocytes into mature adipocytes, characterized in that, in one embodiment, preadipocytes are cultured in a suspended state. The term “preadipocyte” as used herein is defined as a cell that has differentiated from preadipocytes and encapsulates a large number of small lipid droplets. The term “mature adipocyte” as used herein is defined as a globular cell that is similar to the adipocyte found in vivo and is predominantly composed of unicellular lipid droplets. Unless otherwise specified, other terms shall have the meanings commonly used in the field.

[0011] Preadipocytes can be obtained by means and methods known to those skilled in the art. For example, those isolated from visceral adipose tissue or subcutaneous adipose tissue, adipose precursor cells, or mesenchymal stem cells, or those induced to differentiate from stem cells such as ES cells may be used, but are not limited thereto. For example, DMEM / F-12 containing 10% FCS, 66 nM insulin, 250 nM dexamethasone, 0. ImM IBMX, 0.1 mg / L PPAR-γ agonist well known in the art as a medium for differentiation of preadipocytes It may be preadipocytes induced to differentiate from preadipocytes using a medium. Separation and acquisition of preadipocytes can also be obtained by means and methods known to those skilled in the art.

[0012] The animal species from which the preadipocytes are derived is also not particularly limited, and preferably, for example, mammals including mice, rats, rabbits, dogs, cats, horses, horses, etc., more preferably humans. is there. For example By using human preadipocytes, it is possible to study the pathogenesis of metabolic syndrome, etc., or use self-matured adipocytes differentiated and proliferated in vitro for reconstruction after mastectomy in breast cancer patients It is also possible to do so.

[0013] The "suspended state" used in the present invention means placing cells in a released state by preventing or suppressing the adhesion or aggregation with a culture vessel or other cells. Cell floating can be performed by various known means and methods. For example, cells may be placed in a floating state using a culture vessel or device that has been treated to prevent or inhibit cell adhesion, or made of a material that prevents or inhibits cell adhesion. . Examples of such a culture vessel or apparatus include a low-adhesion culture vessel such as a silicon-treated culture vessel (for example, a siliconized flask) or a low-adhesion culture dish. Alternatively, the cells may be cultured in a suspended state by using a hanging drop culture method. In addition, known means and methods may be used in combination as appropriate at the start of floating or in order to continue floating. Examples of methods that can be used are as follows. Alternatively, after culturing the cells in a temperature-responsive culture equipment for cell recovery (for example, CellSeed), the cells are detached by, for example, incubation at 20 ° C. for 30 minutes.

[0014] In another aspect, the present invention provides a mature fat cell obtainable by the above-described differentiation culture method. Such mature adipocytes are very advantageous in that they have morphologically and physiologically similar properties to the fat cells in the animal body. For example, using mature adipocytes obtainable by the differentiation culture method of the present invention, it is possible to conduct experiments closer to the living body at the in vitro port.

[0015] In a further aspect, the present invention is characterized by administering to a subject mature adipocytes obtainable by the above-described culture method for differentiating preadipocytes into mature adipocytes. The present invention relates to a method for treating or preventing diseases such as lipodystrophy. For example, treatment can be performed by performing the above method using preadipocytes derived from a subject with lipodystrophy and transplanting the resulting mature adipocytes into the subject. [0016] In a still further aspect, the present invention uses a mature adipocyte obtainable by the above-described culture method for differentiating preadipocytes into mature adipocytes. It is related with the treatment method. This method can be applied to, for example, breast reconstruction and various cosmetic surgery. Preferably, problems such as side effects can be avoided by using mature adipocytes differentiated from autologous preadipocytes.

[0017] In another aspect, the present invention relates to a method for screening for a substance that affects the differentiation process from preadipocytes to mature adipocytes using the culture method described above. Here, to influence the differentiation process means to suppress or stop the differentiation from preadipocytes to mature adipocytes, or to promote a certain level. Specifically, decreasing the differentiation rate from preadipocytes to mature adipocytes, delaying the differentiation, incompletely or completely stopping the differentiation, reducing the number of mature adipocytes, Or, conversely, increasing the differentiation rate, increasing the number of mature adipocytes, and further immature differentiated mature adipocytes.

[0018] The screening method of the present invention is performed by adding a candidate substance to the differentiation culture system and examining differentiation from preadipocytes to mature adipocytes. There are various candidate substances, for example, the ability to include analogs, derivatives or mutants of ECGC (epicaro tectin gallate), which are known to suppress differentiation into preadipocyte force mature adipocytes. It is not limited to.

[0019] As a means for examining differentiation, for example, calculating the number of mature adipocytes by microscopic observation, observing the size, number, fat content, etc. of fat droplets by oil red staining, from preadipocytes Substances whose expression decreases or increases upon differentiation into mature adipocytes, such as aP2, CD36, adiponectin, resistin, GLUT4, TNF-s, PAI-1, etc. measured by quantitative PCR or immunoplot etc. Is mentioned. In the screening method of the present invention, as a control, the differentiation from preadipocytes to mature adipocytes in the differentiation culture system in the case where no candidate substance is added is also examined, and compared with the differentiation when the candidate substance is added. If the differentiation is suppressed in a candidate substance-added culture system, the candidate substance is considered to be a substance that suppresses the differentiation. If conversion is promoted, the candidate substance is considered to be a substance that promotes the differentiation.

[0020] If, for example, a substance that suppresses differentiation into mature adipocytes using human-derived preadipocytes has been found by the screening method of the present invention, it may be obesity, diabetes, or 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, lupus, and lipodystrophy. Since the screening method of the present invention uses, for example, the culture method of the present invention having the advantages as described above, it is an easy, efficient and economical method for finding such useful substances. Furthermore, the use of the screening method of the present invention using patient-derived cells makes it 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 consumer needs by efficiently using adipocyte-derived preadipocytes to induce adipocytes at the desired site. .

[0021] Therefore, in a further aspect, the present invention relates to a substance that can be obtained by the above screening method and that influences the pre-adipocyte force differentiation process into mature adipocytes. For example, a substance that promotes differentiation from preadipocytes into mature adipocytes may be used in the culture method of the present invention to increase the number of mature adipocytes obtained.

[0022] In yet another embodiment, the present invention provides a kit for screening a substance that affects the differentiation process from preadipocytes to mature adipocytes, characterized by using the culture method described above. provide. The kit of the present invention may contain a container, an instrument, a medium, etc. for carrying out the suspension culture of the present invention. Usually, an instruction manual is attached to the kit.

[0023] In the screening method or kit of the present invention, means for facilitating screening and promoting efficiency 'method, for example, oil red O staining solution for confirming differentiation into mature adipocytes, quantitative PCR Primers and / or probes, ELIS A, antibodies for immunoplots, etc. may be used.

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 [0025] Differentiation of preadipocytes derived from preadipocytes into mature adipocytes Human adipose tissue-derived preadipocytes were added to a basic medium (DMEMZF-12 medium containing 10% FCS) at a concentration of 100,000 / mL. Then, 10 mL of this suspension was seeded on a 10 cm culture dish. The basal medium was changed the next day, and every 3 days thereafter, and adipose precursor cells were grown until confluent. Next, the basal medium was changed to a differentiation medium (DMEM / F-12 medium containing 10% FCS, 66ηm insulin, 250nM dexamethasone, 0. ImM IBMX, 0.1mg / L pioglitazone (Takeda Pharmaceutical)), Pre-adipocytes were obtained by culturing at 37 ° C for 3 days. The differentiation medium was aspirated and the preadipocytes were washed with PBS. Next, 1 mL of trypsin / EDTA solution was added and incubated at 37 ° C for 1 minute. 9 mL of basic medium was added and pipetted, and the resulting cell suspension was transferred to a 15 mL tube. The resulting cell pellet was centrifuged at 440 g for 2 minutes at 4 ° C. Medium) Suspended in 12 mL. 2 mL of the suspension was seeded in a 6-well low-adhesion culture dish (Coaster / Coming), and suspension culture was performed (Fig. 1). 4 days force On day 7 mature lipid cells with lipid droplets were obtained (Fig. 2). On the other hand, when cultured for 4 days under the conventional culture dish adhesion conditions, mature adipocytes with single lipid droplets were not obtained (Fig. 3). Floating culture was performed in a siliconized flask or using the hanging drop culture method, and similar results were obtained.

Example 2

[0026] Inhibition of preadipocyte differentiation into mature adipocytes by ECGC

According to the method described in Example 1, the prepared preadipocytes were suspended and cultured in a medium containing ECGC for 4 days. Next, RNA is extracted, and PCR (internal standard: GAPDH) is performed using the TaqMan [registered trademark] probe (Applied Bio Systems) with the strong RNA as a saddle type, and expression of adiponectin and PAI-1 Was quantified. It was confirmed that the expression of adiponectin in mature adipocytes obtained by culturing in a medium containing ECGC was increased compared to cells cultured in a medium containing ECGC (Fig. 4). . Furthermore, it was confirmed that the expression of PAI-1 in mature adipocytes obtained by culturing in a medium containing ECGC was decreased compared to cells cultured in a medium not containing ECGC (Fig. 5). ). From these results, the book It was found that substances that affect the differentiation process from preadipocytes to mature adipocytes can be screened in the suspension culture of the invention.

Industrial applicability

According to the present invention, a culture method for differentiating preadipocytes into mature adipocytes encapsulating monocystic lipid droplets, which are similar to adipocytes of visceral adipose tissue and subcutaneous adipose tissue, and substances that affect the differentiation process Can be used in the field of pharmaceuticals, for example, in the field of development, manufacturing, or health foods for prophylactic, therapeutic or diagnostic agents for diseases such as deceitful syndromes. .

Claims

The scope of the claims

[1] A culture method for differentiating preadipocytes into mature adipocytes, comprising culturing preadipocytes in a suspended state.

[2] The method according to claim 1, wherein the culture in a suspended state is performed in a silicon-treated culture vessel or a low adhesion culture vessel.

[3] The method according to claim 1, wherein the culture in a suspended state is performed using a hanging drop culture method.

4. The method according to claim 1, wherein the cell is derived from a human.

[5] A mature adipocyte obtainable by the method according to any one of claims 1 to 4.

[6] A method for screening for a substance that affects the differentiation process from preadipocytes to mature adipocytes, wherein the culture method according to claim 1 or 2 is used.

7. The method according to claim 6, wherein the cell is derived from a human.

[8] A substance obtainable by the method according to claim 6 or 7, wherein the preadipocyte force also affects the differentiation process into mature fat cells.

[9] A kit for screening for a substance that affects the differentiation process from preadipocytes to mature adipocytes, wherein the culture method according to claim 1 or 2 is used.

10. The kit according to claim 9, wherein the cell is derived from a human.