WO2014038864A1 - Composition for preventing and treating immune disease using human fetal cartilage-derived cells - Google Patents

Composition for preventing and treating immune disease using human fetal cartilage-derived cells Download PDF

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WO2014038864A1
WO2014038864A1 PCT/KR2013/008016 KR2013008016W WO2014038864A1 WO 2014038864 A1 WO2014038864 A1 WO 2014038864A1 KR 2013008016 W KR2013008016 W KR 2013008016W WO 2014038864 A1 WO2014038864 A1 WO 2014038864A1
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cells
immune
derived
fetal
derived cells
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PCT/KR2013/008016
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French (fr)
Korean (ko)
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민병현
박소라
이수정
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아주대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

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  • the present invention relates to a novel immunomodulatory composition for regenerating damaged tissues, and to a composition for the prevention and treatment of immune diseases using fetal cartilage-derived cells with superior proliferative power or cell phenotype maintenance than known adult stem cells.
  • Adult stem cells are stem cells capable of differentiation into ground, cartilage, bone, muscle cells, etc., and are highly proliferated and have high clinical value for tissue regeneration.
  • certain adult stem cells such as mesenchymal stem cells, have immunosuppressive ability without causing an immune response. (Placenta-Derived Multipotent Cells Exhibit Immunosuppressive Properties That Are Enhanced in the Presence of Interferon- ⁇ , STEM CELLS, 2006), but there are no reports on stem cells with immunomodulatory capacity.
  • Immunomodulators known as drugs that enhance the immune function of living organisms, enhance the immune function of cancer patients by using immunostimulators in the treatment based on the immunological background of cancer patients who have proven to be impaired in immune response at various stages. So-called nonspecific immunotherapy, which also induces immunity against, has been widely practiced. In the case of using live bacteria, cell wall skeleton (CWS) is frequently used as a purified cell component because of its stability and side effects. Immunomodulators are mainly used for cancer treatment.
  • CWS cell wall skeleton
  • An object of the present invention is to provide a composition for the prevention or treatment of immune diseases using fetal cartilage-derived cells that can suppress the proliferation of activated lymphocytes without causing an immune response.
  • the present inventors have found that cells isolated from fetal chondrocytes can overcome the limitations of existing stem cells by regulating the immune response that may occur during transplantation with regeneration of damaged tissue. It has led to the development of immunomodulatory compositions that can be used as an alternative.
  • the present invention provides a composition for the prevention or treatment of immune diseases containing fetal cartilage-derived cells as an active ingredient.
  • the fetal chondrocyte-derived cells may express the surface antigen proteins of stem cells, and in one embodiment, the fetal chondrocyte-derived cells express CD29, CD44, CD90 and CD105, which are specific surface antigens of adult bone marrow-derived mesenchymal stem cells. In addition, embryonic stem cell-specific surface antigens can express SSEA4 and Oct-4.
  • the fetal cartilage-derived cells can control the expression of immune cytokines produced in activated lymphocytes.
  • the fetal cartilage-derived cells can inhibit the expression of TNF- ⁇ , IL-13, IFN- ⁇ related to immune or inflammatory responses.
  • the fetal cartilage-derived cells can modulate immune activity by not affecting or increasing the expression of anti-inflammatory cytokine IL-10.
  • the immunomodulatory composition may include fetal cartilage-derived cell extracts or secreted molecules.
  • the fetal cartilage-derived cells are (a) separating and culturing chondrocytes from fetal cartilage tissue; (b) separating the cells expressing the surface antigen proteins of the stem cells from the cells cultured in step (a); And (c) it can be obtained through the step of verifying the proliferation capacity and multi-differentiation capacity of the cells isolated in step (b) through fetal cartilage derived stem cells.
  • the immunomodulation of fetal cartilage-derived cells is (a) a method for analyzing the proliferation of lymphocytes for cells when cultured together with cells isolated from fetal cartilage tissue and lymphocytes isolated from allogeneic whole blood, and (b) interferon known as immunointerferon. Analysis of the characteristics of cells isolated from fetal chondrocytes reacted with gamma, or (c) to determine the immunoregulatory cytokines secreted during the culturing in step (a) or one or more of the methods. Can be.
  • Fetal cartilage-derived cells according to the present invention do not cause an immune response, but rather have the property of inhibiting the proliferation of activated lymphocytes and controlling the expression of immune cytokines produced in the activated lymphocytes, and proliferating ability and various cell tissues.
  • the differentiation ability of is superior to that of adult stem cells, and can be usefully used as an immunomodulator to control the immune response that may occur during transplantation with regeneration of damaged tissues.
  • fetal cartilage-derived cells do not induce an immune response as a result of analyzing the proliferation of lymphocytes after mixed culture of lymphocytes isolated from fetal cartilage-derived cells and allogeneic blood that inhibited proliferation.
  • Figure 2 shows that fetal chondrocyte-derived cells and adult chondrocytes that inhibited proliferation were treated with mixed lymphocyte reactions using ConA, respectively, and the proliferation of lymphocytes was analyzed. Is to show.
  • Figure 3 shows the immunogenicity of fetal cartilage-derived cells with or without interferon stimulation by flow cytometry shows that the fetal cartilage-derived cells are not activated by interferon gamma (IFN- ⁇ ).
  • IFN- ⁇ interferon gamma
  • Figure 5 shows the mitigating effect of fetal cartilage-derived cells using an animal model.
  • the present invention provides a composition for the prevention or treatment of immune diseases using fetal cartilage tissue-derived cells as an active ingredient that can overcome the limitations of the existing stem cells by regulating the immune response that may occur during transplantation with damaged tissue regeneration. do.
  • the composition for the prevention or treatment of clinically available immune diseases was confirmed by confirming that the immune control ability was not caused. to provide.
  • the fetal cartilage-derived cells are (a) confirming the proliferation of lymphocytes to cells when cultured together with cells isolated from fetal chondrocytes and lymphocytes isolated from allogeneic whole blood, or (b) reacted with interferon gamma known as immunointerferon.
  • the immunomodulatory function can be confirmed by analyzing the characteristics of the cells isolated from fetal cartilage tissue, or (c) identifying the immunoregulatory cytokines secreted during the culturing in step (a). Analysis of whether the immunomodulatory function of can be analyzed by conventional methods known to those skilled in the art, so is not limited thereto.
  • the fetal cartilage-derived cells can express the bone marrow-derived mesenchymal stem cell-specific surface antigens CD29, CD44, CD90, and CD105 in adults, while expressing the embryonic stem cell-specific surface antigens SSEA4 and Oct-4.
  • the fetal cartilage-derived cells are (a) separating and culturing chondrocytes from fetal cartilage tissue; (b) separating the cells expressing the surface antigen proteins of the stem cells from the cells cultured in step (a); And (c) it can be obtained through the step of verifying the proliferation capacity and multi-differentiation capacity of the cells isolated in step (b) through fetal cartilage derived stem cells.
  • Chondrocytes isolated from fetal chondrocytes can be obtained from 12-15 weeks of aborted fetus using collagenase (Collagenase) or obtained through trituration.
  • the fetal chondrocyte-derived cells may express the surface antigen proteins of stem cells, and in one embodiment, the fetal chondrocyte-derived cells express CD29, CD44, CD90 and CD105, which are specific surface antigens of adult bone marrow-derived mesenchymal stem cells. In addition, embryonic stem cell-specific surface antigens can express SSEA4 and Oct-4.
  • the fetal cartilage-derived cells can control the expression of immune cytokines produced in activated lymphocytes. More specifically, the fetal cartilage-derived cells can inhibit the expression of TNF- ⁇ , IL-13, IFN- ⁇ related to immune or inflammatory responses. In addition, the fetal cartilage-derived cells can modulate immune activity by not affecting or increasing the expression of anti-inflammatory cytokine IL-10.
  • the simultaneous expression of the surface antigens of mesenchymal stem cells and the surface antigens of embryonic stem cells in the fetal chondrocyte-derived cells are characterized by Fluorescent-Assisted Cell Sorting (FACS) and Magnetic-Activated Cells. It can be analyzed by immunochemical separation methods including Cell Sorting (MACS) method.
  • FACS Fluorescent-Assisted Cell Sorting
  • MCS Cell Sorting
  • the fetal cartilage-derived cells include musculoskeletal tissue cells, including cartilage, bone, muscle, skin and fat cells; Nerve cell; Hepatocytes; Pancreatic cell; Cardiomyocytes; And it may have a multipotent ability to differentiate into any one of the vascular cells.
  • fetal cartilage-derived cells of the present invention do not stimulate allogeneic lymphocytes, and when lymphocyte activation is induced, the proliferation of lymphocytes is reduced, in particular TNF- ⁇ and IL-13 associated with immune responses.
  • IL-10 which is known as an anti-inflammatory cytokine, inhibits the expression of IFN- [gamma] but does not affect or increase the immune activity.
  • the immunomodulatory composition according to the present invention includes a cell therapeutic agent, but is not limited thereto and may be utilized in various therapeutic forms. Specifically, it may include fetal cartilage-derived cell extracts or secreted molecules. Fetal cartilage-derived cell extracts or secreted molecules may mean any form that can be obtained from fetal cartilage-derived cells, as well as the cells themselves, suspensions or cultures thereof, or concentrates or pastes thereof. It may mean a product, a dry product, a dilution or any product made through metabolism of the cells.
  • the immune disease may be an autoimmune disease, an inflammatory disease and a transplant rejection disease of cells, tissues or organs, and specifically, arthritis, autoimmune hepatitis, rheumatoid arthritis, osteoarthritis, insulin dependent diabetes mellitus, ulcerative colitis, Crohn's disease, Multiple sclerosis, autoimmune myocarditis, scleroderma, myasthenia gravis, multiple myositis / skin myositis, Hashimoto's disease, autoimmune cytopenia, Sjogren's syndrome, vasculitis syndrome, and systemic lupus erythematosus.
  • arthritis autoimmune hepatitis, rheumatoid arthritis, osteoarthritis, insulin dependent diabetes mellitus, ulcerative colitis, Crohn's disease, Multiple sclerosis, autoimmune myocarditis, scleroderma, myasthenia gravis, multiple myositis / skin myositis, Hashimoto's disease
  • composition for preventing or treating immunological diseases of one embodiment of the present invention may be prepared in various mixtures with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier for example, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavorings and the like can be used in combination as pharmaceutically acceptable carriers.
  • buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers and the like can be used in combination.
  • injectables may be prepared in unit dosage ampoules or in multiple dosage forms.
  • bases, excipients, lubricants, preservatives and the like can be used during topical administration.
  • the present invention is not limited thereto, and those skilled in the art may form a composition for preventing or treating the immune disease in various forms, and may include various carriers or additives. I will understand.
  • composition of the present invention can be administered by a variety of routes to mammals, such as rats, mice, livestock, humans. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.
  • dosage of the active ingredient in the composition may vary depending on the route of administration, the degree of disease, the age, sex, and weight of the patient, and may be administered once to several times daily.
  • stem cells are cells that have the ability to differentiate into various kinds of body tissues, that is, undifferentiated cells.
  • the stem cells include adult stem cells (multipotent stem cells) such as embryonic stem cells (pluripotent stem cells) that can be made using human embryos, and bone marrow cells that constantly make blood cells. .
  • Embryonic Stem Cell is an embryonic cell less than 14 days old, and can be differentiated into all cells and tissues constituting the human body in the future, 'almighty cell' or 'pluripotent cell' It is called Fertilized eggs form blastocysts through cell division, inside which there is a mass of cells called inner cell masses, which form embryos. When the cells of this inner cell mass are isolated from the blastocyst and cultured, they become embryonic stem cells that do not differentiate but still have differentiation capacity.
  • “Adult Stem Cell” is extracted from umbilical cord blood (umbilical cord blood) or mature adult bone marrow and blood, and the raw material immediately before differentiation into cells of specific organs such as bone, liver, and blood. It's a cell. These include hematopoietic stem cells, mesenchymal stem cells, and neural stem cells, which are in the spotlight of regenerative medicine. In addition, there are stem cells in the liver, epidermis, and pancreas. The adult stem cells are difficult to proliferate and tend to be easily differentiated. Instead, the adult stem cells can be regenerated according to the characteristics of each organ after transplantation by using various types of adult stem cells as well as the regeneration of organs required by actual medicine. It has characteristics that can be.
  • MSC meenchymal stem cells
  • bone bone
  • cartilage cartilage
  • fat fat
  • tendon tendon
  • nerve tissue nerve tissue
  • fibroblast fibroblast
  • multipotent progenitors before they are differentiated into cells of specific organs such as muscle cells.
  • the "cell therapeutic agent” is a medicine (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared through isolation, culture, and special chewing from humans. It refers to a medicine used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating, selecting, or otherwise changing the biological characteristics of a living autologous, allogeneic, or heterologous cell in vitro.
  • Cell therapy agents are largely classified into somatic cell therapy and stem cell therapy according to the degree of differentiation of cells, and the present invention relates in particular to stem cell therapy.
  • fetal cartilage-derived cells were carried out as described in detail in patent application 2013-0090233 [Fetal Cartilage Tissue-Derived Stem Cell Sources and Cell Therapeutic Agents Comprising the Same]. That is, the collected cartilage tissue is washed several times with phosphate buffered saline (PBS) and finely chopped and cultured with 0.1% collagenase and 1% bovine serum (FBS). After treatment, the cells were left for 12 hours in an incubator at 37 ° C. and 5% CO 2 . Cells isolated by collagenase were washed with a culture medium containing 10% FBS and the method was as follows.
  • PBS phosphate buffered saline
  • FBS bovine serum
  • the cells suspended in the culture medium was centrifuged at 1700 rpm for 10 minutes, the supernatant was removed, the collected cells were obtained, and then suspended again in the culture medium. At this time, after staining some cells with trypan blue (trypan Blue) the cell number was measured using a hemocytometer under an optical microscope.
  • the cells suspended in the culture medium were dispensed into a culture vessel at a concentration of 8 ⁇ 10 4 / cm 2 and then cultured in an incubator at 37 ° C. and 5% CO 2 . Subculture was done when the cells filled 80-90% of the culture vessel.
  • the obtained fetal chondrocyte-derived cells were analyzed by flow cytometry (FACScantoII), and CD29 (Integrin ⁇ 1 chain), CD44, which is widely known as a cell surface factor of bone marrow-derived mesenchymal stem cells.
  • FACScantoII flow cytometry
  • CD29 Integrin ⁇ 1 chain
  • CD44 which is widely known as a cell surface factor of bone marrow-derived mesenchymal stem cells.
  • HA receptor HA receptor
  • CD 90 Thy-1
  • CD105 Endoglin
  • peripheral blood-derived lymphocytes generally used a method of separating lymphocytes from whole blood using Ficoll.
  • lymphocytes isolated from allogeneic blood were divided into 10 5 cells on the same plate and cultured together for 4 days.
  • the ratio of cell division between fetal cartilage-derived cells and lymphocytes was 1: 1000, 1: 100 and 1:10.
  • concanavalin A T cell mitogen
  • 10 ⁇ g / ml was added together with 10 ⁇ g / ml and cultured for 4 days in order to induce activity in the lymphocyte-only group.
  • the suspended lymphocytes and supernatants were collected together and subjected to BrdU assay (Cell proliferation ELISA, BrdU, Roche Diagnostice, Germany).
  • Lymphocytes treated with concanavalin A showed increased proliferation than lymphocytes not treated. It was found that the isolated lymphocytes are normally activated. And it was confirmed that lymphocytes do not proliferate in the group treated with fetal cartilage-derived cells (see FIG. 1). It was determined that fetal cartilage-derived cells do not activate lymphocytes.
  • Isolation and culturing of fetal cartilage-derived cells were performed in the same manner as in the previous example, and the separation of peripheral blood-derived lymphocytes was generally performed using Ficoll to separate lymphocytes from whole blood.
  • lymphocyte proliferation was performed to confirm the inhibition of lymphocyte proliferation of fetal chondrocytes.
  • fetal cartilage-derived cells were treated with gamma rays (3000rad, 5 minutes) to inhibit proliferation, and the cells were divided into 10 3 and 10 4 cells in 96 well plates.
  • lymphocytes isolated from allogeneic blood were dispensed in 10 5 aliquots on the same plate and cultured together for 4 days.
  • the ratio of cell division between fetal cartilage-derived cells and lymphocytes was 1: 100 and 1:10.
  • conkanavalin A T cell mitogen
  • 10 ⁇ g / ml a cell mitogen
  • the suspended lymphocytes and supernatants were collected together and subjected to BrdU assay (Cell proliferation ELISA, BrdU, Roche Diagnostice, Germany).
  • BrdU assay Cell proliferation ELISA, BrdU, Roche Diagnostice, Germany.
  • the adult chondrocytes used as a comparison group were also experimented using the same method.
  • Lymphocytes treated with concanavalin A showed increased proliferation than lymphocytes not treated.
  • the proliferation of lymphocytes decreased as the number of fetal cartilage-derived cells increased (see FIG. 2).
  • Fetal cartilage-derived cells were determined to have properties that could inhibit the proliferation of lymphocytes activated with Concanavalin A.
  • lymphocytes were activated and proliferated in adult chondrocytes. It can be seen that adult chondrocytes do not have the ability to inhibit lymphocyte proliferation.
  • Interferon gamma IFN- ⁇
  • IFN- ⁇ Interferon gamma
  • HLA human leukocyte antigens
  • CD80 CD86 (B7.1, B7.2), which are activated co-stimulatory molecules when inducing signaling to T cells of antigen-presenting cells in the immune response, it was confirmed that there was no change in CD80. (See FIG. 3).
  • fetal cartilage-derived cells do not have antigens that can trigger immune responses and inhibit the proliferation of allogeneic lymphocytes.
  • mixed lymphocyte reaction was performed to confirm the cytokine production of fetal cartilage-derived cells.
  • fetal cartilage-derived cells were treated with gamma rays (3000rad, 5 minutes) to inhibit proliferation, and then divided into 12 well plates with 10 5 and 10 6 cells. After 16 hours, isolated lymphocytes were dispensed for 10 6 minutes. At this time, the ratio of cell division between fetal cartilage-derived cells and lymphocytes was 1:10 and 1: 1.
  • concanavalin A T cell mitogen
  • allogeneic lymphocytes, lymphocytes treated with concanavalin A, and fetal cartilage-derived cells were cultured. After 4 days of culture, the supernatant was collected and centrifuged at 2000 rpm for 5 minutes to remove suspended lymphocytes, and then the supernatant was collected and subjected to cytokine-ELISA analysis (eBioscience, Ready-SET-Go ELISA set).
  • Tumor necrosis factor-alpha (TNF- ⁇ ), a cytokine in acute immune and inflammatory reactions, was produced in the group of lymphocytes activated by concanavalin A, but incubated with fetal cartilage-derived cells. It was confirmed that no generation was made in the group (see FIG. 4 (A)).
  • interleukin-13 IL-13
  • IL-13 interleukin-13
  • IFN- ⁇ immunostimulation gamma
  • Interleukin-10 produced by regulatory T cells or dendritic cells is a cytokine that is used as an anti-inflammatory agent by inducing immune tolerance by inhibiting immune cells that attack autologous tissues.
  • IL-10 Interleukin-10
  • lymphocytes When fetal cartilage-derived cells and lymphocytes were reacted, it was confirmed that the production of interleukin-10 increased as the ratio of fetal cartilage-derived cells increased (see FIG. 4 (D)).
  • Rats of the fetal cartilage-derived cells injected with immunomodulatory capacity were confirmed to have reduced edema by decreasing knee circumference from day 7 (see FIG. 5).
  • the known immunomodulatory agent has a limitation that can not play a role in the treatment of tissue transplantation or tissue regeneration, fetal cartilage-derived cells of the present invention is more proliferative and differentiating ability than adult stem cells, which are widely studied as cell therapy, homogeneous
  • the immunomodulatory composition according to the present invention can be utilized in various therapeutic forms, including cell therapy.

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Abstract

The present invention relates to a composition for preventing or treating an immune disease using human fetal cartilage-derived cells, which can suppress/control an immune response to a tissue transplant. More specifically, the human fetal cartilage-derived cells do not cause an immune response but rather suppress proliferation of activated lymphocytes, control the expression of immune cytokine, which is generated in the activated lymphocytes, and have better proliferation and differentiation capability into a variety of cell tissues compared to adult stem cells, and thus can be useful in regenerating damaged tissue and as an immune control agent for controlling an immune response to a transplant.

Description

태아연골유래 세포를 이용한 면역질환의 예방 및 치료용 조성물Composition for the prevention and treatment of immune diseases using fetal cartilage-derived cells
본 발명은 손상된 조직을 재생하기 위한 새로운 면역조절용 조성물로서, 기존에 알려진 성체 줄기세포보다 증식력이나 세포 표현형 유지가 뛰어난 태아연골유래 세포를 이용한 면역질환의 예방 및 치료용 조성물에 관한 것이다.The present invention relates to a novel immunomodulatory composition for regenerating damaged tissues, and to a composition for the prevention and treatment of immune diseases using fetal cartilage-derived cells with superior proliferative power or cell phenotype maintenance than known adult stem cells.
난치성 조직 손상에 대한 치료 방법으로서 최근 줄기세포 및 조직공학을 이용한 이식치료법이 활발히 개발되고 있다. 하지만, 이러한 이식치료 기법으로는 치료효과가 뛰어나고 인체의 면역반응을 일으키지 않는 안정한 세포치료제를 개발하기 어렵다는 문제가 있다. 그 동안 여러 가지 성체 줄기세포를 개발하여 세포치료제로써 활용하기 위한 시도들이 진행되고 있지만, 면역반응이 없다는 것만 밝혀졌을 뿐 면역조절능력에 대한 확인은 아직 부분적으로만 진행되고 있다. Recently, as a treatment method for intractable tissue damage, transplantation therapy using stem cells and tissue engineering has been actively developed. However, there is a problem that it is difficult to develop a stable cell therapy which is excellent in therapeutic effect and does not cause an immune response by the transplantation technique. Attempts have been made to develop various adult stem cells and use them as cell therapies. However, it has been found that there is no immune response, but the identification of immunomodulatory capacity is still in progress.
성체줄기세포는 지반, 연골, 골, 근육세포 등으로 분화가 가능한 줄기세포로 증식이 잘되어 조직재생을 위한 임상적 가치가 높은 세포이다. 기존에 중간엽줄기세포(mesenchymal stem cells)와 같은 특정 성체줄기세포가 자체로 면역반응을 일으키지 않고 면역억제능력이 있다는 보고(Placenta-Derived Multipotent Cells Exhibit Immunosuppressive Properties That Are Enhanced in the Presence of Interferon-γ, STEM CELLS,2006)가 나오고 있지만, 아직까지 면역조절능력을 가진 줄기세포에 대한 연구보고는 없는 상황이다. Adult stem cells are stem cells capable of differentiation into ground, cartilage, bone, muscle cells, etc., and are highly proliferated and have high clinical value for tissue regeneration. In the past, certain adult stem cells, such as mesenchymal stem cells, have immunosuppressive ability without causing an immune response. (Placenta-Derived Multipotent Cells Exhibit Immunosuppressive Properties That Are Enhanced in the Presence of Interferon-γ , STEM CELLS, 2006), but there are no reports on stem cells with immunomodulatory capacity.
생체의 면역작용을 강화하는 약물로 알려진 면역조절제는 여러 단계에서 면역응답기능에 장애가 생긴다고 증명된 암환자의 면역학적 배경을 바탕으로 치료에 면역촉진제 등을 사용하여 암환자의 면역기능을 증강하고 암에 대한 면역저항성도 유도하는, 이른바 비특이적 면역요법이 널리 시행되고 있다. 생균을 사용하는 경우, 역가의 안정성이나 부작용도 많으므로 정제한 균체성분으로서 세포벽 골격성분(CWS ; cell wall skeleton)이 자주 쓰인다. 면역조절제는 주로 암치료를 목적으로 사용되고 있다. Immunomodulators, known as drugs that enhance the immune function of living organisms, enhance the immune function of cancer patients by using immunostimulators in the treatment based on the immunological background of cancer patients who have proven to be impaired in immune response at various stages. So-called nonspecific immunotherapy, which also induces immunity against, has been widely practiced. In the case of using live bacteria, cell wall skeleton (CWS) is frequently used as a purified cell component because of its stability and side effects. Immunomodulators are mainly used for cancer treatment.
따라서, 줄기세포 및 조직공학을 이용한 이식치료법을 시행 시 인체 내의 불필요한 면역반응을 선별적으로 조절할 수 있는 바람직한 면역조절제의 개발이 매우 중요한 실정이다. Therefore, it is very important to develop a desirable immunomodulator that can selectively regulate unnecessary immune responses in the human body when performing transplantation therapy using stem cells and tissue engineering.
본 발명의 목적은 면역반응을 일으키지 않고, 오히려 활성화된 림프구의 증식을 억제할 수 있는 태아연골유래 세포를 이용한 면역질환의 예방 또는 치료용 조성물을 제공하는 데에 있다.An object of the present invention is to provide a composition for the prevention or treatment of immune diseases using fetal cartilage-derived cells that can suppress the proliferation of activated lymphocytes without causing an immune response.
상기 목적을 달성하기 위하여, 본 발명자는 태아연골조직으로부터 분리한 세포가 손상된 조직의 재생과 함께 이식 시 발생할 수 있는 면역반응을 조절함으로써 기존의 줄기세포의 한계점을 극복할 수 있다는 점을 밝혀내어 임상적으로 활용 가능한 면역조절용 조성물을 개발하기에 이르렀다.In order to achieve the above object, the present inventors have found that cells isolated from fetal chondrocytes can overcome the limitations of existing stem cells by regulating the immune response that may occur during transplantation with regeneration of damaged tissue. It has led to the development of immunomodulatory compositions that can be used as an alternative.
본 발명은 태아연골유래 세포를 유효성분으로 함유하는 면역질환의 예방 또는 치료용 조성물을 제공한다.The present invention provides a composition for the prevention or treatment of immune diseases containing fetal cartilage-derived cells as an active ingredient.
상기 태아연골유래 세포는 줄기세포의 표면항원 단백질을 발현할 수 있으며, 일실시예로는 상기 태아연골유래 세포는 성인의 골수유래 중간엽줄기세포 특이 표면항원인 CD29, CD44, CD90 및 CD105를 발현하면서도, 배아줄기세포 특이 표면항원인 SSEA4와 Oct-4를 발현할 수 있다.The fetal chondrocyte-derived cells may express the surface antigen proteins of stem cells, and in one embodiment, the fetal chondrocyte-derived cells express CD29, CD44, CD90 and CD105, which are specific surface antigens of adult bone marrow-derived mesenchymal stem cells. In addition, embryonic stem cell-specific surface antigens can express SSEA4 and Oct-4.
상기 태아연골유래 세포는 활성화 림프구에서 생성되는 면역 싸이토카인의 발현을 제어할 수 있다.The fetal cartilage-derived cells can control the expression of immune cytokines produced in activated lymphocytes.
보다 상세하게는, 상기 태아연골유래 세포는 면역반응 또는 염증반응에 관련된 TNF-α, IL-13, IFN-γ의 발현은 억제할 수 있다. 또한, 상기 태아연골유래 세포는 항염증 싸이토카인인 IL-10의 발현은 영향을 주지 않거나 증가시킴으로써 면역활성을 조절할 수 있다.More specifically, the fetal cartilage-derived cells can inhibit the expression of TNF-α, IL-13, IFN-γ related to immune or inflammatory responses. In addition, the fetal cartilage-derived cells can modulate immune activity by not affecting or increasing the expression of anti-inflammatory cytokine IL-10.
상기 면역 조절용 조성물은 태아연골유래 세포 추출물(cell extracts) 또는 분비물(secreted molecules)을 포함할 수 있다.The immunomodulatory composition may include fetal cartilage-derived cell extracts or secreted molecules.
상기 태아연골유래 세포는 (a) 태아연골조직에서 연골세포를 분리하여 배양하는 단계; (b) 상기 (a) 단계에서 분리 배양된 세포에서 줄기세포의 표면항원 단백질을 발현하는 세포를 분리하는 단계; 및 (c) 상기 (b) 단계에서 분리된 세포의 증식능 및 다분화 능력을 확인하여 태아연골유래 줄기세포를 검증하는 단계를 통해 수득할 수 있다.The fetal cartilage-derived cells are (a) separating and culturing chondrocytes from fetal cartilage tissue; (b) separating the cells expressing the surface antigen proteins of the stem cells from the cells cultured in step (a); And (c) it can be obtained through the step of verifying the proliferation capacity and multi-differentiation capacity of the cells isolated in step (b) through fetal cartilage derived stem cells.
상기 태아연골유래 세포의 면역조절은 (a) 태아연골조직에서 분리한 세포와 동종 전혈에서 분리한 림프구를 함께 배양 시 세포에 대한 림프구의 증식여부를 분석하는 방법, (b) 면역인터페론으로 알려진 인터페론 감마에 반응시킨 태아연골조직에서 분리한 세포의 특성을 분석하는 방법, 또는 (c) 상기 (a) 단계에서 배양 시 분비되는 면역조절 싸이토카인을 확인하는 방법 중 어느 하나 또는 둘 이상의 방법을 통해 분석할 수 있다. The immunomodulation of fetal cartilage-derived cells is (a) a method for analyzing the proliferation of lymphocytes for cells when cultured together with cells isolated from fetal cartilage tissue and lymphocytes isolated from allogeneic whole blood, and (b) interferon known as immunointerferon. Analysis of the characteristics of cells isolated from fetal chondrocytes reacted with gamma, or (c) to determine the immunoregulatory cytokines secreted during the culturing in step (a) or one or more of the methods. Can be.
본 발명에 따른 태아연골유래 세포는 면역반응을 일으키지 않고, 오히려 활성화된 림프구의 증식을 억제하고, 상기 활성화 림프구에서 생성되는 면역 싸이토카인의 발현을 제어하는 특성을 지니고 있고, 증식 능력과 다양한 세포조직으로의 분화능력이 성체줄기세포에 비해 월등하여 손상된 조직의 재생과 함께 이식 시 발생할 수 있는 면역반응을 조절할 수 있는 면역조절제로서 유용하게 사용할 수 있다.Fetal cartilage-derived cells according to the present invention do not cause an immune response, but rather have the property of inhibiting the proliferation of activated lymphocytes and controlling the expression of immune cytokines produced in the activated lymphocytes, and proliferating ability and various cell tissues. The differentiation ability of is superior to that of adult stem cells, and can be usefully used as an immunomodulator to control the immune response that may occur during transplantation with regeneration of damaged tissues.
도 1은 증식을 억제시킨 태아연골유래 세포와 동종의 혈액에서 분리한 림프구을 혼합배양 후 림프구의 증식을 분석한 결과 태아연골유래 세포가 면역반응을 유발하지 않음을 보여주는 것이다.1 shows that fetal cartilage-derived cells do not induce an immune response as a result of analyzing the proliferation of lymphocytes after mixed culture of lymphocytes isolated from fetal cartilage-derived cells and allogeneic blood that inhibited proliferation.
도 2는 증식을 억제시킨 태아연골유래 세포와 성인연골세포를 각각 ConA를 이용한 혼합림프구반응에 처리한 후 림프구의 증식을 분석한 결과 태아연골유래 세포가 콘카나발린 A에 의한 림프구의 활성화를 억제함을 보여주는 것이다.Figure 2 shows that fetal chondrocyte-derived cells and adult chondrocytes that inhibited proliferation were treated with mixed lymphocyte reactions using ConA, respectively, and the proliferation of lymphocytes was analyzed. Is to show.
도 3은 인터페론 자극 유무에 따른 태아연골유래 세포의 면역원성을 유세포분석기를 통하여 분석한 결과 태아연골유래 세포가 인터페론 감마(IFN-γ)에 의해 면역원성이 활성화되지 않음을 보여주는 것이다.Figure 3 shows the immunogenicity of fetal cartilage-derived cells with or without interferon stimulation by flow cytometry shows that the fetal cartilage-derived cells are not activated by interferon gamma (IFN-γ).
도 4는 증식을 억제시킨 태아연골유래 세포와 성인연골세포를 각각 콘카나발린 A를 이용한 혼합림프구 반응에 처리한 결과 태아연골유래 세포가 림프구에서 발현이 증가되는 면역 싸이토카인의 발현을 조절함을 보여주는 것이다.4 shows that fetal cartilage-derived cells and adult chondrocytes that inhibited proliferation were treated with mixed lymphocyte responses using Concanavalin A, respectively, to show that fetal cartilage-derived cells regulate the expression of immune cytokines with increased expression in lymphocytes. will be.
도 5는 동물모델을 이용한 태아연골유래 세포의 염증 완화 효과를 보여주는 것이다.Figure 5 shows the mitigating effect of fetal cartilage-derived cells using an animal model.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 손상된 조직의 재생과 함께 이식 시 발생할 수 있는 면역반응을 조절하여 기존의 줄기세포의 한계점을 극복할 수 있는 태아연골조직유래 세포를 유효성분으로 이용한 면역질환의 예방 또는 치료용 조성물을 제공한다.The present invention provides a composition for the prevention or treatment of immune diseases using fetal cartilage tissue-derived cells as an active ingredient that can overcome the limitations of the existing stem cells by regulating the immune response that may occur during transplantation with damaged tissue regeneration. do.
즉, 태아연골조직으로부터 세포를 분리하여 동종의 림프구와 반응시켜 면역반응을 분석한 결과, 면역반응을 일으키지 않고 면역 조절 능력이 있음을 확인함으로써 임상적으로 활용 가능한 면역질환의 예방 또는 치료용 조성물을 제공한다.In other words, by separating the cells from fetal cartilage tissue and reacting with lymphocytes of the same type, the immune response was analyzed. As a result, the composition for the prevention or treatment of clinically available immune diseases was confirmed by confirming that the immune control ability was not caused. to provide.
상기 태아연골유래 세포는 (a) 태아연골조직에서 분리한 세포와 동종 전혈에서 분리한 림프구를 함께 배양 시 세포에 대한 림프구의 증식여부를 확인하거나, (b) 면역인터페론으로 알려진 인터페론 감마에 반응시킨 태아연골조직에서 분리한 세포의 특성을 분석하거나, 또는 (c) 상기 (a) 단계에서 배양 시 분비되는 면역조절 싸이토카인을 확인하는 등의 방법으로 면역조절 기능을 확인할 수 있지만, 태아연골조직유래 세포의 면역조절 기능 여부 분석은 당업자에게 알려진 통상의 방법에 의해서도 분석 가능하므로, 이에 한정되는 것은 아니다.The fetal cartilage-derived cells are (a) confirming the proliferation of lymphocytes to cells when cultured together with cells isolated from fetal chondrocytes and lymphocytes isolated from allogeneic whole blood, or (b) reacted with interferon gamma known as immunointerferon. The immunomodulatory function can be confirmed by analyzing the characteristics of the cells isolated from fetal cartilage tissue, or (c) identifying the immunoregulatory cytokines secreted during the culturing in step (a). Analysis of whether the immunomodulatory function of can be analyzed by conventional methods known to those skilled in the art, so is not limited thereto.
상기 태아연골유래 세포는 성인의 골수유래 중간엽줄기세포 특이 표면항원인 CD29, CD44, CD90 및 CD105를 발현하면서도, 배아줄기세포 특이 표면항원인 SSEA4와 Oct-4를 발현할 수 있다.The fetal cartilage-derived cells can express the bone marrow-derived mesenchymal stem cell-specific surface antigens CD29, CD44, CD90, and CD105 in adults, while expressing the embryonic stem cell-specific surface antigens SSEA4 and Oct-4.
상기 태아연골유래 세포는 (a) 태아연골조직에서 연골세포를 분리하여 배양하는 단계; (b) 상기 (a) 단계에서 분리 배양된 세포에서 줄기세포의 표면항원 단백질을 발현하는 세포를 분리하는 단계; 및 (c) 상기 (b) 단계에서 분리된 세포의 증식능 및 다분화 능력을 확인하여 태아연골유래 줄기세포를 검증하는 단계를 통해 수득할 수 있다.The fetal cartilage-derived cells are (a) separating and culturing chondrocytes from fetal cartilage tissue; (b) separating the cells expressing the surface antigen proteins of the stem cells from the cells cultured in step (a); And (c) it can be obtained through the step of verifying the proliferation capacity and multi-differentiation capacity of the cells isolated in step (b) through fetal cartilage derived stem cells.
상기 태아연골조직에서 분리된 연골세포는 12-15주의 유산된 태아로부터 채취하여 콜라게나아제(Collagenase)를 사용하거나 마쇄(trituration)를 통해 수득할 수 있다. Chondrocytes isolated from fetal chondrocytes can be obtained from 12-15 weeks of aborted fetus using collagenase (Collagenase) or obtained through trituration.
상기 태아연골유래 세포는 줄기세포의 표면항원 단백질을 발현할 수 있으며, 일실시예로는 상기 태아연골유래 세포는 성인의 골수유래 중간엽줄기세포 특이 표면항원인 CD29, CD44, CD90 및 CD105를 발현하면서도, 배아줄기세포 특이 표면항원인 SSEA4와 Oct-4를 발현할 수 있다.The fetal chondrocyte-derived cells may express the surface antigen proteins of stem cells, and in one embodiment, the fetal chondrocyte-derived cells express CD29, CD44, CD90 and CD105, which are specific surface antigens of adult bone marrow-derived mesenchymal stem cells. In addition, embryonic stem cell-specific surface antigens can express SSEA4 and Oct-4.
상기 태아연골유래 세포는 활성화 림프구에서 생성되는 면역 싸이토카인의 발현을 제어할 수 있다. 보다 상세하게는, 상기 태아연골유래 세포는 면역반응 또는 염증반응에 관련된 TNF-α, IL-13, IFN-γ의 발현은 억제할 수 있다. 또한, 상기 태아연골유래 세포는 항염증 싸이토카인인 IL-10의 발현은 영향을 주지 않거나 증가시킴으로써 면역활성을 조절할 수 있다.The fetal cartilage-derived cells can control the expression of immune cytokines produced in activated lymphocytes. More specifically, the fetal cartilage-derived cells can inhibit the expression of TNF-α, IL-13, IFN-γ related to immune or inflammatory responses. In addition, the fetal cartilage-derived cells can modulate immune activity by not affecting or increasing the expression of anti-inflammatory cytokine IL-10.
일실시예로서, 상기 태아연골유래 세포에서의 중간엽줄기세포의 표면항원과 배아줄기세포의 표면항원의 동시 발현은 유세포분석(Fluorescent-Assisted Cell Sorting; FACS) 및 자기활성세포선별(Magnetic-Activated Cell Sorting; MACS) 방법을 포함한 면역화학적 분리 방법을 통해 분석할 수 있다.In one embodiment, the simultaneous expression of the surface antigens of mesenchymal stem cells and the surface antigens of embryonic stem cells in the fetal chondrocyte-derived cells are characterized by Fluorescent-Assisted Cell Sorting (FACS) and Magnetic-Activated Cells. It can be analyzed by immunochemical separation methods including Cell Sorting (MACS) method.
상기 태아연골유래 세포는 연골, 골, 근육, 피부 및 지방 세포를 포함하는 근골격계 조직세포; 신경세포; 간세포; 췌장세포; 심근세포; 및 혈관세포 중 어느 하나로 분화하는 다분화 능력을 가질 수 있다.The fetal cartilage-derived cells include musculoskeletal tissue cells, including cartilage, bone, muscle, skin and fat cells; Nerve cell; Hepatocytes; Pancreatic cell; Cardiomyocytes; And it may have a multipotent ability to differentiate into any one of the vascular cells.
기존에 알려진 면역조절제는 주로 암치료에 국한되어 사용되고 있어 조직 이식이나 조직재생에 관한 치료제 역할은 수행할 수 없는 한계가 있는데, 본 발명의 태아연골유래 세포는 세포치료제로써 많이 연구되는 성체줄기세포보다 증식력이나 분화능이 뛰어나며, 동종에 면역반응을 일으키지 않고 면역반응을 조절할 수 있는 능력이 있다. 즉, 일 실시예에 따르면, 상기 태아연골유래 세포는 동종의 림프구에 자극을 주지 않고, 림프구 활성화를 유도할 경우에는 오히려 림프구의 증식이 감소되며, 특히 면역반응에 연관된 TNF-α, IL-13, IFN-γ의 발현은 억제하나 항염증 싸이토카인으로 알려진 IL-10의 발현은 영향을 주지 않거나 증가시킴으로써 면역활성이 조절되는 특성을 가지고 있다.Known immunomodulators are mainly used to treat cancer, so there is a limit that can not play a role in the treatment of tissue transplantation or tissue regeneration, fetal cartilage-derived cells of the present invention than adult stem cells studied as a cell therapy It has excellent proliferative and differentiating ability, and has the ability to control the immune response without causing an immune response. That is, according to one embodiment, the fetal cartilage-derived cells do not stimulate allogeneic lymphocytes, and when lymphocyte activation is induced, the proliferation of lymphocytes is reduced, in particular TNF-α and IL-13 associated with immune responses. In addition, the expression of IL-10, which is known as an anti-inflammatory cytokine, inhibits the expression of IFN- [gamma] but does not affect or increase the immune activity.
본 발명에 따른 면역조절용 조성물은 세포치료제를 포함하지만, 이에 한정되지 않고 다양한 치료제 형태로 활용될 수 있다. 상세하게는 태아연골유래 세포 추출물(cell extracts) 또는 분비물(secreted molecules)을 포함할 수도 있다. 태아연골유래 세포 추출물(cell extracts) 또는 분비물(secreted molecules)은 태아연골유래 세포로부터 얻어질 수 있는 모든 형태의 것을 의미할 수 있으며, 세포 자체뿐만 아니라, 그 현탁액 내지 배양액 또는 이들의 농축물, 페이스트상물, 건조물, 희석물 또는 상기 세포의 대사를 통해 만들어지는 모든 생성물을 의미할 수 있다.The immunomodulatory composition according to the present invention includes a cell therapeutic agent, but is not limited thereto and may be utilized in various therapeutic forms. Specifically, it may include fetal cartilage-derived cell extracts or secreted molecules. Fetal cartilage-derived cell extracts or secreted molecules may mean any form that can be obtained from fetal cartilage-derived cells, as well as the cells themselves, suspensions or cultures thereof, or concentrates or pastes thereof. It may mean a product, a dry product, a dilution or any product made through metabolism of the cells.
상기 면역질환은 자가면역질환, 염증성 질환 및 세포, 조직 또는 기관의 이식거부 질환일 수 있고, 상세하게는 관절염, 자가면역성 간염, 류마티스성 관절염, 골관절염, 인슐린 의존성 당뇨병, 궤양성 대장염, 크론병, 다발성 경화증, 자가면역성 심근염, 피부경화증, 중증근육무력증, 다발성 근육염/피부 근육염, 하시모토병, 자가면역 혈구감소증, 쇼그렌 증후군, 혈관염 증후군 및 전신홍반루푸스일 수 있지만, 이에 제한되지는 않는다.The immune disease may be an autoimmune disease, an inflammatory disease and a transplant rejection disease of cells, tissues or organs, and specifically, arthritis, autoimmune hepatitis, rheumatoid arthritis, osteoarthritis, insulin dependent diabetes mellitus, ulcerative colitis, Crohn's disease, Multiple sclerosis, autoimmune myocarditis, scleroderma, myasthenia gravis, multiple myositis / skin myositis, Hashimoto's disease, autoimmune cytopenia, Sjogren's syndrome, vasculitis syndrome, and systemic lupus erythematosus.
본 발명의 일실시예의 면역질환의 예방 또는 치료용 조성물은 약제학적으로 허용가능한 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 결합제, 활택제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 약제학적으로 허용가능한 담체로서 혼합하여 사용할 수 있다. 주사제의 경우는 완충제, 보존제, 무통화제, 가용화제, 등장화제, 안정화제 등을 혼합하여 사용할 수 있다. 주사제의 경우에는 단위 투약 앰플 또는 다수화 투약 형태로 제조할 수 있다. 또한, 국소 투여 시에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다. 그러나, 본 발명은 이에 제한되는 것이 아니며, 본 발명의 기술분야에 속하는 당업자라면 다양한 형태로 본 발명의 면역질환의 예방 또는 치료용 조성물이 제제화될 수 있으며, 다양한 담체나 첨가제들을 포함할 수 있음을 이해할 것이다.The composition for preventing or treating immunological diseases of one embodiment of the present invention may be prepared in various mixtures with a pharmaceutically acceptable carrier. For example, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavorings and the like can be used in combination as pharmaceutically acceptable carriers. In the case of injections, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers and the like can be used in combination. Injectables may be prepared in unit dosage ampoules or in multiple dosage forms. In addition, during topical administration, bases, excipients, lubricants, preservatives and the like can be used. However, the present invention is not limited thereto, and those skilled in the art may form a composition for preventing or treating the immune disease in various forms, and may include various carriers or additives. I will understand.
상기 조성물은 면역질환의 증상 정도에 따라 투여 방법이 결정되는데, 본 발명의 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내 (intracerebroventricular) 주사에 의해 투여될 수 있다. 또한, 상기 조성물 중 유효성분의 투여량은 투여경로, 질병의 정도, 환자의 나이, 성별, 체중 등에 따라 달라질 수 있으며, 일일 1회 내지 수회 투여할 수 있다.The composition is determined by the method of administration depending on the degree of symptoms of the immune disease, the composition of the present invention can be administered by a variety of routes to mammals, such as rats, mice, livestock, humans. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection. In addition, the dosage of the active ingredient in the composition may vary depending on the route of administration, the degree of disease, the age, sex, and weight of the patient, and may be administered once to several times daily.
본 발명에서 있어서, "줄기세포"란 여러 종류의 신체 조직으로 분화할 수 있는 능력을 가진 세포, 즉 미분화세포이다. 상기 줄기세포에는 사람의 배아를 이용해 만들 수 있는 배아줄기세포(전분화능줄기세포; pluripotent stem cells)와 혈구세포를 끊임없이 만드는 골수세포와 같은 성체줄기세포(다분화능줄기세포; multipotent stem cells)가 있다.In the present invention, "stem cells" are cells that have the ability to differentiate into various kinds of body tissues, that is, undifferentiated cells. The stem cells include adult stem cells (multipotent stem cells) such as embryonic stem cells (pluripotent stem cells) that can be made using human embryos, and bone marrow cells that constantly make blood cells. .
본 발명에서 있어서, "배아줄기세포(Embryonic Stem Cell)"란 수정한지 14일이 안된 배아기의 세포로서, 장차 인체를 이루는 모든 세포와 조직으로 분화할 수 있기 때문에 '전능세포' 혹은 '만능세포'로 불린다. 수정란은 세포분열을 통해 배반포를 형성하는데 그 안쪽에 내세포괴라는 세포덩어리가 있어 이것이 배아를 형성한다. 이 내세포괴의 세포를 배반포로부터 분리하여 배양하면, 분화는 일어나지 않지만 분화 능력은 여전히 가지는 배아줄기세포가 된다. In the present invention, "Embryonic Stem Cell" is an embryonic cell less than 14 days old, and can be differentiated into all cells and tissues constituting the human body in the future, 'almighty cell' or 'pluripotent cell' It is called Fertilized eggs form blastocysts through cell division, inside which there is a mass of cells called inner cell masses, which form embryos. When the cells of this inner cell mass are isolated from the blastocyst and cultured, they become embryonic stem cells that do not differentiate but still have differentiation capacity.
본 발명에서 있어서, "성체줄기세포(Adult Stem Cell)"란 제대혈(탯줄혈액)이나 다 자란 성인의 골수와 혈액 등에서 추출해낸 것으로, 뼈와 간, 혈액 등 구체적 장기의 세포로 분화되기 직전의 원시세포다. 여기에는 조혈모세포(Hematopoietic Stem Cell)와 재생의학의 재료로 각광받고 있는 중간엽줄기세포(Mesenchymal Stem Cell), 신경줄기세포(Neural stem cell) 등이 있다. 이밖에 간이나 표피, 췌장 등에도 줄기세포가 있다. 상기 성체 줄기세포는 증식이 어렵고 쉽게 분화되는 경향이 강한 대신에 여러 종류의 성체 줄기세포를 사용하여 실제 의학에서 필요로 하는 장기 재생을 할 수 있을 뿐 아니라 이식된 후 각 장기의 특성에 맞게 분화할 수 있는 특성을 지니고 있다.In the present invention, "Adult Stem Cell" is extracted from umbilical cord blood (umbilical cord blood) or mature adult bone marrow and blood, and the raw material immediately before differentiation into cells of specific organs such as bone, liver, and blood. It's a cell. These include hematopoietic stem cells, mesenchymal stem cells, and neural stem cells, which are in the spotlight of regenerative medicine. In addition, there are stem cells in the liver, epidermis, and pancreas. The adult stem cells are difficult to proliferate and tend to be easily differentiated. Instead, the adult stem cells can be regenerated according to the characteristics of each organ after transplantation by using various types of adult stem cells as well as the regeneration of organs required by actual medicine. It has characteristics that can be.
본 발명에 있어서, "중간엽 줄기세포(mesenchymal stem cell, MSC)"는 뼈(bone), 연골(cartilage), 지방(fat), 힘줄(건), 신경 조직(nerve tissue), 섬유아세포(fibroblast) 및 근육 세포(muscle cell) 등 구체적인 장기의 세포로 분화되기 전의 다분화 전구 세포 (multipotent progenitor)를 말한다.In the present invention, "mesenchymal stem cells (MSC)" is bone (bone), cartilage (cartilage), fat (fat), tendon (tendon), nerve tissue (nerve tissue), fibroblast (fibroblast) And multipotent progenitors before they are differentiated into cells of specific organs such as muscle cells.
본 발명에서 있어서, "세포치료제"는 사람으로부터 분리, 배양 및 특수한 저작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품(미국 FDA규정)으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종세포를 체외에서 증식, 선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 치료, 진단 및 예방의 목적으로 사용되는 의약품을 지칭한다. 세포치료제는 세포의 분화정도에 따라 크게 체세포치료제, 줄기세포치료제로 분류되며 본 발명은 특히 줄기세포치료제에 관한 것이다.In the present invention, the "cell therapeutic agent" is a medicine (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared through isolation, culture, and special chewing from humans. It refers to a medicine used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating, selecting, or otherwise changing the biological characteristics of a living autologous, allogeneic, or heterologous cell in vitro. Cell therapy agents are largely classified into somatic cell therapy and stem cell therapy according to the degree of differentiation of cells, and the present invention relates in particular to stem cell therapy.
이하, 본 발명을 실시예에 기초하여 보다 상세히 기술한다. 본 발명의 하기 실시예는 본 발명을 구체화하기 위한 것일 뿐 본 발명의 권리범위를 제한하거나 한정하는 것이 아님은 물론이다. 본 발명의 상세한 설명 및 실시예로부터 본 발명이 속하는 기술분야의 전문가가 용이하게 유추할 수 있는 것은 본 발명의 권리범위에 속하는 것으로 해석된다. 본 발명에 인용된 참고문헌은 본 발명에 참고로서 통합된다.Hereinafter, the present invention will be described in more detail based on examples. The following examples of the present invention are not intended to limit or limit the scope of the present invention only to embody the present invention. From the detailed description and examples of the present invention, those skilled in the art to which the present invention pertains can easily be interpreted as belonging to the scope of the present invention. References cited in the present invention are incorporated herein by reference.
실시예 1. 태아연골유래 세포에 의한 림프구의 반응 확인Example 1. Confirmation of lymphocyte response by fetal chondrocytes
태아연골유래 세포와 인간말초혈액유래 림프구의 분리 및 배양을 위해 아주대학교 병원의 윤리위원회(IRB) 승인을 득하였으며, 태아연골유래 세포는 산본제일병원 또는 아주대병원의 산모로부터 수술 시 동의서를 받은 후 태아의 연골을 채취하였고, 인간말초혈액은 기증자 모집공고 후 건강한 기증자로부터 채혈하여 림프구를 분리하였다.Isolation and culture of fetal cartilage-derived cells and human peripheral blood-derived lymphocytes were approved by the Ethics Committee (IRB) of Ajou University Hospital, and fetal cartilage-derived cells were obtained from the mother of Sanbon Cheil Hospital or Ajou University Hospital during surgery. Fetal cartilage was harvested, and human peripheral blood was collected from healthy donors after donor recruitment announcement to separate lymphocytes.
태아연골유래 세포의 분리 방법 및 배양은 이전에 특허출원된 특허출원 제2013-0090233호[태아연골조직유래 줄기세포원 및 이를 포함하는 세포치료제]에 상세 기술된 내용과 같이 수행하였다. 즉, 채취된 연골 조직은 인산완충염식염수(PBS)로 수 차례 세척 후 잘게 잘라 0.1% 콜라게나아제(collagenase), 1% 소혈청(fetal bovine serum, FBS)이 함유된 배양배지(DMEM)를 처리하여 37℃, 5% CO2 조건의 인큐베이터에서 12시간 방치하여 세포를 분리하였다. 콜라게나아제에 의해 분리된 세포는 10% FBS가 함유된 배양배지로 세척하였으며 그 방법은 다음과 같다. 배양배지에 부유된 세포를 1700 rpm에서 10분간 원심분리 후 상층액을 제거하고 모아진 세포를 획득한 후 배양배지에 다시 부유시켰다. 이때 일부 세포를 트립판 블루(trypan Blue)로 염색 후 광학 현미경 하에서 혈구계수판을 이용하여 세포수를 측정하였다. 배양배지에 부유된 세포를 배양용기에 8×104/cm2의 농도로 분주한 후 37℃, 5% CO2 조건의 인큐베이터에서 배양하였다. 계대배양은 세포가 배양용기의 80~90% 정도를 채웠을 때 이루어졌다. The method and culture of fetal cartilage-derived cells were carried out as described in detail in patent application 2013-0090233 [Fetal Cartilage Tissue-Derived Stem Cell Sources and Cell Therapeutic Agents Comprising the Same]. That is, the collected cartilage tissue is washed several times with phosphate buffered saline (PBS) and finely chopped and cultured with 0.1% collagenase and 1% bovine serum (FBS). After treatment, the cells were left for 12 hours in an incubator at 37 ° C. and 5% CO 2 . Cells isolated by collagenase were washed with a culture medium containing 10% FBS and the method was as follows. The cells suspended in the culture medium was centrifuged at 1700 rpm for 10 minutes, the supernatant was removed, the collected cells were obtained, and then suspended again in the culture medium. At this time, after staining some cells with trypan blue (trypan Blue) the cell number was measured using a hemocytometer under an optical microscope. The cells suspended in the culture medium were dispensed into a culture vessel at a concentration of 8 × 10 4 / cm 2 and then cultured in an incubator at 37 ° C. and 5% CO 2 . Subculture was done when the cells filled 80-90% of the culture vessel.
특허출원 제2013-0090233호에 개시된 바와 같이, 상기 얻어진 태아연골유래 세포는 유세포분석기 (FACScantoⅡ)를 이용한 분석 결과, 골수유래 중간엽 줄기세포의 세포표면인자로 널리 알려진 CD29(Integrin β1 chain), CD44(HA receptor), CD 90(Thy-1), CD105(Endoglin)를 높은 수준으로 발현하였으며, 또한 배아유래 줄기세포의 특이 마커인 SSEA-4와 Oct-4을 발현하는 것으로 확인되었다.As disclosed in Patent Application No. 2013-0090233, the obtained fetal chondrocyte-derived cells were analyzed by flow cytometry (FACScantoII), and CD29 (Integrin β1 chain), CD44, which is widely known as a cell surface factor of bone marrow-derived mesenchymal stem cells. (HA receptor), CD 90 (Thy-1), CD105 (Endoglin) was expressed at high levels, and it was confirmed that the expression of SSEA-4 and Oct-4, which are specific markers of embryonic stem cells.
그리고, 말초혈액유래 림프구의 분리는 피콜(Ficoll)을 사용하여 일반적으로 전혈에서 림프구를 분리하는 방법을 사용하였다. In addition, the separation of peripheral blood-derived lymphocytes generally used a method of separating lymphocytes from whole blood using Ficoll.
태아연골유래 세포에 의한 림프구의 반응을 확인하기 위하여 두 세포를 함께 배양하였다. 이때 태아연골유래 세포는 감마선(3000rad, 5분)을 처리하여 증식을 억제한 후 96well plate에 세포수를 102, 103 그리고 104개로 하여 분주하였다. 태아연골유래 세포 배양 16시간 후에 동종혈액에서 분리한 림프구를 같은 plate에 105개로 분주하여 4일간 함께 배양하였다. 이때, 태아연골유래 세포와 림프구의 세포 분주 비율을 1:1000, 1:100 그리고 1:10로 하였다. 그리고 분리된 림프구가 정상적으로 활성하는지를 확인하는 방법으로 림프구만 넣은 그룹 중 활성을 유도할 수 있도록 콘카나발린 A(concanavalin A; T cell mitogen)를 10㎍/㎖ 함께 넣어 4일간 배양하였다. 그 후에 부유해 있는 림프구와 상층액을 함께 채취하여 BrdU assay(Cell proliferation ELISA, BrdU, Roche Diagnostice, Germany)를 진행하였다.Two cells were cultured together to confirm the response of lymphocytes to fetal cartilage-derived cells. At this time, fetal cartilage-derived cells were treated with gamma rays (3000rad, 5 minutes) to inhibit proliferation, and the cells were divided into 10 2 , 10 3 and 10 4 cells in 96well plates. After 16 hours of culturing fetal cartilage-derived cells, lymphocytes isolated from allogeneic blood were divided into 10 5 cells on the same plate and cultured together for 4 days. At this time, the ratio of cell division between fetal cartilage-derived cells and lymphocytes was 1: 1000, 1: 100 and 1:10. As a method of confirming that the isolated lymphocytes are normally activated, concanavalin A (T cell mitogen) was added together with 10 µg / ml and cultured for 4 days in order to induce activity in the lymphocyte-only group. After that, the suspended lymphocytes and supernatants were collected together and subjected to BrdU assay (Cell proliferation ELISA, BrdU, Roche Diagnostice, Germany).
콘카나발린 A를 처리한 림프구는 처리하지 않는 림프구보다 증식이 증가됨을 보였다. 이것은 분리된 림프구가 정상적으로 활성화됨을 알 수 있었다. 그리고 태아연골유래 세포를 함께 처리한 그룹의 경우 림프구가 증식하지 않는다는 것을 확인하였다(도 1 참조). 이는 태아연골유래 세포에 의해서는 림프구를 활성화시키지 않는 특성을 지닌 것으로 판단되었다.Lymphocytes treated with concanavalin A showed increased proliferation than lymphocytes not treated. It was found that the isolated lymphocytes are normally activated. And it was confirmed that lymphocytes do not proliferate in the group treated with fetal cartilage-derived cells (see FIG. 1). It was determined that fetal cartilage-derived cells do not activate lymphocytes.
실시예 2. 태아연골유래 세포에 의한 말초혈액유래 림프구의 증식 감소/억제확인Example 2 Confirmation of Proliferation Reduction / Inhibition of Peripheral Blood-Derived Lymphocytes by Fetal Cartilage-Derived Cells
태아연골유래 세포, 성인연골세포와 인간말초혈액유래 림프구의 분리 및 배양을 위해 아주대학교 병원의 윤리위원회(IRB) 승인을 득하였으며, 태아연골유래 세포는 산본제일병원 또는 아주대병원의 산모로부터 수술 시 동의서를 받은 후 태아의 연골을 채취하였고, 성인연골세포는 아주대병원 정형외과에서 인공연골 치환술을 받는 환자로부터 수술 시 동의서를 받은 후 연골조직을 채취하였다. 인간말초혈액은 기증자 모집공고 후 건강한 기증자로부터 채혈하여 림프구를 분리하였다.Isolation and culture of fetal cartilage-derived cells, adult chondrocytes and human peripheral blood-derived lymphocytes were approved by the Ethics Committee (IRB) of Ajou University Hospital, and fetal cartilage-derived cells were operated from the mother of Sanbon Cheil Hospital or Ajou University Hospital. The fetal cartilage was collected after receiving the informed consent, and adult cartilage cells were obtained from the cartilage tissue at the Ajou University Hospital. Human peripheral blood was collected from healthy donors after notification of donor recruitment, and lymphocytes were isolated.
태아연골유래 세포의 분리 방법 및 배양은 앞선 실시예와 동일하게 수행하였으며, 말초혈액유래림프구의 분리는 피콜(Ficoll)을 사용하여 일반적으로 전혈에서 림프구를 분리하는 방법을 사용하였다. Isolation and culturing of fetal cartilage-derived cells were performed in the same manner as in the previous example, and the separation of peripheral blood-derived lymphocytes was generally performed using Ficoll to separate lymphocytes from whole blood.
태아연골유래 세포의 림프구 증식 억제를 확인하기 위한 방법으로 혼합림프구 반응을 시행하였다. 이때 태아연골유래 세포는 감마선(3000rad, 5분)을 처리하여 증식을 억제한 후 96 well plate에 세포수를 103 그리고 104개로 하여 분주하였다. 태아연골유래 세포 배양 16시간 후에 동종혈액에서 분리한 림프구를 같은 plate에 105개 분주하여 4일간 함께 배양하였다. 이때, 태아연골유래 세포와 림프구의 세포 분주 비율을 1:100과 1:10으로 하였다. 그리고 림프구의 활성을 유도할 수 있도록 콘카나발린 A (T cell mitogen)를 10㎍/㎖ 함께 넣어 4일간 배양하였다. 그 후에 부유해 있는 림프구와 상층액을 함께 채취하여 BrdU assay(Cell proliferation ELISA, BrdU, Roche Diagnostice, Germany)를 진행하였다. 그리고, 비교군으로 사용된 성인연골세포 또한 같은 방법을 사용하여 실험을 진행하였다.Mixed lymphocyte response was performed to confirm the inhibition of lymphocyte proliferation of fetal chondrocytes. At this time, fetal cartilage-derived cells were treated with gamma rays (3000rad, 5 minutes) to inhibit proliferation, and the cells were divided into 10 3 and 10 4 cells in 96 well plates. After 16 hours of culturing fetal cartilage-derived cells, lymphocytes isolated from allogeneic blood were dispensed in 10 5 aliquots on the same plate and cultured together for 4 days. At this time, the ratio of cell division between fetal cartilage-derived cells and lymphocytes was 1: 100 and 1:10. To induce lymphocyte activity, conkanavalin A (T cell mitogen) was added together with 10 µg / ml and cultured for 4 days. After that, the suspended lymphocytes and supernatants were collected together and subjected to BrdU assay (Cell proliferation ELISA, BrdU, Roche Diagnostice, Germany). In addition, the adult chondrocytes used as a comparison group were also experimented using the same method.
콘카나발린 A를 처리한 림프구는 처리하지 않는 림프구보다 증식이 증가됨을 보였다. 그리고 태아연골유래 세포를 함께 처리한 그룹의 경우 태아연골유래 세포의 수가 증가할수록 림프구의 증식이 감소한 것을 확인하였다(도 2 참조). 태아연골유래 세포가 콘카나발린 A로 활성화된 림프구의 증식을 억제할 수 있는 특성을 지닌 것으로 판단되었다. 반면에 성인연골세포의 경우 림프구가 활성화되어 증식됨을 보였다. 이는 성인연골세포의 경우 림프구 증식을 억제하는 능력이 없다는 것을 알 수 있다.Lymphocytes treated with concanavalin A showed increased proliferation than lymphocytes not treated. In addition, in the group treated with fetal cartilage-derived cells, it was confirmed that the proliferation of lymphocytes decreased as the number of fetal cartilage-derived cells increased (see FIG. 2). Fetal cartilage-derived cells were determined to have properties that could inhibit the proliferation of lymphocytes activated with Concanavalin A. On the other hand, lymphocytes were activated and proliferated in adult chondrocytes. It can be seen that adult chondrocytes do not have the ability to inhibit lymphocyte proliferation.
실시예 3. 태아연골유래 세포의 면역원성 확인Example 3. Confirmation of immunogenicity of fetal cartilage-derived cells
면역인터페론으로 알려진 인터페론 감마(IFN-γ)를 200 Units/ml로 7일간 함께 처리함으로써 태아연골유래 세포에 세포매개성 면역반응을 유도한 후 태아연골유래 세포 표면의 특성을 유세포분석기를 통하여 확인하였다. Interferon gamma (IFN-γ), also known as immunointerferon, was treated with 200 Units / ml for 7 days to induce cell-mediated immune responses in fetal cartilage-derived cells, and then the characteristics of fetal cartilage-derived cell surfaces were confirmed by flow cytometry. .
백혈구의 1형으로 장기이식 시 타인의 장기가 체내에 들어오면 인체에서 이물로 인식하여 체외로 방출하려고 하며 심한 거부반응을 일으키는 것으로 알려진 사람백혈구항원(human leukocyte antigen, HLA) ABC와 DR을 분석하였다. 그 결과 태아연골유래 세포는 인터페론 자극에 상관없이 사람 백혈구항원이 나타나지 않는 것을 확인하였다(도 3 참조).When the organs of other people enter the body as a type 1 leukocyte, human leukocyte antigens (HLA) ABC and DR, which are known to cause foreign body's rejection, are recognized by the human body as foreign substances. . As a result, it was confirmed that fetal chondrocyte-derived cells did not show human leukocyte antigen regardless of interferon stimulation (see FIG. 3).
또한, 면역반응에서 항원제시세포의 T세포로 신호전달 유도 시 활성화되는 공동자극 분자인 CD80, CD86 (B7.1, B7.2)의 활성이 나타나는지를 분석한 결과, CD80에서는 변화가 없음을 확인하였다(도 3 참조).In addition, as a result of analyzing the activity of CD80, CD86 (B7.1, B7.2), which are activated co-stimulatory molecules when inducing signaling to T cells of antigen-presenting cells in the immune response, it was confirmed that there was no change in CD80. (See FIG. 3).
실시예 4. 태아연골유래 세포의 싸이토카인 생성Example 4 Cytokine Production of Fetal Cartilage-Derived Cells
앞선 결과들을 통하여 태아연골유래 세포가 면역반응을 일으킬 수 있는 항원을 가지고 있지 않고, 동종 림프구의 증식을 억제하는 것을 확인하였다. 다음 실험으로는 태아연골유래 세포의 싸이토카인 생성을 확인하기 위하여 혼합림프구 반응을 시행하였다. 이때 태아연골유래 세포는 감마선(3000rad, 5분)을 처리하여 증식을 억제한 후 12 well plate에 세포수를 105 그리고 106개로 하여 분주하였다. 16시간 후에 분리한 림프구를 106 분주하였다. 이때, 태아연골유래 세포와 림프구의 세포 분주 비율을 1:10 그리고 1:1로 하였다. 그리고 림프구의 활성을 유도할 수 있도록 콘카나발린 A(T cell mitogen)를 10㎍/㎖ 함께 넣어 4일간 배양하였다. 비교군으로는 동종 림프구와 콘카나발린 A를 처리한 림프구, 그리고 태아연골유래 세포를 배양한 것으로 하였다. 배양 4일 후 상층액을 모은 후 부유해 있는 림프구를 제거하기 위하여 2000rpm, 5분간 원심분리 후 다시 상층액을 모아 싸이토카인-ELISA 분석(eBioscience, Ready-SET-Go ELISA set)을 진행하였다.The previous results confirmed that fetal cartilage-derived cells do not have antigens that can trigger immune responses and inhibit the proliferation of allogeneic lymphocytes. In the next experiment, mixed lymphocyte reaction was performed to confirm the cytokine production of fetal cartilage-derived cells. At this time, fetal cartilage-derived cells were treated with gamma rays (3000rad, 5 minutes) to inhibit proliferation, and then divided into 12 well plates with 10 5 and 10 6 cells. After 16 hours, isolated lymphocytes were dispensed for 10 6 minutes. At this time, the ratio of cell division between fetal cartilage-derived cells and lymphocytes was 1:10 and 1: 1. To induce lymphocyte activity, concanavalin A (T cell mitogen) was added together with 10 µg / ml and cultured for 4 days. In the comparison group, allogeneic lymphocytes, lymphocytes treated with concanavalin A, and fetal cartilage-derived cells were cultured. After 4 days of culture, the supernatant was collected and centrifuged at 2000 rpm for 5 minutes to remove suspended lymphocytes, and then the supernatant was collected and subjected to cytokine-ELISA analysis (eBioscience, Ready-SET-Go ELISA set).
급성면역반응이나 염증반응에 나타나는 싸이토카인인 종양괴사인자-알파(Tumor necrosis factor-alpha, TNF-α)는 콘카나발린 A에 의하여 활성화된 림프구 그룹에서는 생성이 되었지만, 태아연골유래 세포와 함께 배양한 그룹에서는 생성되지 않는 것을 확인하였다(도 4(A) 참조). Tumor necrosis factor-alpha (TNF-α), a cytokine in acute immune and inflammatory reactions, was produced in the group of lymphocytes activated by concanavalin A, but incubated with fetal cartilage-derived cells. It was confirmed that no generation was made in the group (see FIG. 4 (A)).
또한, 활성화 보조성 T세포(helper T cell)에서 생성하는 림포카인인 인터루킨-13(interleukin-13, IL-13)과 염증반응이나 면역자극에 의해 T세포에서 생성되는 인터페론-감마(interferon-gamma, IFN-γ)의 경우, 태아연골유래 세포의 수와 림프구 수의 비율이 1:10보다 1:1의 경우 생산량이 감소하는 것을 확인하였다(도 4(B), 도 4(C) 참조).In addition, interleukin-13 (IL-13), a lymphokine produced in activation helper T cells, and interferon-gamma produced in T cells by inflammatory reactions or immunostimulation gamma, IFN-γ), the ratio of the number of fetal cartilage-derived cells and lymphocytes of 1: 1 than 1:10 was confirmed to decrease the production (see Figure 4 (B), Figure 4 (C)) ).
조절 T세포(regulatory T cell)이나 수지상세포에서 생성하는 인터루킨-10(interleukin-10, IL-10)은 자기조직을 공격하는 면역세포를 억제시켜 면역관용을 유도하여 항염증제로 활용이 되는 싸이토카인으로서, 태아연골유래 세포와 림프구를 반응시킬 경우 태아연골유래 세포의 비율이 커질수록 인터루킨-10의 생산량이 증가됨을 확인하였다(도 4(D) 참조). Interleukin-10 (IL-10) produced by regulatory T cells or dendritic cells is a cytokine that is used as an anti-inflammatory agent by inducing immune tolerance by inhibiting immune cells that attack autologous tissues. When fetal cartilage-derived cells and lymphocytes were reacted, it was confirmed that the production of interleukin-10 increased as the ratio of fetal cartilage-derived cells increased (see FIG. 4 (D)).
동일한 방법으로 성인의 골수에서 분리한 세포를 림프구와 반응시킬 경우, 태아연골유래 세포와 동일한 경향으로 인터페론-감마의 생성이 감소됨을 확인하였다(도 4(E) 참조). 하지만 면역관용에 작용하는 인터루킨-10의 생성은 세포 수가 증가할수록 감소함을 보였다(도 4(F) 참조).In the same way, when the cells isolated from the adult bone marrow reacted with lymphocytes, it was confirmed that the production of interferon-gamma was reduced in the same tendency as the fetal cartilage-derived cells (see FIG. 4 (E)). However, the production of interleukin-10, which acts on immune tolerance, was shown to decrease with increasing cell number (see FIG. 4 (F)).
실시예 5. 태아연골유래 세포의 염증 부종 완화 효과Example 5 Inflammatory Edema Reduction Effect of Fetal Cartilage-Derived Cells
앞선 결과들을 통하여 태아연골유래 세포의 면역 관용을 확인할 수 있었고, 항염증인자인 인터루킨-10(interleukin-10, IL-10)이 증가되고 염증인자인 종양괴사인자-알파(Tumor necrosis factor-alpha, TNF-α)와 인터페론-감마(Interferon-gamma, IFN-γ)가 감소하는 것을 보였다. 다음 실험으로는 동물모델을 이용하여 태아연골유래 세포의 염증 완화 효과를 확인하였다. 랫트(Rat)의 무릎 관절강 내에 세포성 면역반응을 유도하는 물질인 완전프로인트항원보강제(Complete Freund’s adjuvant, CFA)를 인슐린 주사기를 이용하여 100㎍의 함량인 100㎕를 주입하여 염증을 유도하였다. 완전프로인트항원보강제 주입 후 4일째 태아연골유래 세포 5x106 cells를 50㎕으로 만들어 인슐린 주사기를 이용하여 동일한 위치에 주입하였다. 비교군으로는 염증을 유도하지 않은 정상 랫트, 완전프로인트항원보강제와 태아연골유래 세포가 아닌 식염수(Phosphate buffered saline, PBS)를 주입한 랫트, 완전프로인트항원보강제 주입하여 염증을 유도한 후 아무것도 넣지 않은 랫트, 그리고 동일방법으로 염증 유도 후 임상에서 사용되는 염증 치료제인 트리암시놀론(Triamcinolone)을 0.5mg을 주입한 랫트로 하였다. 이때 모든 그룹에서는 동일한 양을 주입하였다. 이후 매일 랫트의 무릎 둘레를 측정하여 부종의 정도를 관찰하였다.The previous results confirmed the immune tolerance of fetal cartilage-derived cells, and increased the anti-inflammatory factor interleukin-10 (IL-10) and the tumor necrosis factor-alpha (TNF). -α) and interferon-gamma (IFN-γ) were shown to decrease. In the next experiment, animal model was used to examine the inflammatory relieving effects of fetal cartilage-derived cells. Inflammation was induced by injecting 100 μl of 100 μg of a complete Freund's adjuvant (CFA), a substance that induces a cellular immune response in rat knee cavity of rats, using an insulin syringe. On day 4 after the injection of complete Freund's adjuvant, fetal cartilage-derived 5x10 6 cells were made into 50 µl and injected at the same location using an insulin syringe. In comparison, normal rats that did not induce inflammation, rats injected with phosphate buffered saline (PBS) instead of fetal cartilage-derived cells, and rats injected with complete Freund antigen adjuvant did not induce inflammation. Rats were not injected, and rats were injected with 0.5 mg of Triamcinolone, an inflammatory agent used in the clinic after induction of inflammation. The same amount was injected in all groups. After that, the rats' knee circumference was measured daily to observe the degree of edema.
완전프로인트항원보강제를 주입한 후 4일째 랫트의 무릎 둘레를 측정한 결과 정상 랫트에 비하여 둘레가 증가, 부종이 생긴 것을 확인하였다. 염증 완화를 위하여 관절내 주사를 하는 트리암시놀론은 스테로이드제로써 이를 처리한 그룹의 랫트는 약물 처리 후 2일째부터 무릎 둘레가 줄어듦으로써 부종이 감소함을 확인하였다(도 5 참조). As a result of measuring the knee circumference of the rat on day 4 after injecting the complete Freund antigen adjuvant, it was confirmed that the circumference was increased and edema occurred compared to the normal rat. Triamcinolone intraarticular injection for relieving inflammation was confirmed that the rats of the group treated with steroids reduced swelling by reducing the circumference of the knee from day 2 after drug treatment (see FIG. 5).
면역조절 능력을 가지고 있는 태아연골유래 세포를 주입한 그룹의 랫트에서는 7일째부터 무릎 둘레가 줄어듦으로써 부종이 감소함을 확인하였다(도 5 참조)Rats of the fetal cartilage-derived cells injected with immunomodulatory capacity were confirmed to have reduced edema by decreasing knee circumference from day 7 (see FIG. 5).
기존에 알려진 면역조절제는 조직 이식이나 조직재생에 관한 치료제 역할은 수행할 수 없는 한계가 있는데, 본 발명의 태아연골유래 세포는 세포치료제로써 많이 연구되는 성체줄기세포보다 증식력이나 분화능이 뛰어나며, 동종에 면역반응을 일으키지 않고 면역반응을 조절할 수 있는 능력이 있어 본 발명에 따른 면역조절용 조성물은 세포치료제를 포함한 다양한 치료제 형태로 활용될 수 있다.The known immunomodulatory agent has a limitation that can not play a role in the treatment of tissue transplantation or tissue regeneration, fetal cartilage-derived cells of the present invention is more proliferative and differentiating ability than adult stem cells, which are widely studied as cell therapy, homogeneous The ability to modulate the immune response without causing an immune response, the immunomodulatory composition according to the present invention can be utilized in various therapeutic forms, including cell therapy.

Claims (10)

  1. 태아연골유래 세포를 유효성분으로 함유하는 면역질환의 예방 또는 치료용 조성물.A composition for preventing or treating immune diseases containing fetal cartilage-derived cells as an active ingredient.
  2. 청구항 1에 있어서, 상기 태아연골유래 세포는 줄기세포의 표면항원 단백질을 발현하는 것을 특징으로 하는 면역질환의 예방 또는 치료용 조성물.The method of claim 1, wherein the fetal cartilage-derived cells for the prevention or treatment of immune diseases, characterized in that for expressing the surface antigen protein of the stem cells.
  3. 청구항 2에 있어서, 상기 태아연골유래 세포는 성인의 골수유래 중간엽줄기세포 특이 표면항원인 CD29, CD44, CD90 및 CD105를 발현하면서도, 배아줄기세포 특이 표면항원인 SSEA4와 Oct-4를 발현하는 것을 특징으로 하는 면역질환의 예방 또는 치료용 조성물.The method according to claim 2, wherein the fetal chondrocyte-derived cells express the adult bone marrow-derived mesenchymal stem cell-specific surface antigens CD29, CD44, CD90 and CD105, while expressing the embryonic stem cell-specific surface antigens SSEA4 and Oct-4 Composition for the prevention or treatment of immune diseases, characterized in that.
  4. 청구항 1에 있어서, 상기 태아연골유래 세포는The method of claim 1, wherein the fetal cartilage-derived cells
    (a) 태아연골조직에서 연골세포를 분리하여 배양하는 단계;(a) separating and culturing chondrocytes from fetal chondrocytes;
    (b) 상기 (a) 단계에서 분리 배양된 세포에서 중간엽줄기세포와 배아줄기세포의 표면항원 단백질을 동시에 발현하는 세포를 분리하는 단계; 및(b) separating the cells expressing the surface antigen proteins of mesenchymal stem cells and embryonic stem cells simultaneously from the cells cultured in the step (a); And
    (c) 상기 (b) 단계에서 분리된 세포의 증식능 및 다분화 능력을 검증하여 태아연골조직유래 줄기세포원을 수득하는 단계를 거쳐 수득되는 것을 특징으로 하는 면역질환의 예방 또는 치료용 조성물.(c) The composition for preventing or treating immune diseases, characterized in that obtained through the step of obtaining the fetal cartilage tissue-derived stem cell source by verifying the proliferative capacity and multi-differentiation ability of the cells isolated in step (b).
  5. 청구항 1에 있어서, 상기 태아연골유래 세포는 활성화 림프구에서 생성되는 면역 싸이토카인의 발현을 제어하는 것을 특징으로 하는 면역질환의 예방 또는 치료용 조성물.The composition for preventing or treating immune diseases according to claim 1, wherein the fetal chondrocyte-derived cells control the expression of immune cytokines produced in activated lymphocytes.
  6. 청구항 1에 있어서, 상기 태아연골유래 세포는 면역반응 또는 염증반응에 관련된 TNF-α, IL-13, IFN-γ의 발현을 억제하는 것을 특징으로 하는 면역질환의 예방 또는 치료용 조성물.The method of claim 1, wherein the fetal cartilage-derived cells for the prevention or treatment of immune diseases, characterized in that to suppress the expression of TNF-α, IL-13, IFN-γ involved in the immune response or inflammatory response.
  7. 청구항 1에 있어서, 상기 태아연골유래 세포는 항염증 싸이토카인인 IL-10의 발현에 영향을 주지 않거나 IL-10의 발현을 증가시킴으로써 면역활성을 조절하는 것을 특징으로 하는 면역질환의 예방 또는 치료용 조성물. The method of claim 1, wherein the fetal cartilage-derived cells do not affect the expression of anti-inflammatory cytokine IL-10 or increase the expression of IL-10 by controlling the immune activity, characterized in that the composition for the prevention or treatment of immune diseases .
  8. 청구항 1에 있어서, 상기 조성물은 태아연골유래 세포 추출물 또는 분비물을 포함하는 것을 특징으로 하는 면역질환의 예방 또는 치료용 조성물.The method of claim 1, wherein the composition is a composition for the prevention or treatment of immune diseases, characterized in that the fetal cartilage-derived cell extract or secretion.
  9. 청구항 1에 있어서, 상기 면역질환은 자가면역질환, 염증성 질환 및 세포, 조직 또는 기관의 이식거부 질환으로 이루어진 군에서 선택된 것을 특징으로 하는 면역질환의 예방 또는 치료용 조성물.The method of claim 1, wherein the immune disease is selected from the group consisting of autoimmune diseases, inflammatory diseases and rejection diseases of cells, tissues or organs.
  10. 청구항 1에 있어서, 상기 면역질환은 관절염, 자가면역성 간염, 류마티스성 관절염, 골관절염, 인슐린 의존성 당뇨병, 궤양성 대장염, 크론병, 다발성 경화증, 자가면역성 심근염, 피부경화증, 중증근육무력증, 다발성 근육염/피부 근육염, 하시모토병, 자가면역 혈구감소증, 쇼그렌 증후군, 혈관염 증후군 및 전신홍반루푸스로 이루어진 군에서 선택된 것을 특징으로 하는 면역질환의 예방 또는 치료용 조성물.The method of claim 1, wherein the immune disease is arthritis, autoimmune hepatitis, rheumatoid arthritis, osteoarthritis, insulin-dependent diabetes, ulcerative colitis, Crohn's disease, multiple sclerosis, autoimmune myocarditis, skin sclerosis, myasthenia gravis, multiple myositis / skin Myositis, Hashimoto's disease, autoimmune thrombocytopenia, Sjogren's syndrome, vasculitis syndrome and systemic lupus erythematosus composition for the prevention or treatment of immune diseases, characterized in that.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004043401A2 (en) * 2002-11-13 2004-05-27 Wackvom Limited Method of preparing anti-angiogenic drug from cartillage and chondrocytes and methods of use
KR20080063406A (en) * 2005-10-13 2008-07-03 안트로제네시스 코포레이션 Immunomodulation using placental stem cells
KR20100084142A (en) * 2009-01-15 2010-07-23 코아스템(주) Pharmaceutical compositions for treatment of bone disease or anti-inflammatory compositions comprising cartilage stem cell
WO2011064669A2 (en) * 2009-11-30 2011-06-03 Pluristem Ltd. Adherent cells from placenta and use of same in disease treatment

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004043401A2 (en) * 2002-11-13 2004-05-27 Wackvom Limited Method of preparing anti-angiogenic drug from cartillage and chondrocytes and methods of use
KR20080063406A (en) * 2005-10-13 2008-07-03 안트로제네시스 코포레이션 Immunomodulation using placental stem cells
KR20100084142A (en) * 2009-01-15 2010-07-23 코아스템(주) Pharmaceutical compositions for treatment of bone disease or anti-inflammatory compositions comprising cartilage stem cell
WO2011064669A2 (en) * 2009-11-30 2011-06-03 Pluristem Ltd. Adherent cells from placenta and use of same in disease treatment

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHANG, C. -J. ET AL.: "Placenta-derived multipotent cells exhibit immunosuppressive properties that are enhanced in the presence of interferon-gamma", STEM CELLS, vol. 24, 2006, pages 2466 - 2477 *

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