KR101019877B1 - Composition of medium for differentiation of adipocyte - Google Patents

Abstract

In the present invention, the adipocyte differentiation medium composition comprises insulin, DHEA (dehydroepiandrosterone), and histamine (histamine). The present invention relates to a method for differentiating preadipocytes into adipocytes. The adipocyte differentiation medium composition of the present invention has the advantage of maximizing the survival rate by allowing the distribution of differentiated adipocytes evenly and stably adhering the differentiated adipocytes compared to the adipocyte differentiation medium compositions used in general. have. In addition, DHEA (dehydroepiandrosterone) and histamine (histamine) used in the adipocyte differentiation medium composition of the present invention is economically advantageous because it is cheaper than dexamethasone and isobutylmethylxanthine.

Adipocyte, differentiation, media, composition

Description

Medium composition for adipocyte differentiation {Composition of medium for differentiation of adipocyte}

In the present invention, the adipocyte differentiation medium composition comprises insulin, DHEA (dehydroepiandrosterone), and histamine (histamine). The present invention relates to a method for differentiating preadipocytes into adipocytes.

At present, there is an active global movement to differentiate adult stem cells into specific cells. This is because the adult stem cells are safe for medical use. Specifically, the adult stem cells have no problem even when transplanted into the body for long-term regeneration, and have a tissue-specific differentiation ability to differentiate themselves according to the characteristics of surrounding tissues. have. In addition, if any damage occurs in the body tissue, stem cells in other organs are swollen and have the flexibility of differentiation into damaged tissues, and it is characterized by self-reproduction in the injected body. In addition, the immune rejection reaction may not occur in that it is able to self-transplant its own cells because it is in an adult body. In this regard, adult stem cells are expected to be helpful for the resolution of almost all kinds of incurable diseases.

Adipose progenitor cells, one of the adult stem cells, also have the same advantages as above, and are known to differentiate into adipocytes, chondrocytes, osteoblasts, neurons, and epithelial cells according to differentiation induction. Especially The method of differentiating adipocytes into adipocytes using fat precursor cells has the advantage of using the differentiated adipocytes as a molding material. In particular, the method has a high engrafting period and engraftment rate compared to the conventional molding method in which only fat is separated and injected. Therefore, there is an urgent need for a method for effectively differentiating fat cells from fat precursor cells.

In order to differentiate the adipocytes from the conventional adipocytes, a medium composition for adipocyte differentiation mainly containing insulin, dexamethasone, isobutylmethylxanthine, etc. was mainly used ( The Journal of Biological chemistry , 2001, 276 (30), 28430-28435; Korean Patent Publication No. 2004-0082068). However, these components are relatively expensive ( Method Mol. Biol ., 2001 (155); 239-47), and rapidly change the morphology of adipocytes, as well as growth arrest and clone expansion. It is known to cause clonal expansion, differentiation related gene changes, and lipid storage changes ( Exp. Biol. Med., (Maywood). 2001.12; 226 (11): 997-1002). Also, it is known that differentiated fat-soluble adipocytes grow in water-soluble medium, which leads to an increase in intracellular fat accumulation rate, thereby causing the adipocytes to float over the medium, resulting in loss of stability and damage ( J. Lipid Res). , 1995 Apr; 36 (4): 868-75.)

Accordingly, the present inventors manufacture a fat cell differentiation medium composition comprising insulin (insulin), DHEA (dehydroepiandrosterone) and histamine (histamine) in a fat cell differentiation medium composition. The present invention was completed by confirming that the differentiation of adipocytes into the adipocytes and the distribution of the differentiated adipocytes are uniform and the differentiated adipocytes are stably fixed and thus the survival rate is significantly higher.

Accordingly, an object of the present invention is to provide an adipocyte differentiation medium composition comprising insulin, DHEA (dehydroepiandrosterone) and histamine in his adipocyte differentiation medium composition.

Still another object of the present invention is to provide a method for differentiating adipocytes (preadipocytes) into adipocytes (adipocytes) using the medium composition.

In order to achieve the above object, the present invention provides a fat cell differentiation medium composition, characterized in that containing the insulin (insulin), DHEA (dehydroepiandrosterone) and histamine (histamine) in the adipocyte differentiation medium composition. do.

The present invention also provides a method for differentiating adipocytes (preadipocyte) into adipocytes (adipocyte) using the medium composition.

Hereinafter, the present invention will be described in detail.

The adipocyte differentiation medium composition of the present invention is characterized in that, in the adipocyte differentiation medium composition, insulin (insulin), DHEA (dehydroepiandrosterone) and histamine (histamine) are contained.

The adipocyte differentiation medium composition refers to a medium composition used to differentiate adipocytes (preadipocyte) into mature adipocytes containing fat.

Adipocyte differentiation medium composition of the present invention is characterized in that it contains insulin (insulin), DHEA (dehydroepiandrosterone) and histamine (histamine).

The DHEA (dehydroepiandrosterone) and histamine (histamine) is a substance that has never been used as a medium composition for adipocyte differentiation, and the DHEA (dehydroepiandrosterone) is a kind of steroid hormone produced by the adrenal gland and is known to be made from cholesterol. . In addition, the histamine (histamine) is known to produce histamine by the histidine decarboxylase acts on the histidine amino acid. In addition, histamine is stored in large granules mainly in mast cells, and is known to play an important role in the body's defense against foreign injuries or their invasion. However, it is not known at all what role the DHEA (dehydroepiandrosterone) and histamine (histamine) play in adipocyte differentiation.

In particular, the conventional adipocyte differentiation medium composition is mainly used by mixing insulin (insulin), dexamethasone (dexamethasone), isobutylmethylxanthine (isobutylmethylxanthine), but the adipocyte differentiation medium composition of the present invention is insulin (insulin), DHEA (dehydroepiandrosterone) ) And histamine are mixed.

In addition, the adipocyte differentiation medium composition of the present invention may additionally contain indomethacin, biotin and pentothenate in insulin, deHEAepiandrosterone and histamine. have. It may also contain further media composition ingredients known to those skilled in the art, if necessary.

The media composition for adipocyte differentiation of the present invention contains insulin, DHEA (dehydroepiandrosterone), histamine, histamine, indomethacin, biotin and pentothenate, Although not limited, insulin is preferably 0.1-10 ug / ml, DHEA (dehydroepiandrosterone) is 0.01-10 uM, histamine is 1-10 uM, indomethacin is 0.1-10 mM, biotin The 20 to 40 uM and pentothenate (panthothenate) refers to containing 10 to 30 uM. Most preferably, 1 ug / ml of insulin, 1 uM of dehydroepiandrosterone (DHEA), 7 uM of histamine, 0.2 mM of indomethacin, 33 uM of biotin, and panthothenate May contain 17 uM.

In one embodiment of the present invention, the adipocyte differentiation medium composition of the present invention is insulin (insulin) 1ug / ㎖, DHEA (dehydroepiandrosterone) 1uM, histamine 7uM, indomethacin (indomethacin) 0.2mM, Biotin was prepared to contain 33 uM and penthothenate 17 uM. In the meantime, the adipocyte differentiation medium composition commonly used as a control group was 1ug / ml of insulin, 1uM of dexamethasone, 0.5mM of isobutylmethylxanthine, and 0.2mM of indomethacin. Biotin was prepared to contain 33 uM and penthothenate 17 uM (see <Example 1>).

The fat precursor cells cultured in <Reference Example 1> were induced to differentiate into adipocytes using the adipocyte differentiation medium composition of the present invention prepared as described above.

As a result of observing the morphology of the differentiated adipocytes using the adipocyte differentiation medium composition of the present invention, it was found that the distribution of the differentiated adipocytes was evenly and stably fixed than the control group (Experimental Example 1-). 1>). In addition, it was found that the survival rate of the adipocytes differentiated in the medium composition of the present invention was much higher than that of the control group during all measurement periods (see <Experimental Example 1-2>). On the other hand, the media composition of the present invention was found to be significantly higher than the glucose uptake compared to the control, which means that the adipocytes absorb glucose to convert to fat, so that the larger the uptake of glucose, the more fat cells were differentiated. (See <Experimental Example 1-3>). In addition, in the medium composition of the present invention, the amount of fat production was higher than that of the control group, and particularly, the initial production amount was much higher, indicating that the adipocyte differentiation was well induced (see <Experimental Example 1-4>).

Therefore, the adipocyte differentiation medium composition of the present invention has the advantage of maximizing the survival rate by allowing a uniform distribution of the differentiated adipocytes and stably fixing the differentiated adipocytes, as compared to the adipocyte differentiation medium compositions used in general. There is this. In addition, DHEA (dehydroepiandrosterone) and histamine (histamine) used in the adipocyte differentiation medium composition of the present invention is economically advantageous because it is cheaper than dexamethasone and isobutylmethylxanthine.

On the other hand, the method for differentiating the adipocytes (preadipocyte) of the present invention into adipocytes (adipocyte) is characterized in that using the medium composition of the present invention.

The progenitor cells can be used separately from adult stem cells according to a known method, but can be used by purchasing a commercial product. In addition, if necessary, the fat precursor cells may be used by culturing according to a conventional culture method, but not limited thereto. The fat precursor cells may be inoculated in a culture medium and cultured at 30 to 45 ° C and 1 to 10% of CO ₂ . . As a medium for culturing the fat precursor cells, but not limited thereto, DMEM (Dulbecco's Modified Eagle Medium), MEM (Minimum Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), α-MEM, RPMI 1640 medium, etc. may be used. The type of serum added to this may be elegant serum (BCS, Bovine calf serum) or fetal bovine serum (FBS, Fetal bovine serum) and the like. In one embodiment of the present invention was used incubated at 37 ° C, CO ₂ 5% in DMEM (Dulbecco's Modified Eagle's Medium) medium containing 10% bovine calf serum (BCS). See example 1).

Differentiating the cultured adipocytes into adipocytes is to inoculate the adipocytes into a medium containing the adipocyte differentiation medium composition of the present invention and differentiate them according to methods known to those skilled in the art. Say. Although not limited thereto, the fat precursor cells are inoculated directly to the medium composition of the present invention, or the medium composition of the present invention is added to the medium in which the fat precursor cells are cultured, and then cultured at 30 to 45 ° C. and CO _{2 to} 1 to 10%. By this, differentiation can be induced. In addition, after inducing the differentiation, a composition consisting of, but not limited to, insulin, biotin, indomethacin, and pentothenate may be additionally added, and also cell differentiation. Alternatively, an additional selection may be made by selecting or combining elegant serum (BCS, Bovine calf serum) or fetal bovine serum (FBS) used for propagation. Most preferably, elegant serum (BCS, Bovine calf serum) is added. First, fetal bovine serum (FBS) can be added.

In one embodiment of the present invention, differentiation was started when the distribution of fat precursor cells to the media area was 95% or more, and at this time, carbon dioxide concentration was maintained at 5.0 ± 0.5% at 37 ° C., and 2 days after the differentiation was induced. After two days, a medium containing only 1 μg / ml of insulin, Biotin, Indomethacin and Pentothenate was additionally supplied.

The adipocyte differentiation medium composition of the present invention has the advantage of maximizing the survival rate by allowing the distribution of differentiated adipocytes evenly and stably adhering the differentiated adipocytes compared to the adipocyte differentiation medium compositions used in general. have. In addition, DHEA (dehydroepiandrosterone) and histamine (histamine) used in the adipocyte differentiation medium composition of the present invention is economically advantageous because it is cheaper than dexamethasone and isobutylmethylxanthine.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Reference Example 1

Cell culture

Adipose progenitor cells (3T3-L1) were purchased from ATCC (catalog no: CL-173) using mouse embryo fibroblasts. The cells were cultured at 37 ° C, 5% CO ₂ in DMEM (Dulbecco's Modified Eagle's Medium) medium containing 10% bovine calf serum (BCS) and passaged every 3 days.

&Lt; Example 1 >

Induction of differentiation of adipocytes into adipocytes using media composition of the present invention

First, in order to differentiate the adipocytes into adipocytes, a media composition of the present invention was prepared as shown in Table 1 below.

Composition of the Medium Composition of the Present Invention Composition content Insulin 1 µg / ml Dehydroepiandrosterone (DHEA) 1 uM Histamine 7 uM Indomethacin 0.2mM Biotin 33uM Penthothenate 17 uM

Differentiation of adipocytes cultured in <Reference Example 1> into adipocytes was induced using the medium composition of the present invention prepared as described above. In other words, differentiation was induced when the distribution of fat bulb over 95% of the medium area was maintained, and the carbon dioxide concentration was maintained at 5.0 ± 0.5% at 37 ℃. Two days after the differentiation was induced, a medium containing only 1 μg / ml of insulin, Biotin, Indomethacin, and Pentothenate was added once every two days.

Meanwhile, in order to induce differentiation of adipocytes into adipocytes as a control group, a media composition commonly used ( The Journal of Biological chemistry 2001, 276 (30); 28430-28435) was prepared as shown in Table 2 below.

Composition of the control group Composition content Insulin 1ug / ml Dexamethasone 1 uM Isobutylmethylxanthine 0.5mM Indomethacin 0.2mM Biotin 33uM Penthothenate 17 uM

Differentiation of adipocytes cultured in <Reference Example 1> into adipocytes under the same conditions as above was induced using the control prepared as described above. Two days after the differentiation was induced, a medium containing only 1 μg / ml of insulin, Biotin, Indomethacin, and Pentothenate was added once every two days.

Experimental Example 1

Effect of the media composition of the present invention on adipocyte differentiation

<1-1> Formation of Adipocyte Differentiation

In order to observe the morphology of the differentiated adipocytes in the media composition of the present invention, after removing the culture medium in the dish containing the differentiated adipocytes of <Example 1>, and washed with PBS (phosphate-buffered saline) The solution was fixed for 1 hour by administering a 10% formaldehyde solution, and then the solution was removed and mixed with an oil red o solution (100 mg isopropanol in 5 mg / ml of oil red o reagent and distilled water 3: 2). Solution) for about 10 minutes. After the staining, washing with water and observing the adipocyte differentiation form using a microscope compared to the control and the results are shown in FIG.

As shown in FIG. 1, in the media composition of the present invention, the differentiation of adipocytes into adipocytes was higher than that of the control group, and the distribution of differentiated adipocytes was uniformly and stably fixed.

<1-2> fat cell survival rate measurement

In order to measure the viability of the differentiated adipocytes in the media composition of the present invention, 40ul of alamar blue solution per 100ul of the medium containing the differentiated adipocytes of <Example 1> was administered at 37 ° C. After reacting for a time, the fluorescence value was measured using a fluorescence measuring device (excitation 544 nm, emission 590 nm) and the results are shown in FIG. 2.

As shown in FIG. 2, it was found that the survival rate of the differentiated adipocytes in the medium composition of the present invention was significantly higher than that of the control group during all measurement periods.

<1-3> glucose absorption amount measurement

Since adipocytes absorb glucose and convert it to fat, it can be seen that the greater the amount of glucose absorption, the more fat cells are differentiated.

First, to determine the amount of glucose absorbed directly into adipocytes, 0.1 mM 2-NBDG (2- (N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino) -2 -deoxyglucose) sample was treated in medium containing differentiated adipocytes of <Example 1> and left at 37 ° C for 1 hour, followed by trypsin treatment to separate cells and FACS (Fluorescence Activated Cell Sorter) The fluorescence value was measured and the results are shown in FIG. 3 in comparison with the control group.

On the other hand, the amount of glucose remaining in the medium was measured to indirectly measure the glucose uptake of adipocytes. Specifically, the medium containing the differentiated adipocytes of Example 1 was diluted to 1/20, and the cells were divided into 96-well plates, and the glucose oxidase / peroxidase solution was used at room temperature. After reacting for 5 minutes, the absorbance was measured at 540 nm and the results are shown in FIG. 4 in comparison with the control group.

3 and 4, in the media composition of the present invention, the glucose uptake was much higher than that of the control group.

<1-4> fat production amount measurement

Since adipocytes absorb glucose and convert it into fat, the degree of adipocyte differentiation can be determined by measuring the amount of fat produced.

In order to measure the amount of fat produced, the adipocytes stained in Experimental Example 1-1 were dissolved using 100% isopropanol, and absorbance was measured at 490 nm. The results are shown in FIG. 5 compared with the control group.

As described in FIG. 5, the amount of fat produced in the medium composition of the present invention was higher than that of the control group, and the initial amount was particularly high. Therefore, it can be seen that the adipocyte differentiation is better induced in the media composition of the present invention than in the control group.

The adipocyte differentiation medium composition of the present invention has the advantage of maximizing the survival rate by allowing the distribution of differentiated adipocytes evenly and stably adhering the differentiated adipocytes compared to the adipocyte differentiation medium compositions used in general. have. In addition, DHEA (dehydroepiandrosterone) and histamine (histamine) used in the adipocyte differentiation medium composition of the present invention is economically advantageous because it is cheaper than dexamethasone and isobutylmethylxanthine.

1 is a photograph showing the morphology of adipocytes differentiated in the media composition of the present invention through oil red o staining (DIM: when differentiated in the control group, DIH: when differentiated in the media composition of the present invention) .

Figure 2 is a graph showing the survival rate of the adipocytes differentiated in the media composition of the present invention (FBS: fetal bovine serum, DIM: when differentiated in the control, DIH: when differentiated in the media composition of the present invention).

3 is a graph showing the amount of glucose directly absorbed by the adipocytes in the media composition of the present invention (DIM: when differentiated in the control group, DIH: when differentiated in the media composition of the present invention).

Figure 4 is a graph showing the amount of glucose remaining in the medium in the medium composition of the present invention, indirectly measured the absorption of glucose of adipocytes (FBS: fetal calf serum, DIM: DIH: When differentiated in the media composition of the present invention).

5 is a graph showing the absorbance of the amount of fat produced in the media composition of the present invention (FBS: fetal calf serum, DIM: when differentiated in the control, DIH: when differentiated in the medium composition of the present invention).

Claims (4)

An adipocyte differentiation medium composition, the adipocyte differentiation medium composition comprising insulin (insulin), DHEA (dehydroepiandrosterone) and histamine (histamine). The medium composition of claim 1, further comprising indomethacin, biotin and penthothenate in the medium composition. According to claim 2, The insulin (insulin) 0.1 ~ 10ug / ㎖, DHEA (dehydroepiandrosterone) is 0.01 ~ 10uM, histamine (histamine) 1 ~ 10uM, indomethacin (indomethacin) 0.1 ~ 10mM, biotin (biotin) A medium composition, characterized in that 20 ~ 40uM and pentothenate (panthothenate) contains 10 ~ 30uM. A method of differentiating adipocytes (adipocytes) into adipocytes using the media composition of claim 1.

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