WO2023038161A1 - Composition of membrane-free stem cell extract for preventing and treating diabetes - Google Patents

Abstract

The present invention relates to a composition of a membrane-free stem cell extract for preventing and treating diabetes, the composition characterized by using a membrane-free stem cell extract derived from adipose stem cells at a concentration of 0.5-2.5 μg/mL.

Description

Composition for prevention and treatment of diabetes mellitus stem cell extract

The present invention relates to a composition for preventing and treating diabetes of a mucosal stem cell extract, and more particularly, a mucous membrane capable of preventing and treating diabetes through the action of promoting glucose absorption of an extract extracted from stem cells isolated from fat cells. It relates to a composition for preventing and treating diabetes of a stem cell extract.

Diabetes is a metabolic disease caused by a defect in insulin secretion or action, and is a chronic metabolic disease in which blood sugar control in the body is not normally performed, resulting in chronic hyperglycemia. Diabetes is largely divided into type 1 diabetes, which is insulin-dependent diabetes, and type 2 diabetes, which is non-insulin-dependent diabetes, depending on the mechanism of occurrence. More than 90% of diabetic patients appear as non-insulin dependent diabetes mellitus, and non-insulin dependent diabetes mellitus leads to complications such as blindness, cardiovascular disease, and renal failure, and death resulting therefrom.

As a treatment for non-insulin dependent diabetes mellitus, oral hypoglycemic agents are used along with improvement of lifestyle such as diet therapy or exercise therapy. However, the use of these drugs has problems causing side effects such as liver function or gastrointestinal disorder, hypoglycemia and lactic acidemia.

There is a continuous demand for the development of diabetes preventive and therapeutic agents that minimize these side effects, increase therapeutic efficacy, and are safe even during long-term use. Therefore, the present inventors are trying to confirm the possibility of preventing and treating diabetes using stem cells.

Stem cells (stem cells) refers to cells constituting organs, skin, bone cartilage, etc. of the human body that newly induce the production of cells to maintain homeostasis in the body through apoptosis and cell production. Adult stem cells, a type of stem cell, can be harvested from bone marrow, umbilical cord blood, and adipose tissue. As in Korean Registered Patent No. 10-2042084, research is being conducted to use stem cells as a treatment for various diseases such as diabetes, obesity, and heart disease in related fields.

In order to solve the above-mentioned problems, an object of the present invention is to provide a composition for preventing and treating diabetes of a mucosal stem cell extract capable of promoting glucose absorption in adipocytes by using mucosal stem cells and thereby preventing and treating diabetes. is to do

In order to achieve the above object, the present invention composition for preventing and treating diabetes mellitus stem cell extract is characterized by using the mucous membrane stem cell extract derived from adipose stem cells at a concentration of 0.5 to 2.5 μg / mL.

In one embodiment of the present invention, after separating and purifying adipose stem cells from adipose tissue, the membraneless stem cell extract was initially cultured in an incubator at 37° C. under 5% CO ₂ condition, followed by subculture After repeating 6 to 10 times, removing the medium, and removing the cell membrane of the stem cells, it is characterized in that the extract is obtained and used.

The composition for the prevention and treatment of diabetes mellitus stem cell extract of the present invention can promote glucose uptake by increasing 2-DG6P uptake in 3T3-L1 adipocytes, which are mouse-derived preadipocytes.

In addition, the composition for preventing and treating diabetes mellitus stem cell extract of the present invention reduces the protein expression of p-IRS-1 (ser 307) and increases the protein expression of p-Akt, p-AMPK, p-ACC and GLUT4 can increase and regulate glucose transport.

Therefore, the composition for preventing and treating diabetes of the extract of the stem cell extract of the present invention can be actively utilized as a functional material for the composition for preventing and treating diabetes.

However, the effects of the invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 is a measurement of the degree of absorption of 2-deoxyglucose-6-phosphate (2-DG6P), a factor related to glucose absorption, in the case of applying the composition for preventing and treating diabetes of the present invention by concentration. graph shown.

Figure 2 is the measurement of p-IRS-1 / IRS, p-Akt / Akt and GLUT4 protein expression, which are factors related to insulin signaling, when the present invention composition for preventing and treating diabetes of a mucosal stem cell extract was applied by concentration. graph shown.

Figure 3 is p-p-AMPK / AMPK, p-acetyl-CoA carboxylase (ACC, which is a factor related to AMPK (Adenosine monophosphate-activated protein kinase) activity for the case where the present invention composition for preventing and treating diabetes of a membraneless stem cell extract is applied by concentration )/Graph showing the protein expression of ACC measured.

Hereinafter, preferred embodiments of the composition for preventing and treating diabetes mellitus stem cell extract according to the present invention will be described in detail with reference to the accompanying drawings.

1 is a measurement of the degree of absorption of 2-deoxyglucose-6-phosphate (2-DG6P), a factor related to glucose absorption, in the case of applying the composition for preventing and treating diabetes of the present invention by concentration. Figure 2 is a graph showing the expression of p-IRS-1/IRS, p-Akt/Akt and GLUT4 proteins, which are insulin signaling-related factors, when the composition for preventing and treating diabetes of the present invention, a mucosal stem cell extract, was applied by concentration. Figure 3 is a graph showing the measurement of AMPK (Adenosine monophosphate-activated protein kinase) activity-related factors p-p-AMPK / AMPK, It is a graph showing the measured protein expression of p-acetyl-CoA carboxylase (ACC)/ACC.

In the drawings and detailed description of the invention, MFSCE may mean Membrane free stem cell extract from adipose tissue. In addition, MDI may include 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 10 μg/mL insulin.

The composition for preventing and treating diabetes mellitus stem cell extract of the present invention may include a membrane stem cell extract derived from adipose stem cells. Specifically, in one embodiment of the present invention, the composition for preventing and treating diabetes of the mucosal stem cell extract may be used at a concentration of 0.5 to 2.5 μg / mL. More preferably, the composition for preventing and treating diabetes of the mucosal stem cell extract may be one in which the nonmembranous stem cell extract derived from adipose stem cells is used at a concentration of 2.5 μg/mL.

If the composition for preventing and treating diabetes of the present invention contains less than 0.5 μg/mL of the mucosal stem cell extract, the active ingredient of the non-membrane stem cell extract may be insufficient, resulting in reduced effectiveness in preventing and treating diabetes. . In addition, when the composition for preventing and treating diabetes of the mucosal stem cell extract of the present invention contains more than 2.5 μg/mL of the nonmembranous stem cell extract, the toxicity of the mucosal stem cell extract in 3T3-L1 preadipocytes As this increases, cell viability may decrease, and the activity of 3T3-L1 preadipocyte may decrease.

In one embodiment of the present invention, after separating and purifying adipose stem cells from adipose tissue, the membraneless stem cell extract was initially cultured in an incubator at 37° C. under 5% CO ₂ condition, followed by subculture After repeating 6 to 10 times, removing the medium, and removing the cell membrane of the stem cells, the extract may be obtained and used.

The subculture is a method of cell proliferation that is periodically transplanted into a new medium. Specifically, target cells can be cultured and proliferated in such a way that strains are preserved and cell generations are succeeded by transplanting the target cells into a new medium. That is, when stem cells grow on a plate containing a medium and have a density of 80 to 90%, some stem cells are obtained from the plate and transferred to a plate containing another medium, so that the stem cells grow again. In addition, when primary culture is performed, if blood is not removed, the blood can be removed during subculture.

The membraneless stem cells of the present invention may refer to stem cells without a cell membrane by removing the cell membrane of the cultured stem cells after the subculture.

Hereinafter, glucose uptake experiments in fat cells for the prevention and treatment of diabetes of the present invention composition for preventing and treating diabetes of a mucosal stem cell extract will be described in detail.

1. Experiment materials

Dulbecco's modified eagle's medium (DMEM), bovine calf serum (BCS), fetal bovine serum (FBS), penicillinstreptomycin, and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from WelGENE (Gyeongsan, Korea). 3-(4,5-Dimethylthiazol-2-yl)-2,3-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone and insulin were purchased from Sigma Chemical Co. (St.Louis, MO, USA) product was purchased and used. Krebs-Ringer-phosphate-HEPES (KRPH) buffer was purchased from Biosolution Co., Ltd. (Seoul, Korea). Bovine serum albumin (BSA) was purchased from Bioworld Technology (Bloomington, MN, USA). Primary antibodies and secondary antibodies were used by Cell Signaling Technology, Inc. (Danvers, MA, USA). Enhanced chemiluminescence (ECL) solution was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

2. Manufacture of membraneless stem cell extract

The membraneless stem cell extract was directly prepared and used by the present applicant. Specifically, body fat tissue discarded through surgical liposuction for the cosmetic purpose of a healthy obese woman in her twenties (BMI 25-29.9) without other diseases according to the results of blood test and doctor's diagnosis was used for sample preparation. The blood tests performed were hepatitis B virus, hepatitis C virus, human immunodeficiency virus, human T lymphotrophic virus, parvovirus B19, cytomegalovirus, Epstein-Barr virus, and syphilitic creponema.

Prior to liposuction, written consent was obtained from the donor that body fat tissue was used for research purposes according to the donor management procedure notified by the Ministry of Food and Drug Safety, and body fat tissue was obtained according to a protocol approved for clinical trials by the Bioethics Committee.

Adipose tissue was cultured in type 2 collagenase (Sigma-Aldrich Corporation, St. Louis, MO, USA), centrifuged, washed, and resuspended in phosphate-buffered saline (PBS) to collect adipose-derived stem cells.

To purify adipose-derived stem cells from adipose tissue, a monoclonal antibody for each surface marker and a secondary antibody, rabbit fluorescein isothiocyanate-labeled anti-mouse immunoglobulin G, were attached to the fixed cells, which were visualized using a fluorescence microscope. The adipose-derived stem cells were purified by confirming the positive markers CD105 and CD29.

The adipose-derived stem cells purified as above were initially cultured in a 37°C 5% CO ₂ incubator, and subculture was repeated 6 to 10 times. The medium was removed, a certain amount of stem cells were obtained, and cell membranes were peeled off using a physical method such as ultrasound, and cell membrane fragments were removed using a method such as a filter to obtain a membraneless stem cell extract. The aqueous membraneless stem cell extract was lyophilized to form a powder formulation and then stored at 5±2°C.

3. Cell culture

Mouse preadipocytes 3T3-L1 preadipocytes used in the experiment were purchased from American Type Culture Collection (Manassas, VA, USA). 3T3-L1 preadipocytes were cultured in DMEM containing 100 units/mL penicillin streptomycin and 10% BCS at 37°C in a 5% CO ₂ incubator. The cultured cells were washed with PBS (pH 7.4) at 80% confluence, then the attached cells were separated with a mixture of 0.05% trypsin and 0.02% EDTA and centrifuged at 1,000 rpm for 3 minutes.

In order to induce differentiation of 3T3-L1 preadipocytes into adipocytes, the differentiation induction medium containing 0.5 mM IBMX, 1 μM Dex, and 10 μg/mL insulin (MDI) and 10% FBS was replaced after 2 days in a 100% confluent state.

After 2 days, culture medium containing 10% FBS and 10 μg/mL insulin was cultured for 2 days, and then replaced with medium containing 10% FBS every 2 days until differentiation into adipocytes.

4. Cell viability measurement

To confirm the cytotoxicity of the membraneless stem cell extract in 3T3-L1 preadipocytes, cell viability was measured by MTT assay.

When the 3T3-L1 preadipocyte reached 80% confluence, it was seeded in a 24-well plate at 5Х10 ⁴ cells/mL. When the cells are in a confluence state, the concentration of the membraneless stem cell extract is divided into 0.5, 1, 2.5, and 5μg/mL for each sample, and after incubation for 72 hours, 5mg/mL MTT solution is injected into each well. It was re-incubated for 4 hours at 37°C. After the incubation was completed, formazan crystals were dissolved in DMSO, left at room temperature for 30 minutes, and absorbance was measured at 540 nm.

5. Measurement of glucose uptake

In order to confirm the effect of glucose uptake in adipocytes, the amount of glucose uptake was measured by Glucose uptake assay.

When 3T3-L1 preadipocytes reached 80% confluence, they were seeded in a 96-well plate at 1.5Х10 ⁴ cells/mL and used for the experiment. 2 days after confluence, differentiation into adipocytes was induced by replacing the differentiation induction medium, and 2 days after induction, cultured in a medium containing 10% FBS and 10 μg/mL insulin for 2 days, and then cultured for 10 days every 2 days until differentiation into adipocytes. It was replaced with medium containing % FBS.

After differentiation was completed, the medium was replaced with a glucose-free medium and cultured for 16 hours, followed by starvation by culturing with 100 μL KRPH buffer containing 2% BSA for 40 minutes. Each sample was treated with membraneless stem cell extract (0.5, 1, 2.5μg/mL) or 1μM insulin and cultured for 30 minutes, then treated with 10mM 2-deoxyglucose and cultured for 20 minutes to prepare samples. Glucose uptake was measured using a glucose uptake assay kit (Abcam, Cambridge, UK).

6. Intracellular expression protein analysis (Western blot analysis)

To investigate the effect of membraneless stem cell extract on the insulin signal cleavage process, protein expression was measured by Western blot analysis.

Radioimmunoprecipitation assay buffer was added to the cells treated with the membraneless stem cell extract to dissolve them, and the obtained proteins were quantified by centrifugation at 4°C and 12,000 rpm for 30 minutes. Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrotransferred to membranes. After blocking by treating the protein-attached membrane with 5% skim milk, the primary antibody was reacted overnight at 4°C.

After washing with PBS-T, the secondary antibody was reacted at room temperature for 1 hour, washed again with PBS-T, and reacted with ECL solution to express protein using the Davinch-Chemi ^TM Chemiluminescence image system (Core Bio, Seoul, Korea) confirmed.

7. Statistical analysis

All experimental results were repeated three times and expressed as mean±standard deviation. To examine the significance of each experimental result, Statistical Package for the Social Sciences (SPSS; IBM Corp., Armonk, NY, USA) was used. Analysis of variance test and Duncan's multiple range test were performed to test the significant difference between the mean values of the samples at P<0.05.

8. Effect of membraneless stem cell extract on cell survival in 3T3-L1 preadipocytes

In order to confirm the cytotoxicity of the membraneless stem cell extract in 3T3-L1 preadipocytes, the cytotoxicity was examined using MTT assy at a concentration of 0.5 to 5 μg/mL. As a result, cell viability was shown in Table 1 below. Specifically, 0.5, 1, and 2.5μg/mL of membraneless stem cell extract did not significantly affect the viability of 3T3-L1 preadipocytes, but 5μg/mL of membraneless stem cell extract significantly improved the cell viability of 3T3-L1 preadipocytes. Reduced. Therefore, in subsequent experiments, 0.5, 1, and 2.5 μg/mL of the membraneless stem cell extract were set as experimental concentrations.

Treatment (μg/mL)	Cell viability (%)
0	100.00±0.52
0.5	106.51±1.72
One	101.88±1.10
2.5	96.61±0.55
5	84.25±1.51

9. Measurement results and analysis of glucose uptake of the composition

When 3T3-L1 cells were treated with the membraneless stem cell extract at concentrations of 0.5, 1, and 2.5 μg/mL, as shown in FIG. 1, the absorption of 2-deoxyglucose-6-phosphate (2-DG6P) increased as the concentration increased. A dependent increase was confirmed. In particular, compared to the control group not treated with membraneless stem cell extract, it was confirmed that glucose absorption increased by showing about 1.8 times higher 2-DG6P absorption at a concentration of 2.5 μg/mL.

Differentiated 3T3-L1 adipocytes are particularly sensitive to insulin, and insulin is known to promote intracellular glucose transport at about 10-fold speed. Accordingly, 3T3-L1 adipocytes are widely used as an effective model for screening antidiabetic materials through insulin signaling.

As shown in Figure 1, as it was confirmed that the absorption of 2-DG6P increased as the concentration increased when the membraneless stem cell extract was treated, it is thought to exhibit a blood glucose lowering effect by promoting glucose absorption in adipocytes.

10. Measurement results and analysis of insulin signaling-related proteins and glucose transporter 4 (GLUT4) protein expression

After treating the differentiated 3T3-L1 adipocyte with a membrane stem cell extract, the protein expression of insulin receptor substrate-1 (IRS-1) and Akt, which are proteins related to insulin signaling, was measured. As shown in FIG. 2, membrane stem cells As the cell extract treatment concentration increased, the protein expression of p-IRS-1/IRS decreased and the protein expression of p-Akt/Akt increased. Subsequently, as a result of confirming the expression of GLUT4 protein in 3T3-L1 adipocytes treated with the membraneless stem cell extract, it was confirmed that the protein expression of GLUT4 increased when the membraneless stem cell extract was treated compared to the control group.

Glucose uptake in adipocytes is mainly accomplished by GLUT4 through the process of insulin signaling. It is known that the insulin signaling process starts when IRS-1 is phosphorylated by an insulin receptor that has tyrosine kinase activity. At this time, when serine phosphorylation of IRS-1 is promoted, tyrosine phosphorylation of IRS-1 is reduced, resulting in insulin resistance.

In addition, IRS is known to play an important role in evaluating antidiabetic activity by regulating ser307 and tyr612 phosphorylation and participating in phosphatidylinositol 3-kinase (PI3K), AKT, and GLUT4 pathways. IRS-1, which is tyrosine phosphorylated by protein tyrosine kinase, activates PI3K, Akt, and GLUT4 in turn, and in this process, GLUT4 moves from the cytoplasm to the cell membrane to transport glucose into the cell.

That is, based on the results of the Glucose uptake assay, Western blot analysis was conducted to determine the effect of the membraneless stem cell extract on the insulin signal transduction process in 3T3-L1 adipocytes. , the protein expression of p-IRS-1/IRS decreased and the expression of p-Akt/Akt and GLUT4 increased. Therefore, it is thought that the membraneless stem cell extract increases glucose utilization in adipocytes by promoting insulin signaling and increasing the expression of GLUT4.

11. Measurement results and analysis of protein expression related to the adenosine monophosphate-activated protein kinase (AMPK) pathway

As a result of measuring protein expression through Western blot analysis to confirm the activation of AMPK by the membraneless stem cell extract, as shown in Figure 3, the protein expression of p-AMPK / AMPK was increased in a concentration-dependent manner compared to the control group, It was confirmed that the protein expression of p-acetyl-CoA carboxylase (ACC)/ACC was also increased. In particular, the highest p-AMPK/AMPK and p-ACC/ACC protein expression increase was shown at the concentration of 2.5μg/mL compared to the control group not treated with the membraneless stem cell extract.

AMPK is a regulator that maintains intracellular energy homeostasis, and is known to inhibit gluconeogenesis in the liver and promote glucose uptake by increasing the expression of GLUT4 in muscle and fat cells. In particular, AMPK is known to independently activate GLUT4 to increase glucose utilization and is widely studied as a target factor for diabetes treatment. ACC is a substrate of AMPK, which regulates energy balance by phosphorylating it to regulate intracellular fatty acid synthesis.

To confirm the activation of AMPK by the membraneless stem cell extract, protein expression was measured through Western blot analysis. As a result, it was confirmed that p-AMPK/AMPK and p-ACC/ACC protein expressions increased when the membraneless stem cell extract was treated. . Therefore, it was shown that the membraneless stem cell extract activates the AMPK pathway in 3T3-L1 adipocytes, which is thought to affect glucose uptake along with the insulin signaling pathway.

As described above, it was confirmed that the composition for preventing and treating diabetes mellitus stem cell extract of the present invention promotes glucose uptake in 3T3-L1 adipocytes through the AMPK pathway and the insulin signaling pathway. These results suggest that adipose-derived stem cells can be used as a substance for improving diabetes through various treatments.

The membraneless stem cell extract used in this study was manufactured by the applicant's patented technology and is an extract containing various peptides and growth factors after removing the cell membrane of stem cells. When the membraneless stem cell extract was analyzed by nano-liquid chromatography-mass spectrometry analysis, 252 proteins regulating metabolic processes, physiological activities, and cell proliferation were identified (data not shown). Among them, 19 proteins, including integrin alpha-5, integrin beta-1, and -3, are involved in cell adhesion, and 36 proteins are reported to be regenerative factors involved in wound healing.

In addition, 9 proteins exhibit anti-inflammatory activity by regulating NF-κB signaling, toll-like receptor 4-dependent signaling, etc., and 17 proteins including SOD1 and CD36 have been reported to be involved in cell detoxification ( data not shown). Considering these results, it is believed that the peptide and protein components of the mucosal stem cell extract suppress the inflammatory response and regulate the cellular metabolic process, and further research on components that can improve sugar absorption in relation to this study is thought to be necessary.

In addition, mucosal stem cell extract is a substance developed to overcome the 1:1 patient-specific treatment of existing stem cells, and is expected to be a substance capable of 1: multiple treatment and industrialization by removing immunogenicity attached to the cell membrane of stem cells. do.

As such, it will be understood that the technical configuration of the present invention described above can be implemented in other specific forms without changing the technical spirit or essential features of the present invention by those skilled in the art to which the present invention belongs.

Therefore, the embodiments described above should be understood as illustrative and not limiting in all respects, and the scope of the present invention is indicated by the claims to be described later rather than the detailed description, and the meaning and scope of the claims and All changes or modified forms derived from the equivalent concept should be construed as being included in the scope of the present invention.

Claims (2)

1. A composition for preventing and treating diabetes of a membraneless stem cell extract, characterized in that the membraneless stem cell extract derived from adipose stem cells is used at a concentration of 0.5 to 2.5 μg/mL.
2. According to claim 1,

   The membraneless stem cell extract,

   After isolating and purifying adipose stem cells from adipose tissue, after initial culture in an incubator at 37° C. under 5% CO ₂ condition, subculture is repeated 6 to 10 times, the medium is removed, and stem cell A composition for preventing and treating diabetes of a mucosal stem cell extract, characterized in that the extract is obtained after removing the cell membrane of the.

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