KR20120089915A - Composition comprising curcumin for improving stem cell potency in undifferentiated adipocytes - Google Patents

Abstract

PURPOSE: A culture composition containing curcumin for enhancing stemness of undifferentiated adipocytes is provided to enhance telomerase activity and to enhance the expression of stemness acting signals. CONSTITUTION: A culture composition for enhancing stemness of undifferentiated adipocytes contains 0.01-1uM of curcumin. The undifferentiated adipocytes are isolated from mammal and are mouse-derived 3T3-L1 fibroblast. A method for enhancing stemness of undifferentiated adipocytes comprises a step of treating curcumin to the undifferentiated adipocytes and a step of culturing the undifferentiated adipoyctes. A composition for cell therapy contains the undifferentiated adipocytes as an active ingredient.

Description

Composition composition curcumin for improving stem cell potency in undifferentiated adipocytes}

The present invention provides a culture composition for enhancing stem cell ability of undifferentiated adipocytes containing curcumin, a method for enhancing stem cell ability by treating curcumin with undifferentiated adipocytes, and a cell having enhanced stem cell capability obtained by the method and the same It relates to a cell therapy composition comprising.

Stem cells have self renewal and proliferative ability to reproduce themselves, and have pluripotency to differentiate into one or more specialized cells under appropriate circumstances. In addition, human embryonic stem cells show the possibility of treating a variety of existing diseases in that they have the ability to differentiate into all kinds of human cells. Since the first human embryonic stem cell derived from fertilized eggs by Dr. Thomson's team in 1998 in the world, hundreds of human embryonic stem cell lines have been established worldwide, and new embryonic stem cell lines derived from new embryos are still being established. Human embryonic stem cell research is currently being actively conducted such as supporting cell research, establishment of culture conditions excluding animal-derived factors, and differentiation technology into specific cells.

However, until now, it is true that the clinical approach is difficult due to the limitations of embryonic stem cells. When used for therapeutic purposes, the immune rejection of stem cells is problematic, and there is a problem that it is difficult to avoid the bioethics controversy caused by embryo destruction. Currently, research on customized stem cells by nuclear transplantation has been conducted to solve the immune rejection problem, but it has not been successful yet. As an alternative to the ethical problem of egg acquisition, embryonic stem cells by dedifferentiating already differentiated somatic cells The research to produce induced pluripotent cells (iPS), which are similar to the cells, is emerging as a global trend. Such iPS not only can accurately replace the use of cloned embryonic stem cells with somatic cells, but is also an area of great interest in the global stem cell field regardless of the bioethics debate. In 2006, Dr. Sinya Yamanaka of Japan produced iPS from somatic cells using four gene combinations, oct4, sox2, klf4, and c-Myc, which are specifically expressed in mouse embryonic stem cells. James Thomson of the United States has reported that other gene combinations, such as Oct4, Sox2, Nanog and Lin28, can reverse human somatic cells.

Despite many studies on the applicability of such stem cells to treatment, there are not many treatments currently available due to difficulties in controlling the differentiation process of embryonic stem cells. However, the possibility of cell therapy using stem cells can be effective in repairing or replacing damaged organs such as kidney, liver and heart, and in treating degenerative diseases such as diabetes, Parkinson's disease, Alzheimer's disease, and diabetes. In terms of alternatives, the cellular approach is still expected to revolutionize the medical field. In particular, it would be desirable to develop cell reprogramming techniques with low ethical issues and low risk of immune rejection, to make stem cells upside down from various cells, and to treat cells using these stem cells. To this end, it is necessary to develop a new, safe stem cell-promoting substance that can promote or promote stem cell potency or stem cellity of a cell, while not causing cancer to the cell.

On the other hand, curcumin (diferuloylmethane) is Turmeric (Curcuma longa ) is a natural polyphenol derived from a plant known as longa ) and has been used for the treatment of diseases associated with inflammation and oxidative stress. Because of its ability to inhibit inflammation and remove free radicals, curcumin has been studied for cancer chemical prophylaxis and cancer growth inhibition, and has been shown to stop cell proliferation, including cell necrosis and cell death in various cancer cells, and to kill cells. It has been recognized as a promising anticancer agent because of its efficient induction. Curcumin is also a naturally occurring antioxidant and, under certain conditions, affects histone low acetylation, demonstrating its antioxidant properties. Curcumin exerts anti-cancer effects in human leukemia cells by eliminating free radicals at low concentrations, which have various pharmacological effects including apoptosis, anti-inflammatory and anti-cancer, and also protect against apoptosis of tumor-induced T cells and, in In vitro experimental evidence and clinical trials have been shown to be useful in preventing disease of human colon cancer. In addition, curcumin has been reported to reduce cognitive decline and oxidative damage associated with aging, and animal studies have shown that curcumin is effective in neurodegenerative conditions such as Alzheimer's and regional cerebral ischemia. As such, curcumin has received commercial attention as a useful pharmaceutical agent. However, it is not yet disclosed whether curcumin has an effect of enhancing stem cell potency or stem cellity in undifferentiated adipocytes.

Accordingly, the present inventors have treated curcumin in undifferentiated adipocytes, which resulted in increased cell proliferation, increased telomerase activity, increased stem cell activity (stemness acting signals), and cell migration activity. This increase was confirmed that curcumin has an effect of enhancing stem cell ability, and came to complete the present invention.

One object of the present invention is to provide a culture composition for enhancing stem cell capacity of undifferentiated adipocytes, including curcumin.

Another object of the present invention is to provide a method for enhancing the stem cell capacity of undifferentiated adipocytes comprising the step of culturing curcumin in undifferentiated adipocytes.

Yet another object of the present invention is to provide a stem cell-enhanced cell obtained by the method and a cell therapeutic composition comprising the same.

As one aspect for achieving the above object, the present invention relates to a culture composition for enhancing stem cell capacity of undifferentiated adipocytes containing curcumin.

In the present invention, "undifferentiated adipocyte", which is a cell to be enhanced in stem cell ability, refers to a cell committed to differentiate into adipocytes or a cell not yet differentiated. The undifferentiated fat cells of the present invention are human, monkey, pigs, horses, cows, sheep, dogs, cats, mice ), But may be derived from mammals such as rabbits, but preferably the undifferentiated adipocytes may be derived from the mammal's embryo. As an example of the above undifferentiated adipocytes, 3T3-L1 fibroblasts derived from a mouse can be mentioned. The 3T3-L1 fibroblasts in which is derived from the times of the mouse, and the synthesis and secretion of collagen and hyaluronic ruro I, and used for the insulin path, obesity, study of cardiovascular disease Adipose cell model in vitro .

As used herein, the term "enhancement of stem cell potency" refers to an increase in proliferation, an increase in telomerase activity, or stem cell acting signals while maintaining undifferentiated adipocytes. Increase expression or increase cell migration activity, including the appearance of one or more of these features. The stem cell ability is a term familiar to the art, for example, Pittenger, MF, Mackay, AM, Beck, SC, Jaiswal, RK, Douglas, R., Mosca, JD, Moorman, Simonetti, DW, Craig, S ., and Marshak, DR 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284 (5411), 143-147.

As used herein, the term "proliferation" means an increase in the number of cells and is used in the same sense as "growth". In the present invention, the term "undifferentiated proliferation" means that the undifferentiated adipocytes proliferate into cells having the same properties as the original cells without being differentiated into adipocytes. Differentiated states can be clearly distinguished in the usual way: for example, undifferentiated cells have a high (nucleus ratio / cytoplasmic ratio) value and are characterized by prominent nucleoli.

In the present invention, the enhancement effect of the stem cell ability of the undifferentiated adipocytes is represented by curcumin.

The term "curcumin (curcumin)" in the present invention has one of methane (diferuloylmethane) di ferrule of ingredient, turmeric (Curcuma longa ) means natural polyphenols derived from plants.

The present inventors confirmed that the cells enhance the stem cell ability when culturing undifferentiated adipocytes by incorporating the curcumin into the medium, and confirmed the effect of enhancing the stem cell ability as follows.

When culturing undifferentiated adipocytes in a media composition comprising curcumin of the present invention, it is possible to enhance cell proliferation of undifferentiated adipocytes. In a specific embodiment of the present invention, in order to examine the potential of curcumin in relation to cell growth, the proliferation of 3T3-L1 fibroblasts treated with various concentrations of curcumin for 24 hours was investigated. Proliferation was increased, while treatment at high concentrations was found to reduce cell proliferation (see FIG. 1A). In addition, it was found that treatment of 0.02 μM curcumin for 24 hours was the most effective condition for stimulating cell proliferation (see FIGS. 1B and 1C). In addition, as a result of the colonization assay to evaluate the cell proliferation efficiency of curcumin-treated 3T3-L1 fibroblasts, it was found that the curcumin-treated cells had about 1.5-fold increase in colony formation compared to the control cells. (See FIG. 1D).

In the present invention, the telomerase activity may be increased when curcumin is cultured by treating undifferentiated adipocytes. In the present invention, the term "telomerase" is a function of protecting the chromosome by being present at the end rather than carrying special information on the chromosome and maintaining and protecting the length of telomeres, which are shortened every time cells divide. It is an enzyme. These telomerases are expressed in cancer cells and stem cells, which can cause stem cells to divide more than somatic cells. In a specific embodiment of the present invention, in order to determine the effect of curcumin on the elimination of ROS in 3T3-L1 fibroblasts, the telomerase activity was measured after curcumin treatment. About 1.6-fold increased telomerase enzyme activity (see FIG. 2).

Expression of the stem cell factor may be induced when cultured in a medium composition comprising curcumin of the present invention. As used herein, the term "stemness acting signals" refers to those expressed to maintain stem cell characteristics, but is not limited thereto, and preferably refers to oct4, sox2, klf4, or c-Myc. . According to a specific embodiment of the present invention, induction of significant activation of PI3K and its sub-mediators (p-MEK, p-ERK, p-AKT) in curcumin-treated 3T3-L1 fibroblasts, p53 and p21 Inhibition of the expression of the cancer suppressor gene product and the over-expression of c-Myc protein was induced, the activation of p-ERK and p-AKT induced by the curcumin is followed by stem cells such as oct4, sox2, klf4, c-Myc It was confirmed that inducing the expression of transcription factors (see Figure 3). From these results, it can be seen that curcumin acts to increase cell proliferation and expression of stem cell factors in 3T3-L1 fibroblasts.

In addition, when cultured in a culture composition comprising the curcumin of the present invention, it is possible to increase the cell migration activity of undifferentiated adipocytes. In a specific embodiment of the present invention, to evaluate the cell migration effect of curcumin treated 3T3-L1 fibroblasts, in In vitro transwell plates and scratch-inducing cell migration assays showed that curcumin-treated cells significantly increased cell migration efficiency over time compared to control cells. The results were consistent with the overexpression of cell migration transcription factors, including MMP1, MMP2, MMP3, SDF1, and VEGF, mediated by inhibition of expression of TIMP1, TIMP2 and TSP1 after curcumin treatment (see FIG. 4).

As described above, cells with enhanced stem cell ability are not differentiated into adipocytes, but are enhanced to stem cell capacity, that is, into a state capable of transferring or programming to a plurality of types of cells. It has the potential to regression. In addition, in the present invention, it is also meant that cells with enhanced stem cell capacity may be changed to cells with increased pluripotency. Therefore, the cells with enhanced stem cell ability may be capable of differentiation into other cells, including adipocytes, under appropriate conditions. For example, differentiation into other cells means that they can be differentiated into mesenchymal-derived cells, including neurons, chondrocytes and muscle cells, neurons, and insulin producing cells (B cells of Israngelhans Island). Accordingly, the differentiated cells may be used to treat various diseases such as cancer, osteoporosis, arthritis, neurodegenerative diseases and diabetes, but are not limited thereto.

In the present invention, the term "culture media" is in Means a medium that can support cell growth and survival in vitro , and includes all conventional media used in the art suitable for the culture of cells. Depending on the type of cells, medium and culture conditions can be selected. The medium used for the culturing is preferably a cell culture minimum medium (CCMM), and generally includes a carbon source, a nitrogen source and a trace element component. Such cell culture minimal media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI1640, F-10, F-12, α Minimal Essential Medium (GMEM) (Glasgow's Minimal essential Medium) and Iscove's Modified Dulbecco's Medium, but are not limited to these. In addition, the medium may include antibiotics such as penicillin, streptomycin, gentamicin, and the like.

Curcumin may be used to include curcumin in the medium for the purpose of the present invention to promote or enhance the stem cell ability of undifferentiated adipocytes, without particular limitation on the type and culture method of the medium. At this time, various applications are possible, such as using curcumin alone or in combination with one or more differentiation control substances known in the art.

Curcumin of the present invention should be included in the medium at an effective concentration. In the present invention, the term "effective concentration" refers to a sufficient amount of curcumin to induce stem cell capacity, and does not exhibit activity below the effective concentration and toxic to the cells above the effective concentration, so curcumin within the effective concentration Use it. Curcumin is preferably treated at a concentration lower than 10 μM, more preferably 0.01 to 1 μM, and at higher concentrations (50 μM), it is possible to reduce stem cell capacity, so it is treated at a concentration lower than 50 μM. Preferably (see FIG. 1).

In another aspect, the present invention relates to a method for enhancing stem cell ability of undifferentiated adipocytes, comprising the step of culturing curcumin to undifferentiated adipocytes.

The enhancement of stem cell ability is selected from the group consisting of increased cell proliferation, increased telomerase activity, increased expression of stem cell acting signals, and increased cell migration activity, as described above. As described above (see FIGS. 1 to 4). In addition, the curcumin is preferably treated with 0.01 to 1 μM for 12 to 24 hours. In addition, the undifferentiated adipocytes are preferably isolated from a mammal, more preferably 3T3-L1 fibroblasts derived from a mouse.

In another aspect, the present invention relates to a cell-enhanced composition obtained by the method for enhancing stem cell ability as described above and a composition for treating cells of animals comprising the same as an active ingredient.

The disease itself can be treated by using the cells of the present invention having enhanced stem cell ability. The cells can be differentiated into the specific types of cells by injection, infusion, implantation, and implantation into the animal in direct contact with the specific type of cell population. Therefore, the disease can be treated by applying the stem cell ability-enhanced cells themselves to the desired type of tissue, and the types of diseases that can be treated are not limited.

Methods of producing tissue using cells that can differentiate into various types of cells ("tissue engineering") are known in the art. Wang X. et al. Have shown that certain adult pancreatic cells can be converted to hepatocytes in fumaroy-laceto-acetate hydrolase (FAH) deficient mice (Wang X. et al. "Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells "Am. J. Pathol. 158 (2): 571-579). Lagasse et al. Have shown that hematopoietic stem cells from bone marrow can be converted to hepatocytes after in vivo transplantation into FAH deficient mice (Lagasse et al. "Purified hematopoietic stem cells can differentiate into hepatocytes in vivo" Nature Medicine, 6 (11)). 1229-1234).

Stem cell capacity-enhanced cells of the present invention is preferably in the form of a cell composition comprising one or more diluents that protect and maintain the cells and facilitate their use in injection, infusion, and implantation into the tissue of interest. The diluent may include physiological saline, PBS (Phosphate Buffered Saline), buffer solution such as HBSS (Hank's balanced salt solution), plasma or blood components.

Cells with enhanced stem cell capacity of the present invention may be differentiated into cells of the desired type or stored in the medium for several days. In the latter case, it is preferable to add cytokines or LIF (leukemia inhibitory factor) to the medium to prevent loss of multipotency. In addition, by lyophilizing and storing the cells can maintain the multipotent ability.

The stem cell-enhanced cells may be differentiated into mesenchymal-derived cells including bone cells, chondrocytes, and muscle cells, nerve cells, adipocytes, and insulin producing cells (B cells of Israngelhans Island). Accordingly, the differentiated cells may be used to treat various diseases such as cancer, osteoporosis, arthritis, neurodegenerative diseases and diabetes, but are not limited thereto.

The differentiation can be confirmed as follows as an example. Differentiation into bone cells, chondrocytes and muscle cells can be confirmed by a method using a dye that is specifically stained for each cell. Differentiation into bone cells and adipocytes can be confirmed by measuring the degree of formation of bone nodules and lipids. In addition, differentiation into osteocytes is expressed by osteonectin, RXR, osteopontin, differentiation into adipocytes by AP and PPAR-γ, and differentiation into neurons by Tuj, GFAP, MAP2ab, and NF160. Differentiation into insulin producing cells can be confirmed by analyzing the expression level of insulin, respectively.

As described in detail above, incubation with curcumin in undifferentiated adipocytes increases the proliferation of the cells, increases the activity of telomerase, increases the expression of stem acting signals, Curcumin has an effect of enhancing stem cell ability, such as increased cell migration activity. As such, cells that have enhanced stem cell ability using curcumin can be used for stem cell research and further differentiated into various cells by treatment under appropriate conditions. Cancer, osteoporosis, arthritis, and degenerative nerves can be differentiated using such differentiated cells. It can be used to treat various diseases such as diseases and diabetes.

Figure 1 shows the effect of stimulating the active cell proliferation in 3T3-L1 fibroblasts of curcumin. (A): Cell proliferation of 3T3-L1 fibroblasts treated for 24 hours at various concentrations was assessed by trypan blue exclusion. Low concentrations (0.01, 0.02, 0.1 and 1 μM) of curcumin increased cell proliferation, whereas high concentrations (greater than 50 μM) decreased cell proliferation. (B): The cell proliferation effect was evaluated for 72 hours at low concentration. Administration of 0.02 μM curcumin for 24 hours was most effective in stimulating cell proliferation. (C): Phase increase photographs were taken of control cells with increased cell proliferation of curcumin treated cells. (D): The results of the colonization assay indicate that curcumin actively stimulated cell proliferation of 3T3-L1 fibroblasts. Data of the present invention were analyzed using Fisher's test or t-test (* P <0.05, ** P <0.01).
FIG. 2 shows the effect of inducing extended cell growth characteristics such as increased telomerase activity in curcumin 3T3-L1 fibroblasts. Curcumin actively increased telomerase activity in association with overexpression of telomerase related transcription factors such as TR, TERT, and TEP1. Data of the present invention were analyzed using Fisher's test or t-test (* P <0.05, ** P <0.01).
Figure 3 shows that curcumin induces the active expression of cell proliferation-related signal proteins, several functional factors, and stem cell factors in 3T3-L1 fibroblasts. (A): Curcumin induced PI3K activation in 3T3-L1 fibroblasts and significant activation of downstream mediators (p-MEK, p-ERK, and p-AKT). Curcumin has a significant effect on the overexpression of various cell proliferation-related transcription factors, including SP1, CDK1, and CDK2. (B): Curcumin directly attenuated the levels of apoptosis related proteins, p38 and p-SAPK / JNK. Curcumin also induced the inhibition of expression of p53 and p21 cancer suppressor gene products. (C): Curcumin is mediated by overexpression of stem cell genes (oct4, sox2, klf4, c-Myc) and inhibition of expression of cancer suppressor genes of p53 and p21, resulting in cell proliferation-related functional genes (REX1, SP1, Active expression of CDK1, and CDK2).
4 shows that curcumin induces active cell migration in 3T3-L1 fibroblasts. Curcumin to evaluate the efficacy of the treated cell migration 3T3-L1, was carried out in vitro trans-well culture plate and scratch-inducing cell migration assays. (A): Transwell plate analysis showing that curcumin significantly increased the cell migration efficacy of 3T3-L1 fibroblasts in time dependent compared to control cells. (B): To obtain clearer evidence of vigorous cell migration in curcumin treated cells, a simple cell scraped wound model assay was performed. Pictures of cells moving beyond the designated baseline were taken with a phase contrast microscope. Curcumin treated cells actively crossed the baseline compared to control cells. (C) Curcumin induced vigorous expression of cell migration related transcription factors. Data of the present invention were analyzed using Fisher's test or t-test (* P <0.05, ** P <0.01).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Materials and methods

Cell culture Curcumin  process

Mouse 3T3-L1 fibroblasts (American Type Culture Collection, Rockville, MD, USA) were moistened with 5% CO2 using Dulbecco's modified Eagle medium (DMEM) medium containing 10% bovine serum and 1% penicillin / streptomycin. Incubated in air at 37 ° C. Curcumin (Sigma Chemical Co., St Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) and stored at minus 20 ℃. For curcumin treatment, the cultured 3T3-L1 cells were laid at a density of 5 × 10 ^{5 in a} 10 cm culture dish and stabilized for 24 hours in DMEM with 5% bovine serum added. Cells were then treated with various concentrations of curcumin in serum-free DMEM medium for the indicated times (24 and 72 hours). After treatment, the optimal concentration of curcumin was selected based on cell viability via trypan blue exclusion assay.

Trypan blue  Pigment Exclusion

3T3-L1 fibroblasts were incubated for 24 hours with DMEM containing 5% bovine serum of 7 X 10 ⁴ cells per well in a 6 well culture dish. The medium was then replaced with serum free DMEM and treated with curcumin concentrations specified at 24 and 72 hours. After treatment, cells were harvested and viable cells containing 0.4% Trypan Blue (Gibco) were counted using an erythrocyte counter. In all cell viability assays, triplet wells were used and each experiment was repeated at least three times.

Colony Cell Analysis ( Colony - forming cell  ( CFU ) assay )

3T3-L1 fibroblasts were cultured in a 10 cm culture dish at a density of 3.5 X 10 ³ and stabilized in DMEM containing 5% bovine serum for 24 hours. After stimulating cells with 0.02M curcumin for an additional 24 hours in serum-free DMEM, the medium was replaced with medium containing 10% bovine serum. After 15 days thereafter, cells were fixed with 4% paraformaldehyde (PFA) and stained with 0.1% toluidine blue in 1% PFA, and cell proliferation efficiency was then determined by counting visible colonies. It was.

Telomerase  Activity analysis ( Telomerase Activity Assay )

3T3-L1 fibroblasts were incubated with 5 × 10 ⁵ cells per 10 cm culture dish and stabilized with DMEM containing 5% bovine serum for 24 hours. The cells were then treated with 0.02 μM curcumin for 24 hours in serum free DMEM. Curcumin stimulated cells were harvested and telomerase activity was measured using the telomerase PCR ELISA kit (Roche Diagnostics) according to the manufacturer's instructions.

Cell migration assay ( Cell Migration Assay )

To assess cell migration of curcumin-treated 3T3-L1 fibroblasts, 5 × 10 ⁵ cells were placed in a 10 cm culture dish and cultured with DMEM containing 5% bovine serum for 24 hours in a 37 ° C. carbon dioxide incubator. It was. The cells were further treated with 0.02 μM curcumin for 24 hours. The cultured cells were then passed through Costar transwell membranes and placed in 6 well culture dishes. Below the transwell membrane, curcumin was added to each well with DMEM containing 2% bovine serum. In the upper space cells were incubated for 2 hours at 37 ° C. in DMEM containing 2% bovine serum, and the culture dish was incubated overnight at 37 ° C. in a carbon dioxide incubator. Cells in the subspace were dried in air, cross stained with Harris hematoxylin for about 20 minutes, and then washed. Stained inserts were placed on object slides, and the number of cells on the bottom surface was evaluated 200 times with an inverted bright-field microscope. Each insert was numbered in 10 X 20 fields. Cell migration was expressed as the number of cells per field of spontaneous migration towards the cell bottom. To obtain clear evidence on the role of curcumin in 3T3-L1 fibroblast migration, a simple cell scraped wound model assay was performed. Cells were cultured in DMEM containing the place in 10 cm cell culture dishes to a density ^{of 5} X10 5, 5% BCS. After 24 hours of stabilization, cells drew a straight line across the culture dish and went through three washes with medium. Cells were then incubated with serum free DMEM with 0.02 μM curcumin. Cells traveling to the wounded region were photographed with a 40x microscope.

RNA  Extraction and Reverse transcription PCR

3T3-L1 fibroblasts were treated with curcumin 0.02 μM as described above, recovered, and total RNA was extracted according to the manufacturer's instructions (RNeasy kit, Qiagen, La Jolla, Calif.). The extracted total RNA was subjected to random hexamer by AMV reverse transcriptase (Amersham Corp., Arlington Heights, IL) for complementary DNA (complementary DNA) synthesis. Then, PCR was performed using a primer concentration of 20 pM using a Mastercycler (Eppendorf, Hamburg, Germany) machine. PCR conditions were 1 X (3 minutes at 94 ° C.), 35 X (45 seconds at 94 ° C .; 45 seconds at 56.5 ° C .; and 1 minute at 72 ° C.), and 1 X (10 minutes at 72 ° C.). Was. The amplified PCR product was subjected to electrophoresis on a 1% agarose gel containing 6x gel loading dye (Bioneer) containing EthiBr (ethidium bromide).

Protein extraction and Western Blot  analysis

After treating 3T3-L1 fibroblasts with 0.02 μM curcumin as above, the cells were harvested and lysed, and the protein concentration was determined using the BioRad protein assay (BioRad Lab., Hercules, Calif.). Was quantified. For Western blot analysis, the same amount of protein was placed on an SDS-polyacrylamide gel and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) by electroblotting. Blots were probed with species specific antibodies and incubated overnight at 4 ° C. After 1 hour incubation with diluted Enzyme Link secondary antibody, blots were shown by ECL according to manufacturer's instructions (Amersham). Primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.) And Cell Signaling technology. Anti-rabbit and anti-mouse immune antibodies (immunoglobulins) were purchased from Amersham.

Statistical analysis

All data were obtained from at least five independent experiments and expressed as mean ± standard deviation. Statistical significance between groups was calculated using student's two tailed t-test.

result

One. Curcumin  3 T3 - L1  In fibroblasts Increased Telomerase  Cell proliferation and stem cell gene expression were induced with activity.

To examine the curcumin potential for cell growth, the proliferation of 3T3-L1 fibroblasts treated with various concentrations of curcumin for 24 hours was assessed by trypan blue exclusion. As shown in FIG. 1A, after 24 hours of curcumin treatment, low concentrations (0.01, 0.02, 0.1 and 1 μM) of curcumin increased cell proliferation, whereas high concentrations (50 μM) decreased cell proliferation. Of the concentrations tested, 0.02 μM curcumin was a magnetically effective concentration that stimulated cell proliferation. The proliferative effect of the low concentration of curcumin treated and evaluated for 72 hours showed a better effect on stimulating cell proliferation when treated for 24 hours (FIG. 1B). It was confirmed that the cell proliferation of curcumin-treated cells was very active by observing an increase in the density of cells under a phase contrast microscope of 50 times in FIG. 1C. Therefore, 24 hours treatment with 0.02 μM curcumin appeared to be the optimal treatment condition. Clonogenic assays were also performed to assess cell proliferation efficiency of curcumin treated 3T3-L1 fibroblasts. In the colonization assay, 0.02 uM curcumin treated cells increased about 1.5-fold in colonization compared to control cells (FIG. 1D). To determine the effect of curcumin on the elimination of ROS in 3T3-L1 fibroblasts, telomerase activity was measured 24 hours after curcumin 0.02 μM treatment (FIG. 2). As shown in Figure 2, curcumin treated cells showed about 1.6-fold increase in telomerase enzyme activity.

Expression of gene markers in both control and curcumin treated cells was examined using Western blot and RT-PCR analysis. As shown in FIG. 3, we induced significant activation of PI3K and its sub-mediators (p-MEK, p-ERK, p-AKT) in 3T3-L1 fibroblasts by 0.02 μM curcumin (FIG. 3A). Curcumin, on the other hand, directly attenuated the levels of apoptosis related proteins, p38 and p-SAPK / JNK (FIG. 3B). These results clearly show that curcumin induced 3T3-L1 fibroblast proliferation through the activation of PI3K and MEK signaling pathways with direct inhibition of p38 and p-SAPK / JNK. Curcumin exerted a dominant effect on overexpression of cell proliferation related transcription factors, including REX1, SP1, CDK1, and CDK2 (FIGS. 3A and 3C). Curcumin also induced the inhibition of the expression of p53 and p21 cancer suppressor gene products and overexpression of c-Myc protein (FIG. 3). Activation of p-ERK and p-AKT induced by curcumin in 3T3-L1 fibroblasts then induced expression of stem cell transcription factors such as oct4, sox2, klf4, c-Myc (FIG. 3C). These results indicate that curcumin can induce active cell proliferation and stem cell signals with increased telomerase activity in 3T3-L1 fibroblasts.

2. Curcumin  3 T3 - L1  Induces active cell migration in fibroblasts.

In order to evaluate the cell migration effect of curcumin treated 3T3-L1 fibroblasts, in vitro transwell plates and scratch-inducing cell migration assays were performed. As shown in FIG. 4A, 0.02 μM curcumin treatment significantly increased cell migration efficiency in time dependent manner compared to control cells. To get clearer evidence of vigorous cell migration in curcumin treated cells, a simple cell scratch wound model assay was used, and both curcumin treated cells and controls were scratched to one side from the designated reference line. Then the movement of the cell itself was observed. Photographs of cells above the designated reference line were taken with a phase contrast microscope. As shown in FIG. 4B, curcumin treatment induced active crossing of the baseline compared to the control, which was found to be about 2.5-fold increase compared to the control. These results are consistent with the overexpression of cell migration transcription factors, including MMP1, MMP2, MMP3, SDF1, and VEGF, mediated by inhibition of expression of TIMP1, TIMP2 and TSP1 after curcumin treatment (see Figure 4C).

Claims (14)

Containing curcumin, culture composition for enhancing stem cell capacity of undifferentiated adipocytes. The composition of claim 1, wherein the undifferentiated adipocytes are isolated from a mammal. The composition of claim 2, wherein the undifferentiated adipocytes are 3T3-L1 fibroblasts derived from mouse. The method of claim 1, wherein the enhancement of stem cell capacity is selected from the group consisting of increased cell proliferation, increased telomerase activity, increased expression of stem cell acting signals, and increased cell migration activity. The composition represented by the feature. The composition of claim 1, wherein the curcumin is included in a concentration of 0.01 to 1 μM. The composition of claim 4, wherein the stem cell factor is selected from the group consisting of oct4, sox2, klf4, and c-Myc. Comprising a step of culturing curcumin to undifferentiated adipocytes, a method for enhancing the stem cell capacity of undifferentiated adipocytes. 8. The method of claim 7, wherein said undifferentiated adipocytes are isolated from a mammal. The method of claim 8, wherein the undifferentiated adipocytes are 3T3-L1 fibroblasts derived from mouse. 8. The method of claim 7, wherein the enhancement of stem cell capacity is selected from the group consisting of increased cell proliferation, increased telomerase activity, increased expression of stem cell acting signals, and increased cell migration activity. The method represented by the feature. The method of claim 7, wherein the curcumin is treated with 0.01 to 1 μM for 12 to 72 hours. The method of claim 10, wherein said stem cell factor is selected from the group consisting of oct4, sox2, klf4, and c-Myc. A cell having enhanced stem cell capacity obtained through the method according to any one of claims 7 to 12. Cell therapy composition comprising the cell according to claim 13 as an active ingredient.

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