KR102547244B1 - Composition for Inducing Differentiation of Stem Cells into Chondrocytes Comprising the Extract Of Cumin as an Effective Ingredient - Google Patents

Abstract

The present invention relates to a composition for inducing differentiation of stem cells into chondrocytes containing a cumin extract as an active ingredient, and a method for inducing differentiation of stem cells into chondrocytes, comprising treating the separated stem cells with the cumin extract. , The composition for inducing the differentiation of stem cells into chondrocytes containing the cumin extract according to the present invention as an active ingredient has a remarkable effect of inducing the promotion of stem cells into chondrocytes, which is useful when trying to induce stem cells into chondrocytes. There is an advantage.
In addition, the composition for inducing the differentiation of stem cells into chondrocytes containing the cumin extract according to the present invention as an active ingredient can be used as a preventive and therapeutic agent for bone defects by inducing the differentiation of stem cells into chondrocytes. There is an advantage to being

Description

Composition for Inducing Differentiation of Stem Cells into Chondrocytes Comprising the Extract Of Cumin as an Effective Ingredient}

The present invention relates to a composition for inducing differentiation of stem cells into chondrocytes containing a cumin extract as an active ingredient and a method for differentiating stem cells into chondrocytes.

Cumin ( Cuminum cyminum L.) has been used since ancient times to treat various indications in traditional healing systems over a large geographic area (Johri, RK, 2011, Pharmacogn Rev 5: 63-72). Cumin is a rich source of essential oil and has been actively studied for its chemical composition and biological activity. Cumin has been applied to the treatment of various diseases (Kermani, M., et al., 2012. Chin J Integr Med 2012). Cumin is known to be effective against candida infection (Katiraee, F., et al., 2017. Curr Med Mycol 3: 1-6). Cumin has been extracted and used with various solvents such as water, methanol, ethanol and ethyl acetate. For example, a water-soluble extract of cumin was applied to evaluate the inflammatory response and anti-allergic effect of immune cells, and a methanol extract of cumin was used to evaluate its protective role against biomolecular damage and neuropharmacological activity. Additionally, an ethanol extract of cumin was applied to lower triglycerides in diabetic rats, and an ethyl acetate extract of cumin was evaluated for wound healing applications.

Stem cells have been of great interest for a long time, especially for the treatment of various diseases. Stem cells are known to have various functions (Tae, J.Y., et al., 2019. Exp Ther Med 18: 326-331). It has been reported that stem cells can differentiate into various tissues and secrete various factors, and that this function, called a paracrine effect, affects surrounding tissues (Lee, H., et al. , 2018. Adv Clin Exp Med 27: 971-977). Recently, stem cells have been applied to tissue regeneration (Kang, S.H., et al., 2019. J Periodontal Implant Sci 49: 258-267).

Regulation of mesenchymal stem cells on cartilage differentiation is an important issue in stem cell therapy for articular cartilage defects and osteoarthritis. Meanwhile, it has been reported that low-molecular compounds including ethanol, ascorbic acid, dexamethasone, prostaglandin E2, staurosporine, catogenin, and oxopiperazine derivatives effectively induce chondrogenic differentiation of mesenchymal stem cells. However, effective monomolecular compounds for more excellent cartilage differentiation are continuously being studied.

In particular, techniques for differentiating mesenchymal stem cells into osteoblasts using specific proteins or genes have been reported, and studies on the induction of differentiation into osteoblasts by treating natural product-derived extracts or compounds during stem cell culture have been conducted. being reported

The patents and references mentioned herein are incorporated herein by reference to the same extent as if each document were individually and expressly specified by reference.

Republic of Korea Patent Publication No. 10-2004-0089733 (2004.10.21)

The present inventors studied whether cumin extract can be used for cartilage differentiation of bone marrow mesenchymal stem cells (BMSCs), and when the cumin extract is added to the cartilage differentiation induction medium, bone marrow-derived medium The invention was completed by demonstrating experimentally that the differentiation induction of mesenchymal stem cells into chondrocytes could be promoted.

Accordingly, an object of the present invention is to provide a composition for inducing differentiation of stem cells into chondrocytes, comprising a cumin extract as an active ingredient.

Another object of the present invention is to provide a culture medium for stem cell culture containing cumin extract as an active ingredient.

Another object of the present invention is to provide a method for inducing differentiation of stem cells into chondrocytes, comprising the step of treating the separated stem cells with a cumin extract.

Another object of the present invention is to provide a composition for preventing or treating bone defects comprising chondrocytes differentiated by the composition for promoting cartilage differentiation of the present invention as an active ingredient.

Other objects and technical features of the present invention are presented more specifically by the following detailed description, claims and drawings.

One aspect of the present invention provides a composition for inducing differentiation of stem cells into chondrocytes, comprising a cumin extract as an active ingredient.

The cumin extract, which is an active ingredient of the composition for inducing differentiation of stem cells into chondrocytes of the present invention, increases the induction of differentiation of stem cells into chondrocytes.

According to one embodiment of the present invention, the cumin extract included in the composition for inducing the differentiation of stem cells into chondrocytes includes various organs or parts of cumin (eg, leaves, flowers, fruits, seeds, stems, roots, branches, barks, etc.), and preferably may be extracts obtained from cumin seeds, but is not limited thereto.

According to one embodiment of the present invention, the cumin extract may be extracted using water, an organic solvent, or a mixture thereof as an extraction solvent.

In the present invention, the organic solvent may be selected from the group consisting of C ₁ -C ₄ alcohol, hexane (n-hexane), ether, glycerol, propylene glycol, butylene glycol, ethyl acetate and methyl acetate, but is not limited thereto. Instead, it is preferable to use ethanol or methanol as the C1-C4 alcohol, but it is not limited thereto.

In the present invention, "extract" refers to a substance extracted or separated from a raw material by any method, and includes without limitation an extract extracted from a raw material, a concentrate obtained therefrom, and a dried product and powder of the concentrate.

The extract may be obtained through all known extraction methods such as solvent extraction, ultrasonic extraction, filtration, and reflux extraction, preferably using solvent extraction or reflux extraction.

In the present invention, the cumin extract may be prepared in a powder state by an additional process such as concentration under reduced pressure and freeze drying or spray drying.

In addition, the cumin extract of the present invention can be prepared according to a conventional method known in the art, that is, under a conventional temperature and pressure condition, using a conventional solvent.

The cumin extract obtained through the above process exhibits an effect of inducing differentiation of stem cells into chondrocytes.

According to one embodiment of the present invention, the stem cells may include embryonic stem cells, adult stem cells, and induced pluripotent stem cells (ips cells) in which various differentiated cells are initialized, but are not limited thereto. Preferably, it may be an adult stem cell, and more preferably, it may be a stem cell derived from bone marrow.

In the present invention, "stem cell" refers to cells having self-renewal ability that are pluripotent or totipotent that can differentiate into cells of all tissues of an individual, and embryonic stem cells , induced pluripotent stem cells and adult stem cells.

In the present invention, "embryonic stem cells" refer to embryonic stem cells derived from a fertilized egg produced by combining sperm, which is a male reproductive cell, and egg, which is a female reproductive cell. On the other hand, embryonic stem cells are obtained from the inner cell aggregation of blastocysts before implantation and have the ability to differentiate into cells of all tissues, but are not yet differentiated cells that can proliferate indefinitely in an undifferentiated state if culture conditions are given. says In other words, in a special differentiation culture environment, cell differentiation is induced in the desired direction and can be used as a cell therapy for diseases such as nerve, diabetes, and hematology, so that a single embryonic stem cell line is expected to be used for the treatment of numerous patients. means

In the present invention, "induced pluripotent stem cells" refer to cells induced to have pluripotent differentiation potential through an artificial dedifferentiation process, and are also referred to as induced pluripotent stem cells (iPSCs).

In the present invention, "adult stem cells" are primitive cells isolated from tissues of mammals, including humans, just before differentiation, and have the ability to proliferate indefinitely and various types of cells (eg, fat cells, It is a stem cell that can differentiate into cartilage cells, muscle cells, bone cells, etc.).

In the present invention, "differentiation" refers to a phenomenon in which a less specialized cell develops into a specific cell and changes into a specific type of cell in size or shape, membrane potential, metabolic activity, and response to signals. The "differentiation" mainly occurs during the process of forming a complex tissue in a single zygote by multicellular organisms, or differentiates adult stem cells into specific cells when repairing damaged tissues in adulthood.

According to one embodiment of the present invention, the "differentiation" may be the differentiation of stem cells into chondrocytes.

According to one embodiment of the present invention, the composition for inducing differentiation of stem cells into chondrocytes may include the cumin extract in an amount of 0.0001 to 10 μg/ml, preferably in an amount of 0.0005 to 5 μg/ml. It may be included, and more preferably may be included in an amount of 0.001 to 1 μg / ml, but is not limited thereto.

Another aspect of the present invention provides a stem cell culture medium comprising a cumin extract as an active ingredient.

The cumin extract may be included as one component in a culture medium for stem cell culture.

The stem cell culture medium includes all media commonly used for stem cell culture, for example, DMEM (Dulbeco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F It may be a basic medium such as -10, F-12, MEM (Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), or a medium containing a component capable of inducing cartilage differentiation. , but not limited thereto.

According to one embodiment of the present invention, the cumin extract contained in the stem cell culture medium is extracted from various organs or parts (eg, leaves, flowers, fruits, seeds, stems, roots, branches, bark, etc.) of cumin. It means obtained by doing, and preferably may be an extract obtained from seeds of cumin, but is not limited thereto.

According to one embodiment of the present invention, the stem cells cultured in the stem cell culture medium may include embryonic stem cells, adult stem cells, and induced pluripotent stem cells (ips cells) in which various differentiated cells are initialized. Not limited to this. Preferably, it may be an adult stem cell, and more preferably, it may be a stem cell derived from bone marrow.

According to one embodiment of the present invention, the cumin extract contained in the stem cell culture medium may be included in an amount of 0.0001 to 10 μg/ml, preferably 0.0005 to 5 μg/ml, More preferably, it may be included in an amount of 0.001 to 1 μg/ml, but is not limited thereto.

Another aspect of the present invention provides a method for inducing differentiation of stem cells into chondrocytes, comprising the step of treating the separated stem cells with a cumin extract.

The method of inducing differentiation of stem cells into chondrocytes according to the present invention includes treating the separated stem cells with the cumin extract and culturing the stem cells treated with the cumin extract.

According to one embodiment of the present invention, the stem cells to be promoted for cartilage differentiation may be in vitro, and the cumin extract may be treated with the stem cells in the form of the stem cell culture medium.

According to one embodiment of the present invention, stem cells may be cultured on a three-dimensional scaffold to promote cartilage differentiation. Biocompatible materials may be used as components constituting the scaffold, and osteoblasts cultured and differentiated on the scaffold may be transplanted into the living body in a state attached to the scaffold for the treatment of bone diseases without separately separating from the scaffold.

According to one embodiment of the present invention, the cumin extract included in the method for inducing differentiation of stem cells into chondrocytes is used in various organs or parts of cumin (e.g., leaves, flowers, fruits, seeds, stems, roots, branches, and bark). etc.), and preferably may be an extract obtained from cumin seeds, but is not limited thereto.

According to one embodiment of the present invention, the cumin extract included in the method for inducing differentiation of stem cells into chondrocytes may be extracted using water, an organic solvent, or a mixture thereof as an extraction solvent, and the extraction solvent is preferably may be methanol, but is not limited thereto.

According to one embodiment of the present invention, in the step of treating the cumin extract, the cumin extract may be treated at a concentration of 0.0001 to 10 μg/ml, preferably at a concentration of 0.0005 to 5 μg/ml, More preferably, it may be treated at a concentration of 0.001 to 1 μg/ml, but is not limited thereto.

Another aspect of the present invention provides a composition for preventing or treating bone defects comprising chondrocytes differentiated by the composition for promoting cartilage differentiation of the present invention as an active ingredient.

In the present invention, "bone defect" refers to a bone-related disease requiring bone differentiation and regeneration, and includes traumatic bone damage such as fracture and bone defect occurring after surgery.

The differentiated chondrocytes of the present invention can be used for various cell therapies. Differentiated osteoblasts can be mixed with various biological supplements such as collagen or hydroxyapatite scaffolds and surgically transplanted into bone tissue.

The composition for preventing or treating bone defect according to the present invention may further contain collagen or a bone scaffold as an auxiliary component in addition to the differentiated osteoblasts.

The number of differentiated chondrocytes in the composition for preventing or treating bone defects according to the present invention can be adjusted depending on the type of disease, route of administration, age and sex of the patient, and severity of the disease. As a 5×10 ⁷ cell/cm3 scaffold, it can be administered once or divided into several times.

The composition for inducing the differentiation of stem cells into chondrocytes containing the cumin extract as an active ingredient according to the present invention has a remarkable effect of promoting and inducing stem cells into chondrocytes, which is useful when trying to induce stem cells into chondrocytes. In addition, since the induced chondrocytes can be applied to various regenerative medical markets that require chondrocytes, including the dental field, there is an advantage in that their utilization is high.

In addition, the composition for inducing the differentiation of stem cells into chondrocytes containing the cumin extract according to the present invention as an active ingredient can be used as a preventive and therapeutic agent for bone defects by inducing the differentiation of stem cells into chondrocytes. There is an advantage to being

1 is a schematic diagram showing a method for culturing and analyzing stem cells according to the present invention.
Figure 2 shows the morphological changes of stem cells on days 1, 3, 7, and 14 of culture after treatment with cumin extract at various concentrations (0, 0.001, 0.01, 0.1, and 1 μg/ml) in a cartilage differentiation induction medium under an inverted microscope. It is an observed image.
Figure 3 shows the change in the diameter of cell spheroids on the 1st, 3rd, 5th and 7th days of culture after treatment with cumin extract at various concentrations (0, 0.001, 0.01, 0.1 and 1 μg/ml) in the cartilage differentiation induction medium. .
Figure 4a shows images after Alcian staining of 7 and 14 days of culture after treatment with cumin extract at various concentrations (0, 0.001, 0.01, 0.1 and 1 μg/ml) in the cartilage differentiation induction medium.
Figure 4b shows the quantitative analysis of Alcian staining results by densitometry at 7 and 14 days of culture after treatment with cumin extract at various concentrations (0, 0.001, 0.01, 0.1 and 1 μg/ml) in the cartilage differentiation induction medium. it is shown
Figure 5a shows Sox9 mRNA expression values by quantitative real-time PCR at 14 days of culture after treatment with cumin extract at various concentrations (0, 0.001, 0.01, 0.1 and 1 μg/ml) in the cartilage differentiation induction medium.
Figure 5b shows COL2A1 mRNA expression values by quantitative real-time PCR at 14 days of culture after treatment with cumin extract at various concentrations (0, 0.001, 0.01, 0.1 and 1 μg/ml) in the cartilage differentiation induction medium.
Figure 5c shows COL1A1 mRNA expression values by quantitative real-time PCR at 14 days of culture after treatment with cumin extract at various concentrations (0, 0.001, 0.01, 0.1 and 1 μg/ml) in the cartilage differentiation induction medium.
6 shows the results of Western blot analysis for Sox9, collagen II and collagen I protein expression.

Specific examples described in this specification are meant to represent preferred embodiments or examples of the present invention, and the scope of the present invention is not limited thereby. It will be apparent to those skilled in the art that variations and other uses of the present invention do not depart from the scope of the invention described in the claims.

Example

Experimental Materials and Methods

1. Preparation of cumin extract

Cumin ( Cuminum cyminum L.) of the present invention was collected by Dr. Salah Uddin from Shibgonj sub-district, Bogra District, Rajshahi District, Bangladesh .

A voucher specimen recorded as KRIB 0086021 was deposited at the Plant Herbarium of the Korea Research Institute of Bioscience and Biotechnology.

After drying and grinding the seeds of cumin, the resulting powder (75 g) was subjected to repeated sonication (15 min) and soaking (2 h) in 1 L of 99.9% (v/v) methanol at 45 °C for 3 days. and extracted.

The product was filtered through non-fluorescence cottons, concentrated under reduced pressure at 45°C using a rotary distiller (N-1000SWD, EYELA), and 13.2 g of cumin extract was obtained by freeze-drying.

2. Culture of human bone marrow-derived mesenchymal stem cells (BMSCs)

The protocol of the present invention was reviewed and approved by the Clinical Trial and Research Review Board of Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea. All experiments were performed in accordance with the relevant guidelines and regulations specified in the Declaration of Helsinki.

Figure 1 shows the outline of the stem cell culture and analysis method according to the present invention.

Human bone marrow mesenchymal stem cells (BMSCs) derived from human bone marrow (BMSCs; Catholic MASTER cells) were obtained from Catholic Cell Therapy Corporation (CIC, Seoul, Korea).

Isolation and characterization of BMSCs was performed according to a previously reported method (Jeong, C.H., et al., 2014. Biomed Res Int 2014: 129145). The Catholic Cell Therapy Foundation confirmed that the human bone marrow-derived mesenchymal stem cells expressed CD73 and CD90 at 90% or more.

The culture medium was changed every 2 or 3 days, and the bone marrow-derived mesenchymal stem cells were cultured at 37°C in 95% O ₂ and 5% CO ₂ . grown in an incubator.

3. Analysis of stem cell morphology

The stem cells were plated in 600 μm diameter silicon elastomer-based concave microwells (StemFIT 3D; MicroFIT, Seongnam, Gyeonggi-do) at a density of 1Х10 ⁶ cells/well, and cartilage induction medium (STEMPRO Chondrogenesis Differentiation kit ; Gibco). In addition, the medium was replaced with fresh induction medium every 2-3 days.

The final treatment concentrations of cumin extract were 0, 0.001, 0.01, 0.1 and 1 μg/ml, respectively. Morphological evaluation was performed using an inverted microscope (CKX41SF, Olympus Corporation, Tokyo, Japan) on days 1, 3, 7 and 14 of culture.

4. Alcian blue staining

On days 7 and 14, respectively, cells were rinsed twice with phosphate-buffered saline and fixed with 4% paraformaldehyde at 20 °C for 15 min. Cells were then washed with distilled water, rinsed with 60% isopropanol and covered with alcian blue solution (SigmaAldrich; Merck KGaA, Darmstadt, Germany) for 20 minutes. After incubation, cells were rinsed with 3% acetic acid and washed with distilled water (D.W).

Relative values of chondrogenesis were determined by observing cell morphology using an inverted microscope and measuring the intensity of alcian blue staining using image analysis software (ImageJ, National Institutes of Health, Bethesda, MD).

5. SOX9, COL1A1 and COL2A1 by quantitative real-time PCR

After 14 days of induction of chondrogenesis, spheroids were homogenized in TRIzol Reagent (Invitrogen, Carlsbad, CA) and total RNA was obtained.

Complementary DNA was synthesized by SuperScript II RTase (Invitrogen) according to the manufacturer's instructions.

Real-time PCR was performed on a StepOnePlus™ Real Time PCR System (Applied Biosystems, Waltham, MA) using the SYBR Green PCR Kit (Applied Biosystems).

The mRNA expressions of SOX9 and COL2A1 as chondrogenic marker genes and COL1A1 as a fibrocartilage marker gene were evaluated. Primer sequences are shown in Table 1 below.

Gene Accession no. Primer sequences SOX9 NM 000346.4 forward: 5'-CCCTTCAACCTCCCACACT-3'
reverse: 5'-GAGTTCTGGTGGTCGGTGTA-3' COL2A1 NM 033150.3 forward: 5'-AAGGTTTTCTGCAACATGGA-3'
reverse: 5'-TCTTCTTTGGGAACGTTTGCT-3' COL1A1 NM 000088.4 forward 5'-TACCCCACTCAGCCCAGTGT-3'
reverse: 5'-CCGAACCAGACATGCCTCTT-3' β-actin NM 001101 forward: 5'-AATGCTTCTAGGCGGACTATGA-3'
reverse: 5'-TTTCTGCGCAAGTTAGGTTTT-3'

The reaction was performed at 95 °C for 10 min, 40 cycles of 95 °C for 15 sec and 59 °C for 30 sec. mRNA expression levels were normalized to β-actin levels.

6. Western Blot Analysis

Spheroids cultured under chondrogenic medium were collected on day 14 (number of spheroids = 300) and washed with 1x phosphate buffered saline. Cell lines were lysed with RIPA buffer containing 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1% IGEPAL, 10 mM NaF, 0.1 mM EDTA and protease inhibitor cocktail.

Protein concentration was determined using a protein assay method based on Bradford Assay solution (Bio-Rad, Contra Costa County, CA).

The same amount of protein (10 μg) was loaded on 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide 10% gel electrophoresis) at 110 V for 2 hours, and performed on a PVDF membrane (Merck Millipore, Burlington, MA) for 1.5 hours at 100 V did

-Actin(SC-47778; Santa Cruz Biotechnologies, Dallas, TX)를 4°C에서 밤새 배양하였다.The membrane was blocked in 5% skimmed milk for 1 hour at room temperature, followed by primary antibody 9 (anti-rabbit SOX9 antibody (D8G8H; Cell Signaling Technology, Inc., Danvers, MA)), anti Rabbit collagen II antibody (ab34712; Abcam, Cambridge, MA), anti-rabbit collagen I antibody (ab34710; Abcam) and anti-mouse

-Actin (SC-47778; Santa Cruz Biotechnologies, Dallas, TX) was incubated overnight at 4 °C.

Thereafter, the membrane was coated with a secondary antibody (mouse anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (SC-2357; Santa Cruz Biotechnology)) and an anti-mouse horseradish peroxidase (HRP) conjugated IgG secondary antibody (SC-2357). 516102; Santa Cruz Biotechnology) at a 1:3,000 dilution and incubated for 1 hour at room temperature.

The membrane was visualized using ECL Prime Western Blotting Reagent (RPN2232; GE Heath Care, Chicago, IL) and images were taken using X-ray film for a Chemiluminescence Western System (Advansta, San Jose, CA). .

Relative protein expression levels were normalized to the level of the corresponding loading control β-actin. The experiment was performed three times, and image analysis software (ImageJ) was used for quantification.

7. Statistical Analysis

Experimental data are presented as mean ± standard deviation. Normality test was performed, and one-way analysis of variance was performed using Tukey's post hoc test to identify differences between groups using a commercially available program (SPSS 12 for Windows, SPSS Inc., USA). (ANOVA) was performed. The significance level is 0.05.

Experiment result

1. Evaluation of stem cell morphology

Figure 2 shows images of the morphology of stem cells treated with cumin extract (CCT) at final concentrations of 0.001, 0.01, 0.1, and 1 μg/ml on days 1, 3, 7, and 14. Intact stem cell spheroids were made on day 1 in concave microwells made of silicone elastomer.

The morphology of stem cells treated with cumin extract (CCT) at final concentrations of 0.001, 0.01, 0.1 and 1 μg/ml showed marked changes when compared to untreated controls. In addition, no morphological changes were observed in stem cells when cultured for 3, 7 or 14 days.

2. Evaluation of Spheroid Diameter

Spheroids in all groups maintained their size from day 1 to day 14 (Fig. 3).

The diameters of spheroids at day 1 were 138.8 ± 12.7, 106.1 ± 18.0, 129.6 ± 15.0, 125.2 ± 18.0 and 164.7 respectively according to the treatment concentrations of 0, 0.001, 0.01, 0.1 and 1 μg/ml of cumin extract (CCT). ± 11.1 (P>0.05).

On day 3, the diameters of spheroids were 120.7 ± 12.4, 103.0 ± 5.7, 131.0 ± 13.2, 122.6 ± 9.0 and 148.9 depending on the treatment concentrations of 0, 0.001, 0.01, 0.1 and 1 μg/ml of cumin extract (CCT), respectively. ± 10.5 (P < 0.05).

The diameters of spheroids at day 7 were 129.0 ± 19.5, 123.3 ± 14.8, 115.3 ± 17.7, 140.5 ± 9.5 and 137.6 respectively according to the treatment concentrations of 0, 0.001, 0.01, 0.1 and 1 μg/ml of cumin extract (CCT). ± 6.3 (P>0.05).

The diameters of spheroids at day 14 were 151.7 ± 22.0, 167.3 ± 22.2, 125.8 ± 12.9, 167.8 ± 18.6 and 155.9 respectively according to the treatment concentrations of 0, 0.001, 0.01, 0.1 and 1 μg/ml of cumin extract (CCT). ± 21.9 (P>0.05).

3. Alcian blue staining evaluation

Alcian blue staining was performed on days 7 and 14 to detect the presence of cartilage-specific protein expression (FIG. 4a).

On day 7, the relative expression of cartilage-specific proteins was 100.0 ± 1.3, 93.6 ± 3.7, 80.5 ± 7.9, 71.1 ± 5.6 and 71.4 depending on the treatment concentrations of 0, 0.001, 0.01, 0.1 and 1 μg/ml of cumin extract (CCT), respectively. was ±5.0.

The relative expression of cartilage-specific proteins on day 14 was 110.5 ± 3.5, 99.8 ± 3.3, 72.1 ± 8.6, 73.2 ± 2.2 and 73.2 ± 2.2 respectively according to the treatment concentrations of 0, 0.001, 0.01, 0.1 and 1 μg/ml of cumin extract (CCT). It was 106.8 ± 2.7.

4. Evaluation of SOX9, COL1A1 and COL2A1 by quantitative real-time PCR

According to quantitative real-time polymerase chain reaction, the mRNA level of SOX9 was 1.009 ± 0.172, 1.473 ± 0.130, 1.083 ± 0.047, 1.156 ± 1.156 ± 0.001, 0.01, 0.1, and 1 μg/ml of cumin extract (CCT), respectively. 0.100 and 0.772 ± 0.061 (Fig. 5a) (P < 0.05).

According to quantitative real-time polymerase chain reaction, the mRNA level of COL2A1 was 1.000 ± 0.015, 1.432 ± 0.222, 0.784 ± 0.096, 0.860 ± 0.860 ± 0.001, 0.01, 0.1, and 1 μg/ml of cumin extract (CCT), respectively. 0.304 and 0.649 ± 0.072 (Fig. 5b) (P > 0.05).

According to the quantitative real-time polymerase chain reaction, the mRNA level of COL1A1 was 1.031 ± 0.299, 0.722 ± 0.042, 0.750 ± 0.061, 0.587 ± 0.587 ± 0.750 ± 0.061, respectively according to the treatment concentration of 0, 0.001, 0.01, 0.1 and 1 μg/ml of cumin extract (CCT). 0.037 and 0.538 ± 0.007 (Fig. 5c) (P < 0.05).

5. Western Blot Analysis Evaluation

Western blot analysis was performed to analyze the expression of Sox9, collagen II and collagen I proteins (FIG. 6).

Normalization of protein expression confirmed that Sox9 expression was 245.6, 203.9, 116.3, and 48.1, respectively, according to treatment concentrations of 0, 0.001, 0.01, 0.1, and 1 μg/ml of cumin extract (CCT), when the control group was considered as 100%. .

Collagen II expression levels were similarly low to undetectable levels.

Normalization of protein expression confirmed that the collagen I expression was 85.0, 56.6, 92.1, and 75.0, respectively, according to the processing concentrations of 0, 0.001, 0.01, 0.1, and 1 μg/ml of cumin extract (CCT), considering the control group as 100%. did

From the above results, it can be confirmed that the differentiation of stem cells into chondrocytes was improved as the expression of genes related to the differentiation of stem cells into chondrocytes was significantly increased by the treatment with the cumin extract.

Claims (20)

A composition for inducing differentiation of stem cells into chondrocytes, comprising a cumin extract as an active ingredient. According to claim 1,
The cumin extract is a composition for inducing differentiation of stem cells into chondrocytes, characterized in that extracted from seeds of cumin. According to claim 1,
The cumin extract is a composition for inducing differentiation of stem cells into chondrocytes, characterized in that extracted using water, an organic solvent or a mixture thereof as an extraction solvent. According to claim 3,
The organic solvent is one or more organic solvents selected from the group consisting of C1-C4 alcohol, hexane, ether, glycerol, propylene glycol, butylene glycol, ethyl acetate and methyl acetate. A composition for inducing differentiation. According to claim 4,
The alcohol is a composition for inducing differentiation of stem cells into chondrocytes, characterized in that methanol. According to claim 1,
The stem cell is a composition for inducing differentiation of stem cells into chondrocytes, characterized in that adult stem cells. According to claim 6,
The adult stem cell is a composition for inducing differentiation of stem cells into chondrocytes, characterized in that stem cells derived from bone marrow. According to claim 1,
The composition is a composition for inducing differentiation of stem cells into chondrocytes, characterized in that it comprises a cumin extract in an amount of 0.0001 to 10 μg / ml. delete delete delete delete delete A method for inducing differentiation of stem cells into chondrocytes, comprising the step of treating the separated stem cells with a cumin extract. According to claim 14,
The method of inducing differentiation of stem cells into chondrocytes, characterized in that the cumin extract is extracted from seeds of cumin. According to claim 14,
The method of inducing differentiation of stem cells into chondrocytes, characterized in that the cumin extract is extracted using water, an organic solvent or a mixture thereof as an extraction solvent. According to claim 16,
The organic solvent is one or more organic solvents selected from the group consisting of C1-C4 alcohol, hexane, ether, glycerol, propylene glycol, butylene glycol, ethyl acetate and methyl acetate. Differentiation induction method. According to claim 17,
The method of inducing differentiation of stem cells into chondrocytes, characterized in that the alcohol is methanol. According to claim 15,
The step of treating the cumin extract is a method for inducing differentiation of stem cells into chondrocytes, characterized in that the treatment with the cumin extract at a concentration of 0.0001 to 10 μg / ml. delete

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