WO2013085344A1

WO2013085344A1 - Method for screening material for prevention or treatment of osteoporosis using leptin secretion of mesenchymal stem cells

Info

Publication number: WO2013085344A1
Application number: PCT/KR2012/010640
Authority: WO
Inventors: 노민수
Original assignee: 아주대학교 산학협력단
Priority date: 2011-12-08
Filing date: 2012-12-07
Publication date: 2013-06-13
Also published as: KR101354826B1; KR20130064574A

Abstract

The present invention relates to a method for screening a material for the prevention or treatment of osteoporosis using mesenchymal stem cells, and more specifically, to a method for screening a material for the prevention or treatment of osteoporosis within a short time by measuring the amount of leptin secreted in mesenchymal stem cells. According to the present invention, a method for screening an agent for the prevention or treatment of osteoporosis using mesenchymal stem cells and a method for screening a dietary supplement composition for the prevention or improvement of osteoporosis allow a candidate for an agent for the prevention or treatment of osteoporosis to be screened within three days, thereby remarkably enhancing efficiency compared with conventional screening methods, which need 10 days or more, and are expected to be used for screening a large quantity of materials within a short time due to a simple experimental method.

Description

Screening method for preventing or treating osteoporosis using mesenchymal stem cell leptin secretion

The present invention relates to a method for screening a substance for preventing or treating osteoporosis using mesenchymal stem cells, and more specifically, to prevent osteoporosis in a short time by measuring the amount of leptin secreted from mesenchymal stem cells. Or to a method for screening a therapeutic substance.

Leptin is known to be a hormone that can be used to treat obesity by suppressing appetite by secreting from adipose tissue. Leptin is secreted by adipocytes, suppresses appetite in the central nervous system, and inhibits fat accumulation in some peripheral nervous systems. Mice with abnormalities in the leptin gene or mice with genetic defects in the leptin receptor show obesity. Recently, many studies have been conducted on anti-obesity agents that can increase the amount of leptin in the blood. Leptin is also known to be involved in the production of osteoblasts locally to help bone formation and metabolism. Therefore, the cell-based measurement method for selecting natural products, synthetic compounds, or biological substances that can promote the secretion of leptin is expected to be useful for the development of new drugs and health foods related to the treatment and prevention of anti-obesity, osteoporosis.

Recently, there have been attempts to measure leptin levels using adipocytes, which are known to be mainly secreted by leptin.However, methods for promoting leptin secretion using primary cultured adipocytes are not easy to isolate and culture, and require a long time. Therefore, it is difficult to be used in the preclinical candidate search phase of new drug development. In addition, there is a big difference in the leptin secretion ability or the sensitivity of leptin receptors according to the degree of obesity and the adipose tissue by site. Therefore, the method of evaluating leptin secretion using primary cultured fat cells has limitations in evaluating new drugs and health foods. .

It is estimated that about 160 million people suffer from aging osteoporosis based on the age group of 50 or older in the world's seven major pharmaceutical markets, and the life expectancy is prolonged due to the development of medical technology. The development of effective osteoporosis treatments is expected to create enormous added value in the industrial economy.

Osteoporosis is a systemic degenerative bone disease in which bone minerals decrease and bone strength decreases due to changes in the microstructure of the bones to increase bone fracture. Current treatments for osteoporosis include drugs from biophosphonates such as alendronate (trade name Fosamax), selective estrogen receptor modulators such as raloxifene, or parathyroid hormones that regulate bone formation Derivatives are used. And recently, antibody drugs such as Denosumab, which inhibits the cytokine that destroys bone, have also started to be available.

Oral biophophonate drugs are currently widely used in the treatment of various osteoporosis (osteopenia, osteoporosis and severe osteoporosis), but these drugs suffer from gastrointestinal side effects and complicated medications. In particular, oral biophophonate drugs represented by Posamax have a potential risk because their molecular mechanism of action is not yet clear. Therefore, researches are being actively conducted on methods that can improve the side effects of oral biophosphate drugs, select compounds with high therapeutic efficacy, and reduce the cost of expensive monoclonal and parathyroid hormone preparations.

Oral bisphosphonate drugs commonly prescribed for the treatment of osteoporosis, such as alendronate, risedronate, ibandronic acid, zoledronic acid, and minodronic acid, reduce the activity of osteoclasts by participating in the osteoclast pathway. This is known to reduce bone resorption. However, in bone metabolism, the balance of bone resorption and bone formation is very important. Considering this, the effect of oral bisphosphonate-like drugs on the osteoclast pathway alone has all effects on reducing bone loss. It is difficult to explain.

Selective estrogen receptor modulators such as Raloxifene are mainly prescribed for the prevention and treatment of osteoporosis that occurs after menopause, and are effective against breast cancer prevention and osteopenia. However, compared to oral bisphosphonate drugs, there are problems of low efficacy and high side effects. In addition, except for the selective estrogen receptor modulators, the specific mechanism of action related to the effect of treating osteoporosis is still unclear.

Recently, new drugs for treating osteoporosis, such as parathyroid hormone derivatives, having new mechanisms for promoting bone formation or new drugs that affect the differentiation of osteoblasts have been in the spotlight. As a method for screening such new drugs, methods for treating test substances in osteoblast-like cell lines or mesenchymal stem cells and measuring osteoprotegerin (OPG) and alkaline phosphatase (ALP) are used. When using mesenchymal stem cells, osteoporosis treatment and / or prophylactic agents are treated for at least one week in a culture medium that induces bone differentiation (typically, a medium containing dexamethasone, vitamin C, and beta-glycerol phosphate). Long time is required because bone formation markers should be measured after incubation for up to 3 weeks. Therefore, it is not suitable for the screening of a large amount of substances because of the low efficiency for developing new drug candidates or raw materials for health food development.

As described above, in order to develop an osteoporosis prevention and / or treatment agent having fewer side effects and excellent therapeutic effects, it is required to develop a method for selecting substances having an osteoporosis treatment effect in an easy manner within a short time.

The present invention has been made to solve the above-mentioned problems in the prior art, in order to provide a screening method capable of selecting a substance having a prophylactic and / or therapeutic effect of osteoporosis within a short time, mesenchymal stem cells ( Treating and culturing the test substance in mesenchymal stem cells; Measuring the amount of leptin in the cell culture by enzyme-linked antibody attachment reaction; And a screening method for preventing or improving osteoporosis, and a screening method for a health functional food composition for preventing or improving osteoporosis, comprising selecting a candidate substance which significantly increases the amount of leptin as compared to a control which has not been treated with the test substance. For that purpose.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention comprises the steps of treating and culturing a test substance in mesenchymal stem cells; Measuring the amount of leptin in the cell culture by enzyme-linked antibody attachment reaction; And it provides a screening method of preventing or treating osteoporosis prevention agent comprising the step of selecting a candidate substance to significantly increase the leptin amount compared to the control group was not treated.

In another aspect, the present invention comprises the steps of treating and culturing the test substance in mesenchymal stem cells; Measuring the amount of leptin in the cell culture by enzyme-linked antibody attachment reaction; And it provides a method for screening a health functional food composition for osteoporosis prevention or improvement comprising the step of selecting a candidate substance to significantly increase the amount of leptin compared to the control group was not treated.

In one embodiment of the present invention, the screening method is characterized in that culture within one week.

In another embodiment, the test substance is characterized in that selected from the group consisting of synthetic compounds, microbial cultures or extracts, synthetic peptides, nucleic acids, proteins, antibodies, aptamers and natural extracts.

In another embodiment of the present invention, the mesenchymal stem cells are characterized in that the stem cells derived from bone marrow or adipose tissue.

Screening method for preventing or treating osteoporosis using mesenchymal stem cells according to the present invention, and for screening a health functional food composition for preventing or improving osteoporosis, by selecting a candidate substance for preventing or treating osteoporosis within a week, at least 10 days Compared with the conventional screening method, which is necessary, the efficiency can be significantly increased. In addition, the inventors first revealed that leptin, which can control various physiological activities, is produced in mesenchymal stem cells, and thus, the screening method of the present invention can be used for screening substances for preventing and / or treating osteoporosis, It is expected that the present invention may be applied to screening of prophylactic and / or therapeutic substances for various leptin related diseases.

1 is a schematic diagram showing briefly the features of the present invention.

Figure 2 shows the results of measuring leptin secretion in mesenchymal stem cells, adipocyte differentiation or osteoblast differentiation induced mesenchymal stem cells.

3 is a view showing the effect of osteoporosis therapeutic agent on leptin secretion capacity of mesenchymal stem cells or adipose differentiation mesenchymal stem cells.

The inventors of the present invention have completed the present invention as a result of research on a method for selecting osteoporosis therapeutic substances in an easy manner in a short time in order to develop an osteoporosis prevention agent and / or a therapeutic agent having low side effects and excellent therapeutic effects.

Since leptin is known to control various physiological activities, there have been attempts to screen new drugs using adipocytes, which are known to secrete leptin, but adipocytes are not easy to separate cells. Since the sensitivity of leptin receptors differs among individuals, the method of measuring leptin secretion using primary cultured adipocytes is limited in the screening of new drugs and health foods due to the large deviation.

The present inventors confirmed that leptin is also secreted from mesenchymal stem cells that can be easily separated from bone marrow or adipose tissue, compared to adipocytes, and developed a screening method using leptin secretion ability of mesenchymal stem cells. Mesenchymal stem cells can be easily separated from bone marrow or adipose tissue and are easy to culture. In addition, it was expected that the reliability of the screening method could be improved due to the small variation among individuals.

In one embodiment of the present invention, it was confirmed that mesenchymal stem cells also secrete leptin (see Example 1), and in other embodiments, the amount of leptin secreted from mesenchymal stem cells increases rapidly when the osteoporosis treatment agent is treated. It was confirmed (see Example 2).

From the above results, it was confirmed that the screening method of the present invention can be used to select candidate substances that can be used as a therapeutic or prophylactic agent for osteoporosis in a short time and efficiently.

Accordingly, the present invention comprises the steps of treating and culturing the test substance in mesenchymal stem cells; Measuring the amount of leptin in the cell culture by enzyme-linked antibody attachment reaction; And it provides a screening method of preventing or treating osteoporosis prevention agent comprising the step of selecting a candidate substance to significantly increase the leptin amount compared to the control group was not treated.

In another aspect, the present invention comprises the steps of treating and culturing the test substance in mesenchymal stem cells; Measuring the leptin amount in the cell culture by an enzyme-linked antibody attachment reaction; And it provides a method for screening a health functional food composition for osteoporosis prevention or improvement comprising the step of selecting a candidate substance to significantly increase the amount of leptin compared to the control group was not treated.

There is no restriction on the period of treatment and incubation of the test substance to the mesenchymal stem cells, preferably 1 day to 10 days, more preferably within a week.

In addition, there is no limitation on the type of the test substance. Preferably it may be selected from the group consisting of synthetic compounds, microbial cultures or extracts, synthetic peptides, nucleic acids, proteins, antibodies, aptamers and natural extracts.

[실시예]EXAMPLE

실시예 1. 인간 골수 중간엽 줄기세포의 렙틴 분비능 측정Example 1 Measurement of Leptin Secretion of Human Bone Marrow Mesenchymal Stem Cells

To determine if leptin is secreted from human bone marrow mesenchymal stem cells (hBM-MSCs), 10% fetal bovine serum (FBS) was added to mesenchymal stem cells. Incubated in DMEM (Dulbecco Modified Eagle's Medium) medium at 37 ℃, 5% CO ₂ conditions. In addition, in order to determine whether changes in leptin secretion are differentiated into fat cells and osteoblasts, 1ug / mL of insulin, dexamethasone (DEXA) 100nM, and 0.5 mM of isobutylmethylxanthine (IBMX) was added or mixed, and troglitazone was added as a control to incubate. For osteoblast differentiation, dexamethasone 100 nM, ascorbic acid (Vit. C) 50 uM, and beta-glycerol phosphate (beta-glycerol phosphate) 10 mM were added or mixed, respectively. As a control, cells cultured under conditions inducing adipocyte differentiation (Adipo) were used. Each cultured cell is shown in Figure 2 by measuring the cell differentiation and leptin secretion.

In order to induce differentiation into adipocytes, fat bodies in cells treated with insulin, dexamethasone, and isobutylmethylxanthine were cultured using Oil Red O (ORO) staining.

As shown in Fig. 2 (a), the cells treated with each other had almost no increase in the amount of fat body compared with the cells not treated, but all three were mixed with the cells treated with dexamethasone and isobutylmethylxanthine. Cells treated by was confirmed that the amount of fat body increased by more than five times compared to the cells not treated. On the other hand, as shown in Fig. 2 (c), the amount of leptin in the cells treated with a mixture of dexamethasone and isobutylmethylxanthine induced in differentiation into adipocytes and all three treated cells did not increase much, It was confirmed that the cells treated with dexamethasone alone increased more than 30 times compared with the cells not treated.

To induce differentiation into osteoblasts, alkaline phosphatase (ALP) activity, which is used as an indicator of osteoblast differentiation, was measured using cells cultured with dexamethasone, vitamin C and betaglycerol phosphate.

As shown in Fig. 2 (b), cells treated with only dexamethasone-treated cells and dexamethasone and vitamin C showed approximately twice the ALP activity as compared to untreated cells, and mixed dexamethasone and betaglycerol phosphate. Treated cells showed about three times higher activity. And the cells treated with all three mixtures showed four times more activity, confirming that osteoblast differentiation was promoted. In addition, leptin secretion was increased in cells treated with dexamethasone alone, cells treated with dexamethasone mixed with vitamin C, cells treated with mixed dexamethasone and betaglycerol phosphate, and cells treated with all three (FIG. 2 (d)).

These results confirm that mesenchymal stem cells secrete leptin, adipocyte differentiation does not affect leptin secretion ability, and leptin secretion is increased in osteoblast differentiation-induced mesenchymal stem cells.

실시예 2. 인간 골수 중간엽 줄기세포의 렙틴 분비능을 이용한 골다공증 약물 및 염증매개인자의 렙틴 분비에 대한 영향Example 2 Effect on Leptin Secretion of Osteoporosis Drugs and Inflammatory Mediators Using Leptin Secretion of Human Bone Marrow Mesenchymal Stem Cells

To determine the effects of osteoporosis therapeutics on leptin secretion in mesenchymal stem cells, dexamethasone, alendronate, raloxifene, strontium ranelate, interleukin- 17 (interleukin-17, IL-17) and interleukin-11 (IL-11) were treated for 3 days and then incubated. After incubation for 3 days, the amount of leptip accumulated in the cell culture supernatant was quantified using an enzyme linked immunosorbent assay (ELISA). Adipocyte differentiation mesenchymal stem cells were induced for adipocyte differentiation for 15 days and then treated with osteoporosis treatment and cultured for 3 days to measure the amount of leptin. The results are shown in FIG.

As shown in Figure 3, when treated with osteoporosis treatment on adipose-derived mesenchymal stem cells, it was confirmed that the leptin secretion capacity was increased by about five times compared to the control group, but in mesenchymal stem cells rapidly up to 100 times It was confirmed that the increase. The results indicate that mesenchymal stem cells are more sensitive to osteoporosis therapeutic agents than adipose differentiation mesenchymal stem cells, and thus, the method of measuring leptin secretion of mesenchymal stem cells is shorter than conventional methods. It was confirmed that it can be easily used to select a therapeutic agent for osteoporosis within.

The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it is to be understood that the embodiments described above are exemplary in all respects and not restrictive.

Screening method for the prevention or treatment of osteoporosis using mesenchymal stem cells according to the present invention, and screening method for a health functional food composition for preventing or improving osteoporosis, selection of candidate substances for the prevention or treatment of osteoporosis takes a minimum of 10 days or more Compared with the existing screening method, the efficiency is significantly higher, and thus it is easier to supply a preventive or therapeutic agent for osteoporosis. In addition, the inventors first revealed that leptin, which can control various physiological activities, is produced in mesenchymal stem cells, and the screening method of the present invention can be used for screening substances for preventing and / or treating osteoporosis. It can also be applied to the selection of prophylactic and / or therapeutic substances for various leptin related diseases.

Claims

Treating and culturing the test substance in mesenchymal stem cells;

Measuring the amount of leptin in the cell culture by enzyme-linked antibody attachment reaction; And

A method for screening a prophylactic or therapeutic agent for osteoporosis, comprising selecting a candidate substance which significantly increases the amount of leptin as compared to a control which is not treated with a test substance.
The method of claim 1,

Treating and culturing the test substance to the mesenchymal stem cells, characterized in that the culture within a week.
The method of claim 1,

The test substance is characterized in that selected from the group consisting of synthetic compounds, microbial cultures or extracts, synthetic peptides, nucleic acids, proteins, antibodies, aptamers and natural extracts.
The method of claim 1,

The mesenchymal stem cells, characterized in that the stem cells derived from bone marrow or adipose tissue.
Treating and culturing the test substance in mesenchymal stem cells;

Measuring the amount of leptin in the cell culture by enzyme-linked antibody attachment reaction; And

A method for screening a nutraceutical composition for preventing or improving osteoporosis, comprising selecting a candidate substance that significantly increases the amount of leptin as compared to a control which has not been treated with a test substance.
The method of claim 5,

Treating and culturing the test substance to the mesenchymal stem cells, characterized in that the culture within a week.
The method of claim 5,

The test substance is characterized in that selected from the group consisting of synthetic compounds, microbial cultures or extracts, synthetic peptides, nucleic acids, proteins, antibodies, aptamers and natural extracts.
The method of claim 5,

The mesenchymal stem cells, characterized in that the stem cells derived from bone marrow or adipose tissue.