KR20190107644A - Methods for improving proliferation of stem cell using chenodeoxycholic acid - Google Patents
Methods for improving proliferation of stem cell using chenodeoxycholic acid Download PDFInfo
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Abstract
Description
본 발명은, 케노데옥시콜산(chenodeoxycholic acid)을 포함하는 줄기세포 증식 향상용 배지 조성물, 및 이의 용도에 관한 것이다.The present invention relates to a medium composition for improving stem cell proliferation comprising a chenodeoxycholic acid, and use thereof.
중간엽 줄기세포는 그 다분화능과 함께, 조직의 재생, 치료 및 면역 반응에 관여하는 세포로 알려져 있어, 이와 같은 특성을 이용하여 제대혈, 골수 등으로부터 중간엽 줄기세포를 분리배양하여 다양한 질환의 치료제로 개발하고자 하는 노력이 있어왔으나, 줄기세포를 계대배양함에 따라 노화가 진행되며 세포 분화가 일어나므로 줄기세포능(stemness)을 잃게 되는 문제가 있다.Mesenchymal stem cells are known to be involved in tissue regeneration, treatment, and immune response, as well as their multipotency, and by using such characteristics to separate and culture mesenchymal stem cells from umbilical cord blood, bone marrow, etc. Efforts have been made to develop, but there is a problem that the stem cell capacity (stemness) is lost because aging progresses and cell differentiation occurs by subcultured stem cells.
즉, 윤리적인 문제가 없는 성체줄기세포를 이용한 세포치료제의 개발을 위해서는, 세포의 줄기세포능을 유지하면서 효과적으로 증식시킬 수 있는 방법을 확립하는 것이 필수적이나, 성체줄기세포는 증식율이 낮고, 쉽게 노화되어 한 조직에서 얻을 수 있는 세포의 수가 한정적이라는 한계가 있다.In other words, in order to develop a cell therapy using adult stem cells without ethical problems, it is essential to establish a method that can effectively proliferate while maintaining the stem cell capacity of the adult stem cells, but adult stem cells have a low proliferation rate and easily age. There is a limit to the number of cells that can be obtained from a tissue.
이러한 문제를 해결하여 줄기세포 효율을 증진시키기 위한 방안으로서, 바이러스 벡터를 이용한 유전자 조작이나 특정 단백질 과발현이 제안되어 왔으나 이는 안정성 문제로 인하여 임상 적용에는 한계가 있는 바, 1세대 줄기세포 연구를 통해 특정질환 치료를 위한 임상적용 가능성은 검증되었으나 낮은 효율과 효과기전 규명이 미흡하고 안전성 확보 또한 해결되지 않고 있다.As a way to improve stem cell efficiency by solving these problems, genetic manipulation using viral vectors or overexpression of specific proteins have been proposed, but there are limitations in clinical applications due to stability problems. Although the clinical applicability for the treatment of diseases has been proven, low efficiency and effectiveness mechanisms are insufficient and safety is not solved.
이와 관련하여, 최근 분화세포(differentiated cell)에 비하여 배아줄기세포(ESC)와 유도만능줄기세포(iPS)에서 해당(glycolysis) 대사과정이 증가하고 산화적 인산화(oxidative phosphorylation) 대사과정이 감소되어 있음이 보고됨으로써, 대사과정을 조절하는 리프로그래밍(reprogramming)을 통해 줄기세포의 줄기세포능(stemness) 유지 및 세포 노화억제를 통한 고효율 줄기세포 개발 가능성이 제시되고 있으나, 이에 대한 분자생물학적 기전은 거의 밝혀지지 않았으며, 아직 효율적인 조절기술은 개발되지 못하고 있는 실정이다.In this regard, glycolysis and oxidative phosphorylation have been decreased in embryonic stem cells (ESC) and induced pluripotent stem cells (iPS) compared to differentiated cells. This report suggests the possibility of developing high-efficiency stem cells through the maintenance of stem cells of stem cells and inhibition of cellular aging through reprogramming that regulates metabolic processes. Not yet, efficient control technology has not been developed yet.
이에, 본 발명자는 줄기세포의 노화를 억제하고 세포 생존 및 증식율을 향상시킬 수 있는 방법에 대하여 예의 연구한 결과, 종래 담석용해제로 알려져 있는 케노데옥시콜산(chenodeoxycholic acid)이 줄기세포를 효과적으로 증식시킬 수 있음을 발견함으로써 본 발명을 완성하게 되었다.Therefore, the present inventors have studied intensively about a method for inhibiting aging of stem cells and improving cell survival and proliferation rate. As a result, chenodeoxycholic acid, known as a conventional gallstone dissolving agent, effectively proliferates stem cells. The present invention has been completed by discovering that it can.
따라서, 본 발명은, 케노데옥시콜산을 포함하는, 줄기세포 생존/증식 향상용 배지 조성물, 및 이의 용도를 제공하는 것을 목적으로 한다.Accordingly, an object of the present invention is to provide a stem cell survival / proliferation medium composition comprising kenodeoxycholic acid, and a use thereof.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.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.
본 발명은, 케노데옥시콜산을 포함하는, 줄기세포 증식 향상용 배지 조성물을 제공한다.The present invention provides a medium composition for improving stem cell proliferation, including kenodeoxycholic acid.
본 발명의 일 구체예로서, 상기 케노데옥시콜산은 배지에 1∼100μM의 농도로 포함되어 있는 것을 특징으로 한다.In one embodiment of the present invention, the kenodeoxycholic acid is characterized in that contained in the medium at a concentration of 1 to 100μM.
본 발명의 다른 구체예로서, 상기 배지는 10% 소태아혈청(FBS) 및 50 ug/ml 젠타마이신이 첨가된 α-MEM(Minimum Essential Media) 배지인 것을 특징으로 한다.In another embodiment of the present invention, the medium is characterized in that the medium essential media (α-MEM) added with 10% fetal bovine serum (FBS) and 50 ug / ml gentamycin.
본 발명의 또 다른 구체예로서, 상기 줄기세포는 배아줄기세포 또는 성체줄기세포인 것을 특징으로 한다.In another embodiment of the present invention, the stem cells are characterized in that the embryonic stem cells or adult stem cells.
본 발명의 또 다른 구체예로서, 상기 성체줄기세포는 제대, 제대혈, 골수, 지방, 근육, 신경, 피부, 양막 및 태반으로 구성된 군에서 선택되는 1종 이상의 조직으로부터 유래된 중간엽 줄기세포인 것을 특징으로 한다.In another embodiment, the adult stem cells are mesenchymal stem cells derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane and placenta. It features.
또한, 본 발명은 상기 배지 조성물에 줄기세포를 배양하는 단계를 포함하는, 줄기세포 증식 향상방법을 제공한다.The present invention also provides a method for improving stem cell proliferation, comprising culturing stem cells in the medium composition.
또한, 본 발명은 상기 방법에 의해 얻어진, 증식능이 향상된 줄기세포를 제공한다.The present invention also provides a stem cell obtained by the above method, improved proliferative capacity.
본 발명의 일 구체예로서, 상기 줄기세포는 제대, 제대혈, 골수, 지방, 근육, 신경, 피부, 양막 및 태반으로 구성된 군에서 선택되는 1종 이상의 조직으로부터 유래된 중간엽 줄기세포인 것을 특징으로 한다.In one embodiment, the stem cells are mesenchymal stem cells derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerves, skin, amniotic membrane and placenta. do.
본 발명에 의하면, 유전자 조작이나 바이러스 벡터 등을 사용하지 않으면서 배양환경 조절이라는 간편하고도 안전한 방법을 통하여, 차세대 고효율 줄기세포를 대량 생산할 수 있다.According to the present invention, it is possible to mass-produce the next generation of high efficiency stem cells through a simple and safe method of controlling the culture environment without using genetic manipulations or viral vectors.
또한, 본 발명에 의하면, 줄기세포의 대사 과정을 선택적으로 조절할 수 있는 분자생물학적 기전, 대사조절 마커를 규명함으로써 고효율 줄기세포 개발에 대한 새로운 방법론을 제시할 수 있다.In addition, according to the present invention, it is possible to suggest a new methodology for developing high-efficiency stem cells by identifying molecular biological mechanisms and metabolic control markers that can selectively regulate metabolic processes of stem cells.
도 1은, 줄기세포에 케노데옥시콜산을 처리시 줄기세포 증식효율에 미치는 영향을 확인한 BrdU 어세이 결과이다.
도 2는, 줄기세포에 케노데옥시콜산을 처리시 줄기세포 증식효율에 미치는 영향을 확인한 ATP 어세이 결과이다.1 is a BrdU assay confirming the effect on the stem cell proliferation efficiency when treated with kenodeoxycholic acid on stem cells The result is.
Figure 2, ATP assay confirming the effect on stem cell proliferation efficiency when treatment with kenodeoxycholic acid on stem cells The result is.
본 발명은, 케노데옥시콜산을 포함하는, 줄기세포 증식 향상용 배지 조성물을 제공한다.The present invention provides a medium composition for improving stem cell proliferation, including kenodeoxycholic acid.
종래, 케노데옥시콜산((R)-((3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid)은 일차성 담즙산으로서 담즙염을 콜레스테롤로 전환하는 효소를 억제하는 담석용해제로서의 치료 용도가 알려져 있었으나, 본 발명에서는 케노데옥시콜산의 줄기세포능 향상 용도를 최초로 발견하였다.Conventionally, kenodeoxycholic acid ((R)-((3R, 5S, 7R, 8R, 9S, 10S, 13R, 14S, 17R) -3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta [a] phenanthren-17-yl) pentanoic acid) is a primary bile acid, and the therapeutic use as a gallstone dissolving agent that inhibits the enzyme that converts bile salts into cholesterol is known. It was.
[chenodeoxycholic acid] [chenodeoxycholic acid]
본 발명에서 배지에 포함되는 케노데옥시콜산의 농도에 제한은 없으며, 예를 들면 1∼100uM의 농도로 포함되는 것이 바람직하며, 10uM인 것이 더욱 바람직하다.There is no restriction | limiting in the density | concentration of the kenodeoxycholic acid contained in a medium in this invention, For example, it is preferable that it is contained in concentration of 1-100 uM, and it is more preferable that it is 10 uM.
본 발명에서 세포 배양에 이용되는 배지에 제한은 없으며, 예를 들면 10% 소태아혈청(FBS) 및 50 ug/ml 젠타마이신이 첨가된 α-MEM(Minimum Essential Media) 배지인 것이 바람직하다.There is no limitation on the medium used for cell culture in the present invention, for example, it is preferable to use α-MEM (Minimum Essential Media) medium to which 10% fetal bovine serum (FBS) and 50 ug / ml gentamicin are added.
본 발명에서 '줄기세포'란 미분화된 세포로서 자기 복제 능력을 가지면서 두 개 이상의 서로 다른 종류의 세포로 분화하는 능력을 갖는 세포를 말한다. 본 발명의 줄기세포는 자가 또는 동종 유래 줄기세포일 수 있으며, 인간 및 비인간 포유류를 포함한 임의 유형의 동물 유래일 수 있고, 상기 줄기세포가 성체로부터 유래된 것이든 배아로부터 유래된 것이든 이에 한정되지 않는다.In the present invention, 'stem cell' refers to a cell having an ability to differentiate into two or more different types of cells while having an autologous ability as an undifferentiated cell. Stem cells of the present invention may be autologous or allogeneic stem cells, may be from any type of animal, including humans and non-human mammals, and is not limited to whether the stem cells are derived from adults or embryos Do not.
본 발명의 줄기세포는 배아 줄기세포 또는 성체 줄기세포를 포함하며, 바람직하게는 성체 줄기세포이다. 상기 성체 줄기세포는 중간엽 줄기세포, 인간 조직 유래 중간엽 기질세포(mesenchymal stromal cell), 인간 조직 유래 중간엽 줄기세포, 다분화능 줄기세포 또는 양막상피세포일 수 있으며, 바람직하게는 중간엽 줄기세포이나, 이에 한정되지 않는다. 상기 중간엽 줄기세포는 제대, 제대혈, 골수, 지방, 근육, 신경, 피부, 양막 및 태반 등으로부터 유래된 중간엽 줄기세포일 수 있으나, 이에 한정되지 않는다.Stem cells of the present invention include embryonic stem cells or adult stem cells, preferably adult stem cells. The adult stem cells may be mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, multipotent stem cells or amniotic epithelial cells, preferably mesenchymal stem cells However, the present invention is not limited thereto. The mesenchymal stem cells may be mesenchymal stem cells derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, and placenta, but is not limited thereto.
본 발명에서, '제대-유래 줄기세포(Wharton's Jelly-derived stem cells)'라 함은 제대로부터 분리된 줄기세포를 모두 포함하며, 포유류의 태아가 태반에서 성장할 수 있도록 모체와 배를 연결해주는 줄을 의미할 수 있으며, 일반적으로 와튼 젤리(Wharton's Jelly)로 둘러싸인 3개의 혈관, 즉 2개의 배꼽 동맥과 1개의 배꼽 정맥으로 구성된 조직을 의미할 수 있다. In the present invention, 'Wharton's Jelly-derived stem cells' includes all stem cells that are properly separated from each other, and a line connecting the mother and the embryo so that the mammalian fetus can grow in the placenta. In general, it may mean a tissue composed of three blood vessels surrounded by Wharton's Jelly, that is, two umbilical arteries and one umbilical vein.
본 발명에서, '태반-유래 줄기세포(placenta-derived stem cells)'라 함은 태반으로부터 분리된 줄기세포를 모두 포함하며, 바람직하게는 체외로 분리된 인간의 태반으로부터 분리된 4종류의 줄기세포, 즉 (1) 양막 상피세포(human amniotic epithelial cells, hAEC), (2) 양막에서 유래한 중간엽 줄기세포(human amniotic mesenchymal stromal cells 또는 human amniotic mesenchymal stem cells, hAMSC), 3) 융모막 중간엽 줄기세포(human chorionic mesenchymal stromal cells 또는 human chorionic mesenchymal stem cells, hCMSC), 및 (4) 융모 영양막 세포(human chorionic trophoblastic cells, hCTC)를 포함한다.In the present invention, the term 'placenta-derived stem cells' includes all stem cells separated from the placenta, and preferably, four types of stem cells separated from the human placenta separated in vitro. Ie (1) human amniotic epithelial cells (hAEC), (2) amnion-derived mesenchymal stromal cells or human amniotic mesenchymal stem cells (hAMSC), and 3) chorionic mesenchymal stem cells. Cells (human chorionic mesenchymal stromal cells or human chorionic mesenchymal stem cells (hCMSC)), and (4) human chorionic trophoblastic cells (hCTC).
중간엽줄기세포의 분리는 당업자에게 자명한 방법으로 수행될 수 있으며, 예를 들면, Pittenger 등(Science 284: 143, 1997)와 van 등(J. Clin. Invest., 58: 699, 1976)의 문헌들에 개시된 방법으로 분리될 수 있다.Isolation of mesenchymal stem cells can be performed by methods known to those skilled in the art, for example, by Pittenger et al. (Science 284: 143, 1997) and van et al. (J. Clin. Invest., 58: 699, 1976). It can be separated by the method disclosed in the literature.
본 발명에서 줄기세포 증식 향상이란, 세포노화가 억제되고 세포증식능 및 단백질 항상성이 향상됨으로서 줄기세포 특성이 유지되는 의미를 포함하며, 이에 의해 줄기세포 마커인 Nanog, Oct4 또는 KLF4 유전자 등의 발현이 증가될 수 있다. 이들 유전자는 배아줄기세포에서 많이 발현하는 줄기세포의 전분화능, 즉 줄기세포 특성을 유지하는데 중요한 역할을 하는 유전자로 알려져 있다.Stem cell proliferation improvement in the present invention includes the meaning that stem cell characteristics are maintained by suppressing cell aging and improving cell proliferation ability and protein homeostasis, thereby increasing expression of stem cell markers such as Nanog, Oct4 or KLF4 genes. Can be. These genes are known as genes that play an important role in maintaining the pluripotency of stem cells expressed in embryonic stem cells, that is, stem cell characteristics.
또한, 본 발명은 상기 배지 조성물에 줄기세포를 배양하는 단계를 포함하는 줄기세포 증식 향상방법, 및 이에 의해 얻어진 증식능이 향상된 줄기세포를 제공한다.In addition, the present invention provides a method for improving stem cell proliferation, comprising a step of culturing stem cells in the medium composition, and a stem cell having improved proliferation ability.
또한, 본 발명은 상기 줄기세포를 포함하는, 다양한 질환 치료용 세포 치료제를 제공한다.The present invention also provides a cell therapeutic agent for treating various diseases, including the stem cells.
이하, 본 발명의 이해를 돕기 위하여 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, examples are provided to help understand the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.
[실시예]EXAMPLE
실시예 1. 인간 제대 중간엽 줄기세포의 준비Example 1 Preparation of Human Umbilical Cord Mesenchymal Stem Cells
인간 제대 중간엽 줄기세포는 삼성서울병원의 IRB (IRB# 2015-09-023-003)에 의해 승인된 기준에 따라 탯줄을 확보한 후, 다음의 방법으로 중간엽 줄기세포를 분리하였다. Human umbilical cord mesenchymal stem cells were obtained by umbilical cord according to the criteria approved by IRB (IRB # 2015-09-023-003) of Samsung Seoul Hospital, and then the mesenchymal stem cells were separated by the following method.
우선, 3-4cm의 탯줄 조직을 잘게 자르고, 세포 외 기질을 분해하기 위해 콜라게나아제 용액 (Gibco, USA)을 60-90분 동안 처리한 후, 0.25% 트립신 (Gibco, USA)을 넣고 30분 동안 37℃에서 분해시켰다. 이후, 소태아혈청 (Fetal Bovine Serum, FBS) (Biowest, USA)을 넣고 1000×g에서 10분 동안 원심 분리하여 세포를 얻고, MEM 배지(Minimum Essential Media) (Gibco, USA)에 10% FBS와 50 ug/ml 젠타마이신 (Gibco, USA)이 첨가된 배지를 이용하여 37℃, 5% CO2 환경에서 세포를 배양하여, passage 5 또는 6의 중간엽 줄기세포를 실험에 사용하였다.First, chop the cord tissue of 3-4 cm, treat collagenase solution (Gibco, USA) for 60-90 minutes to decompose the extracellular matrix, add 0.25% trypsin (Gibco, USA) for 30 minutes While at 37 ° C. Subsequently, fetal bovine serum (FBS) (Biowest, USA) was added and centrifuged at 1000 × g for 10 minutes to obtain cells, and 10% FBS and MEM medium (Minimum Essential Media) (Gibco, USA) Cells were cultured in a 37 ° C., 5% CO 2 environment using medium supplemented with 50 ug / ml gentamycin (Gibco, USA), and mesenchymal stem cells of passage 5 or 6 were used for the experiment.
실시예 2. 중간엽 줄기세포 증식 향상 확인Example 2. Confirmation of Mesenchymal Stem Cell Proliferation Improvement
인간 제대 중간엽 줄기세포(1×104 세포)를 96 well plate에 분주하여 24시간 동안 배양한 후, 케노데옥시콜산(10 μM)를 처리하고 72시간 후에 하기의 세포증식 분석을 수행하였다. Human umbilical cord mesenchymal stem cells (1 × 10 4 cells) were aliquoted into 96 well plates and incubated for 24 hours. After treatment with kenodeoxycholic acid (10 μM), the following cell proliferation assay was performed 72 hours later.
2-1. BrdU cell proliferation assay2-1. BrdU cell proliferation assay
세포증식의 정도는 세포의 DNA 합성시에 결합되는 5-bromo-2'-deoxyuridine (BrdU)를 이용한 BrdU cell proliferation assay kit(Cell Signaling Technology, USA)로 분석하였다. 구체적으로는, BrdU를 세포에 첨가하고 24시간 동안 반응시킨 후 세포를 고정하고 DNA를 변성시킨 다음, Anti-BrdU 항체를 처리하고, HRP(horse-radish peroxidase)가 연결된 2차 항체를 사용하여 TMB(Tetramethylbenzidine) 기질과 반응시키고, ELISA reader로 450nm에서 흡광도 수치를 측정하였다.The degree of cell proliferation was analyzed by BrdU cell proliferation assay kit (Cell Signaling Technology, USA) using 5-bromo-2'-deoxyuridine (BrdU) that binds to the cell DNA synthesis. Specifically, BrdU was added to the cells and allowed to react for 24 hours, the cells were fixed, the DNA was denatured, the anti-BrdU antibody was treated, and TMB using a secondary antibody linked to horse-radish peroxidase (HRP). Reaction with (Tetramethylbenzidine) substrate, the absorbance value was measured at 450nm with ELISA reader.
그 결과, 도 1에 나타낸 바와 같이, 케노데옥시콜산 처리군은 미처리 대조군에 비하여 중간엽 줄기세포의 증식률을 현저히 향상시킬 수 있음을 확인하였다.As a result, as shown in Figure 1, it was confirmed that the kenodeoxycholic acid treatment group can significantly improve the proliferation rate of mesenchymal stem cells compared to the untreated control group.
2-2. ATP cell proliferation assay2-2. ATP cell proliferation assay
살아있는 세포 내의 ATP 수준은 일정하게 유지되며, 생존하고 있는 세포의 수를 직접적으로 반영하므로, ATP 수준은 세포 증식을 측정하기 위한 방법으로 사용된다. 본 실시예에서는 ATP 측정을 위하여 CellTiter-Glo Luminescent Cell Viability Assay (Promega Corporation, USA)를 이용하였으며, 발광도의 측정은 mithras LB-943 plate luminometer (Berthold Technologies, Germany)에서 확인하였다. Since ATP levels in living cells remain constant and directly reflect the number of living cells, ATP levels are used as a method to measure cell proliferation. In this example, CellTiter-Glo Luminescent Cell Viability Assay (Promega Corporation, USA) was used for ATP measurement, and luminescence was measured by mithras LB-943 plate luminometer (Berthold Technologies, Germany).
그 결과, 도 2에 나타낸 바와 같이, 케노데옥시콜산 처리군은 미처리 대조군에 비하여 중간엽 줄기세포의 증식률을 현저히 향상시킬 수 있음을 확인하였다.As a result, as shown in Figure 2, it was confirmed that the kenodeoxycholic acid treated group can significantly improve the proliferation rate of mesenchymal stem cells as compared to the untreated control group.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해되어야 한다.The foregoing 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, the embodiments described above are to be understood in all respects as illustrative and not restrictive.
Claims (2)
Kenodeoxycholic acid (chenodeoxycholic acid) comprising the step of culturing the stem cells in a medium containing, the proliferation ability improved stem cell manufacturing method.
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