KR101329524B1 - Methods for Specifically Inducing Cell Death of Undifferentiated Pluripotent Stem Cells - Google Patents

Methods for Specifically Inducing Cell Death of Undifferentiated Pluripotent Stem Cells Download PDF

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KR101329524B1
KR101329524B1 KR1020110048624A KR20110048624A KR101329524B1 KR 101329524 B1 KR101329524 B1 KR 101329524B1 KR 1020110048624 A KR1020110048624 A KR 1020110048624A KR 20110048624 A KR20110048624 A KR 20110048624A KR 101329524 B1 KR101329524 B1 KR 101329524B1
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stem cells
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inhibitor
sirtuin
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김동욱
허용준
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연세대학교 산학협력단
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Abstract

본 발명은 (a) 미분화 전능성 줄기세포 및 분화 세포(differentiated cells)를 포함하는 세포 시료를 준비(preparation)하는 단계; 및 (b) 상기 세포 시료에 시르투인(Sirtuin) 억제제를 처리하여 미분화 전능성 줄기세포를 선택적으로 사멸시키는 단계를 포함하는 미분화 전능성 줄기세포의 선택적 제거방법 및 미분화 전능성 줄기세포가 제거를 위한 세포 조성물에 관한 것이다. 본 발명에 따르면, 전능성 줄기세포의 분화과정 중 미분화로 남아있는 줄기세포를 효율적으로 제거하여 세포 분화 초기 미분화 세포에 의한 분화효율 저해 및 세포 간 분화 정도 차이를 감소시킬 수 있으며 분화과정 중 이식 후 종양생성의 가능성을 지닌 미분화 세포를 제거하여 전능성줄기세포를 임상적용에 있어 안전성 확보가 기대된다.The present invention comprises the steps of (a) preparing a cell sample comprising undifferentiated pluripotent stem cells and differentiated cells (preparation); And (b) treating the cell sample with a sirtuin inhibitor to selectively kill undifferentiated pluripotent stem cells and a cell composition for removing undifferentiated pluripotent stem cells. It is about. According to the present invention, stem cells remaining as undifferentiated during the differentiation of pluripotent stem cells can be efficiently removed, thereby inhibiting the differentiation efficiency by the undifferentiated cells in the early stage of cell differentiation and reducing the difference between the differentiation between cells. By removing undifferentiated cells with potential for production, it is expected to secure safety for clinical application of pluripotent stem cells.

Description

미분화 전능성 줄기세포의 선택적 세포사멸 방법{Methods for Specifically Inducing Cell Death of Undifferentiated Pluripotent Stem Cells}Methods for Specific Inducing Cell Death of Undifferentiated Pluripotent Stem Cells

본 발명은 미분화 전능성 줄기세포의 선택적 제거방법 및 미분화 전능성 줄기세포가 제거를 위한 세포 조성물에 관한 것이다.
The present invention relates to a method for selective removal of undifferentiated pluripotent stem cells and a cell composition for removal of undifferentiated pluripotent stem cells.

배아줄기세포(embryonic stem cells)는 착상 전 배반포의 내부세포괴(inner cell mass: ICM)로부터 확립된 세포로서, 특정배양조건에서 무한히 증식하고 미분화 상태를 유지할 수 있으며 체내에 모든 종류의 세포로 분화가 가능한 전분화능을 가지는 세포를 말한다. 배아줄기세포는 1980년대 초 생쥐에서 처음 만들어졌고 인간의 경우 1998년 미국 위스콘신대학의 Thomson 박사 연구팀에 의해 처음 확립되었다.1 특히 배아줄기세포는 체외(In vitro)에서 적절한 외부환경의 조절에 의해 신경계 세포, 심근 세포, 혈관내피 세포, 조혈모세포, 간세포, 췌장세포 및 생식세포 등의 인체를 구성하는 다양한 종류의 세포로 분화할 수 있다. 이런 특성으로 인해 배아줄기세포는 체외에서 자발적 혹은 유도에 의해 연구자가 원하는 세포로의 분화가 가능하다.1 따라서 배아줄기세포는 현재 여러 난치성 질환들을 치료할 수 있는 재료 및 발생학 연구의 도구 그리고 여러 약물의 탐색 연구 재료로써 사용될 수 있다.2 하지만 성체줄기세포와 달리 자신의 세포가 아닌 세포를 이용해야 하기 때문에 발생하는 면역거부반응이 배아줄기세포의 이용에 있어 가장 큰 단점으로 제기되어 왔고, 이를 극복하기 위해 체세포를 이용하여 배아줄기세포와 유사한 전분화능을 가진 세포를 만드는 몇몇 기술이 확립되었다.3-6 이런 기술들 중 하나가 유도만능줄기세포(Induced pluripotent stem cell: iPS cell)의 확립이며, 이것은 완전히 분화된 체세포에 특정 유전자(예를 들면, Oct4, Nanog, c-Myc, Klf-4), 단백질 또는 화학물을 주입하여 분화가 가능한 미분화 상태를 유지하는 전분화능을 가진 세포이다.7-14 유도만능줄기세포는 배아줄기세포와 더불어 여러 난치성 질환을 치료할 수 있는 재료 및 연구의 도구로 가치를 인정받고 있다.15-16 배아줄기세포와 유도만능줄기세포처럼 전분화능을 가진 줄기세포를 일컬어 전능성줄기세포(pluripotent stem cell)라고 하며 상기 두 가지 세포는 성격이 거의 유사한 것으로 알려져 있다.8-9,11-13,17-18 전능성줄기세포를 이용하기 위해서는 다음과 같은 단계를 거쳐야 한다. Embryonic stem cells are cells established from the inner cell mass (ICM) of blastocysts before implantation. They are able to proliferate indefinitely under certain culture conditions, remain undifferentiated, and differentiate into all kinds of cells in the body. It refers to a cell with possible pluripotency. Embryonic stem cells were first created in mice in the early 1980s, and in humans they were first established in 1998 by Dr. Thomson's team at the University of Wisconsin. 1 Embryonic Stem Cells In particular In vitro ), it can differentiate into various kinds of cells constituting the human body, such as nervous system cells, cardiomyocytes, vascular endothelial cells, hematopoietic stem cells, hepatocytes, pancreatic cells and germ cells by appropriate external environment control. Due to this characteristic, embryonic stem cells can be differentiated into cells desired by the researcher either spontaneously or in vitro. 1 Therefore, embryonic stem cells can be used as a navigation tool in materials research, and many of the current medications materials and embryology research that can cure many incurable diseases. 2 However, unlike adult stem cells, the immune rejection reaction caused by the use of cells other than their own cells has been raised as the biggest disadvantage in using embryonic stem cells. To overcome this, embryonic stem cells using somatic cells Several techniques for making cells with similar pluripotency have been established. 3-6 One of these techniques is the establishment of induced pluripotent stem cells (iPS cells), which are specific genes for fully differentiated somatic cells (eg Oct4, Nanog, c-Myc, Klf-4). ), A cell with pluripotency that maintains an undifferentiated state capable of differentiation by injecting proteins or chemicals. 7-14 Induced pluripotent stem cells, along with embryonic stem cells, have been recognized as a valuable tool for the treatment of various refractory diseases. Stem cells with pluripotency, such as 15-16 embryonic stem cells and induced pluripotent stem cells, are also called pluripotent stem cells, and the two cells are known to be nearly similar in nature. In order to use 8-9,11-13,17-18 pluripotent stem cells, the following steps should be taken.

첫 번째 단계는 유지 배양 단계이다. 전능성줄기세포가 가진 성체줄기세포와 다른 성격 중 하나는 특정 배양조건에서 세포가 미분화 상태를 유지하면서 무한히 증식할 수 있다는 것이며 이로 인해 치료에 필요한 많은 양의 세포를 만들어 낼 수 있다. 이 과정에서 줄기세포의 분화를 막고 고유의 성질을 잃어버리지 않게 하는 기술이 필요하다. 전능성줄기세포는 세포 단독으로 미분화 상태를 유지하기 어려워 일반적인 경우 생쥐의 섬유아세포(fibroblast)와 같은 지지세포(Feeder cell) 위에서 키우게 된다.1 오늘날에는 줄기세포 배양 기술의 발전으로 생쥐 배아줄기세포는 LIF(Leukemia inhibitory factor)의 처리를 통해 지지세포가 없는 조건에서 유지 배양이 가능하다.19 또한 인간 전능성줄기세포의 경우에는 유지배양을 위한 지지세포를 생쥐의 세포로 사용함으로써 발생하는 이종간 오염을 막기 위하여, 인간의 세포를 이용한 지지세포 개발 및 지지세포 없이 특수한 배양액을 사용하여 배양하는 방법을 연구하고 있다.20-22,24 The first step is the maintenance culture step. One of the different characteristics of adult stem cells from pluripotent stem cells is that they can proliferate indefinitely while maintaining their undifferentiated state under certain culture conditions, which can produce large amounts of cells for treatment. In this process, there is a need for a technique that prevents the differentiation of stem cells and does not lose their intrinsic properties. Pluripotent stem cells are difficult to maintain an undifferentiated state by cells alone, and thus are grown on feeder cells such as fibroblasts in mice. 1 Today, with the development of rat stem cell culture techniques embryonic stem cell is capable of maintaining the culture in the absence of feeder cells with the processing conditions of (Leukemia inhibitory factor) LIF. 19 In the case of human pluripotent stem cells, in order to prevent heterogeneous contamination caused by using support cells for maintenance culture as cells of mice, development of support cells using human cells and culturing using a special culture without support cells Is studying. 20-22,24

두 번째 단계는 연구 및 임상에 사용하기 위해 전능성줄기세포를 원하는 세포로 만드는 분화과정이다. 전능성줄기세포는 유지 배양환경에서 특정한 인자 제거(예를 들면, 생쥐의 경우 LIF, 인간의 경우 bFGF(basic fibroblast growth factor))만으로도 자발적으로 삼배엽을 형성하고 여러 가지의 세포 종류로 분화하게 되는데 이러한 과정을 배아체(embryonic body) 형성이라고 한다.34 하지만 연구 및 치료에는 그 목적에 따라 적합한 한가지 종류의 순수한 세포가 필요하다. 목적에 따른 특정 종류의 세포를 만들어 내기 위해서는 배양환경, 배양방법 및 첨가물질(예를 들면, 단백질 및 화학물) 조절 등의 여러 분화기술이 요구된다. 이러한 분화기술은 발생학을 기반으로 하여, 체내 환경(In vivo)에서 발현되는 인자 및 환경을 시험관 내(In vitro)에 적용하고 최적화하여 원하는 종류의 세포로만 분화할 수 있도록 많은 연구들이 진행 중에 있다. The second step is the differentiation process of making pluripotent stem cells into desired cells for use in research and clinical use. Pluripotent stem cells spontaneously form trioderm and differentiate into various cell types by removing specific factors (eg LIF in mice and basic fibroblast growth factor in humans) in maintenance culture. The process is called embryonic body formation. 34 However, research and treatment require one type of pure cell that is suitable for its purpose. In order to produce a specific kind of cells according to the purpose, various differentiation techniques such as control of the culture environment, culture method and additives (eg, proteins and chemicals) are required. Due to this differentiation technique is based on the embryology, the system environment (In vivo) within the agent and the environment to be expressed in vitro (In Many studies are underway to apply and optimize in vitro to differentiate only into the cells of the desired type.

마지막 단계는 원하는 세포로 분화된 세포를 안전하게 이식하는 과정이다. 하지만 성체줄기세포와 달리 전능성줄기세포를 재료로 하는 분화세포의 경우 이식 후 안전성에 엄격한 제한이 있다. 전능성줄기세포의 전분화능 및 무한히 증식할 수 있는 능력은 치료를 위한 이식 후 종양세포를 만들 수 있기 때문이다. 종양형성의 가능성은 전능성줄기세포를 치료에 활용함에 있어서 가장 큰 장벽으로 여겨져 왔다. 따라서 여러 연구자들에 의해 이식 후 종양생성의 가능성을 없애는 연구가 진행되고 있다.35-36 최근 분화기술의 발달로 전능성줄기세포를 이용한 분화세포가 임상적용에 가까워짐에 따라 줄기세포의 유지 및 분화에 있어서 종양생성의 위험성을 없애는 안정성 연구의 중요성은 더욱 커지고 있다.The final step is to safely transplant the differentiated cells into the desired cells. However, unlike adult stem cells, differentiated cells based on pluripotent stem cells have a strict restriction on safety after transplantation. The pluripotency of pluripotent stem cells and the ability to proliferate infinitely is because tumor cells can be made after transplantation for treatment. The possibility of tumor formation has been considered the greatest barrier to the use of pluripotent stem cells in the treatment. Therefore, several researchers are working to eliminate the possibility of tumor formation after transplantation. With the development of differentiation technology, differentiated cells using pluripotent stem cells are approaching clinical applications, and the importance of stability studies to eliminate the risk of tumor formation in the maintenance and differentiation of stem cells is increasing.

배아줄기세포 및 유도만능줄기세포를 난치성 질병 치료 목적으로 이용하기 위해서는 많은 연구를 통한 안전한 유지기술과 원하는 세포로의 분화 그리고 안전한 이식의 과정을 반드시 거쳐야 한다. 하지만 각각의 과정에서 배아줄기세포의 증식 능력 및 전분화 능력은 질병 치료를 위해 반드시 필요한 능력이지만 그로 인해 원치 않는 세포로의 분화로 인한 부작용이 따를 수 있으며, 남아있는 미분화 세포에 의한 테라토마(Teratoma)와 같은 종양형성의 위험성 또한 가지고 있다.35-36 종양형성의 원인은 인간 배아줄기세포 및 유도 만능세포를 이용한 분화유도 상황에서 분화가 되지 않고 전능성줄기세포의 성질을 그대로 유지하려고 하는 세포가 남아있기 때문이다.38 이러한 현상은 세포간 접촉에 의한 분화 신호 전달 억제 및 미분화 신호 전달 유지 또는 줄기세포의 이상에 의한 분화억제반응 등 여러 가지 원인이 있을 수 있다. 또한 유전자 도입 과정 중 이상이 생긴 유도만능줄기세포, 장기간 배양으로 인한 배양적응 줄기세포 등의 문제가 발생한 줄기세포뿐만 아니라 보통의 줄기세포에서도 분화유도 조건에 따라 미분화 세포가 남아 있을 수 있다.31,35-36,39-41 이식 전 종양형성을 일으킬 수 있는 전능성줄기세포 이용에 안전성을 확보하기 위해 몇몇 연구자들은 FACS 및 MACS와 같은 세포분리기구를 이용하여 세포막에 존재하는 항원을 이용해 분화된 세포를 분리하거나, 미분화 마커에 대한 항원을 이용해 미분화 세포를 배제하였다.35,65 하지만 분리기구를 사용하는 동안 세포에 스트레스를 주거나 상처를 입히기 때문에 분리된 세포의 생존에 부정적인 영향을 끼칠 수 있다. 또한 세포를 추출하고 분리하는 과정이 복잡하며 세포분리에 필요한 항체와 장비 등으로 인한 고가의 비용이 요구되는 점도 문제점으로 지적되어 왔다. 따라서 앞으로 세포치료제로서 분화된 전능성줄기세포를 사용하기 위해서 이러한 문제점은 반드시 극복해야 할 과제이다. In order to use embryonic stem cells and induced pluripotent stem cells for the treatment of refractory diseases, a lot of research must be carried out through safe maintenance techniques, differentiation into desired cells, and safe transplantation. However, in each process, the proliferative and pluripotent capacity of embryonic stem cells is necessary for the treatment of disease, but may have side effects due to differentiation into unwanted cells, and teratoma caused by remaining undifferentiated cells. It also has the risk of tumorigenesis. 35-36 Tumor formation is caused by remaining cells that do not differentiate and maintain the properties of pluripotent stem cells in the differentiation-inducing situation using human embryonic stem cells and induced pluripotent cells. 38. This phenomenon can have several causes such as differentiation inhibitory reaction by the above inhibition of the differentiation signal transmission caused by the contact between the undifferentiated cells, and signaling or maintenance stem cells. In addition, undifferentiated cells may remain according to differentiation-inducing conditions in normal stem cells as well as stem cells having problems such as induced pluripotent stem cells which have abnormalities during the gene introduction process and cultured stem cells due to long-term culture. To ensure safety in the use of pluripotent stem cells that can cause tumorigenesis prior to transplantation, some researchers have used cell separation devices such as FACS and MACS to differentiate the antigens present in the cell membrane. Cells were isolated or undifferentiated cells were excluded using antigens against undifferentiated markers. 35,65 However, stress or injury on cells during the use of isolation devices may adversely affect the survival of isolated cells. In addition, it has been pointed out that the process of extracting and isolating cells is complicated and requires expensive costs due to antibodies and equipment required for cell separation. Therefore, in order to use differentiated pluripotent stem cells as cell therapy in the future, these problems must be overcome.

본 연구는 전능성줄기세포가 연구 및 임상에 이용되기 위한 미분화세포의 선택적 세포사멸 유도에 그 목적을 두고 있으며, 전능성줄기세포의 생존유지 및 사멸에 중요한 역할을 하는 주요 유전자 및 단백질 연구를 통해 물리적 방법이 아닌 세포 생존 기작 조절을 가능하게 함으로써 미분화세포의 선택적 세포사멸 유도 방법을 밝혀 종양 형성 위험성을 제거하려 한다. 이 과정에서 전능성줄기세포에서 특이적으로 과발현되어 있는 단백질 중 시르투인(Sirtuin, Sirt1 및 Sirt2)이 p53을 억제하고 있다는 암세포에서 연구되었던 결과를 토대로 시르투인이 전능성줄기세포에서 세포생존 및 사멸에 중요한 역할을 하고 있을 것이라는 가설을 수립하였다.42 따라서 본 연구에서는 전능성줄기세포에서 시르투인 단백질이 세포 사멸에 작용하는 기작을 밝히고, 분화 과정 중 Sirt1, Sirt2의 특이적 억제제를 이용해 활성을 조절하여 전능성줄기세포의 특이적 세포사멸을 유도하고자 한다. The purpose of this study is to induce selective apoptosis of undifferentiated cells for the use of pluripotent stem cells in research and clinical trials, and to study physical methods through major gene and protein studies that play an important role in maintaining and killing pluripotent stem cells. In addition, it is possible to control cell survival mechanisms, thereby revealing a method for inducing selective apoptosis of undifferentiated cells, thereby removing the risk of tumor formation. In this process, the survival and death of sirtuin in pluripotent stem cells was based on the results of studies on cancer cells in which sirtuin (Sirtuin, Sirt1 and Sirt2) among the proteins overexpressed specifically in pluripotent stem cells inhibit p53. We hypothesized that it would play an important role. 42 In this study, we investigated the mechanism by which sirtuin protein acts on apoptosis in pluripotent stem cells, and induces specific apoptosis of pluripotent stem cells by regulating its activity by using specific inhibitors of Sirt1 and Sirt2 during differentiation. do.

전능성줄기세포를 선택적으로 세포사멸을 유도하기 위해서는 그 세포의 특성을 알아야 한다. 분화된 세포와 구분되는 전능성줄기세포의 특성 중 세포사멸에 중요한 역할을 할 수 있는 요소를 사전 문헌조사와 실험을 통해 알아보았다. 그 중 전능성줄기세포에서 Sirt1이 과발현 된다는 것과 Sirt1이 세포 사멸에 중요한 역할을 하는 p53의 활성을 조절 한다는 것을 이전의 연구를 통해 알 수 있었다.42 In order to selectively induce apoptosis of pluripotent stem cells, the characteristics of the cells must be known. The factors that can play an important role in apoptosis among the characteristics of pluripotent stem cells, which are distinguished from differentiated cells, were examined through prior literature studies and experiments. Previous studies have shown that Sirt1 is overexpressed in pluripotent stem cells and that Sirt1 regulates the activity of p53, which plays an important role in cell death. 42

Sirt1은 7가지 종류의 유사단백질로 구성된 시르투인 패밀리(sirtuin family) 중 하나이며, 그 패밀리는 Sirt1, Sirt2, Sirt3, Sirt4, Sirt5, Sirt6 및 Sirt7으로 구분된다. 세포 내 시르투인의 역할은 7가지 단백질이 모두 다르지만 공통적인 기작은 NAD+-의존성 디아세틸라제(deacetylase)로 각각의 목표 단백질의 탈아세틸화을 조절 하는 기능으로 설명할 수 있고 이를 통해 여러 단백질의 활성을 조절함으로써 그 역할을 수행하는 것으로 알려져 있다. 각각의 종류와 영향을 주는 단백질 그리고 그 효과는 다음 표와 같다(표 1, Current Drug Targets , 2010, Vol . 11, No . 10).Sirt1 is one of the sirtuin families consisting of seven kinds of pseudoproteins, which are divided into Sirt1, Sirt2, Sirt3, Sirt4, Sirt5, Sirt6 and Sirt7. The role of sirtuins in cells differs from all seven proteins, but the common mechanism is NAD + -dependent deacetylase, which can be explained as a function of controlling deacetylation of each target protein. It is known to play its role by modulating activity. The types and effects of each type of protein and their effects are shown in the following table (Table 1, Current Drug Targets , 2010, Vol . 11, No. 10 ).

시르투인Siruin 세포내 위치Intracellular location 주요 타겟Main target 기능function Sirt1Sirt1 핵nucleus p53, Ku70, MyoD, PPARγ, PGC1α, NFκB, FOXO, Zyxin, Hsf-1, STAT-3p53, Ku70, MyoD, PPARγ, PGC1α, NFκB, FOXO, Zyxin, Hsf-1, STAT-3 세포의 생존, 대사 및 스트레스 반응 조절Regulate cell survival, metabolism and stress response Sirt2Sirt2 세포질/핵Cytoplasm / nucleus α-튜불린, 히스톤 H4, FOXO3aα-tubulin, histone H4, FOXO3a 미소관 안정의 조절, 이질 염색체 형성, 세포 주기 조절, 산화적 스트레스 조절Regulation of microtubule stability, heterogeneous chromosome formation, cell cycle regulation, oxidative stress regulation Sirt3Sirt3 미토콘드리아Mitochondria AceCS2, complex of the respiratory chain, 글루탐산 탈수소 효소(GDH), 이소시트르산 탈수소 효소 2(ICDH2)AceCS2, complex of the respiratory chain, glutamic acid dehydrogenase (GDH), isocitric acid dehydrogenase 2 (ICDH2) 미토콘드리아 기능 활성화, 열발생 조절, 당분해 외의 에너지 소스 사용, 항산화제의 재생산 Activating mitochondrial function, controlling heat generation, using energy sources other than glycolysis, reproduction of antioxidants Sirt4Sirt4 미토콘드리아Mitochondria 글루탐산 탈수소 효소(GDH)Glutamic Acid Dehydrogenase (GDH) 아미노산에 의한 인슐린 분비의 하향조절Downregulation of insulin secretion by amino acids Sirt5Sirt5 미토콘드리아Mitochondria 시토크롬 c, 카바모일 포스페이트 합성 효소Ⅰ(CPS)Cytochrome c, Carbamoyl Phosphate Synthetase I (CPS) 호흡 및 세포 자멸, CR(calorie restriction) 시 대체 에너지 소스의 사용 Use of alternative energy sources for respiratory and apoptosis, calorie restriction Sirt6Sirt6 핵(이질염색질)Nucleus (heterochromatin) DNA pol β, 히스톤 H3DNA pol β, histone H3 DNA 수선 조절, S-단계 세포의 텔로머의 제어DNA repair regulation, telomer control of S-phase cells Sirt7Sirt7 핵(핵인)Nuclear RNA 폴리머라제 ⅠRNA polymerase I rRNA 합성 및 리보솜 생산의 조절Regulation of rRNA Synthesis and Ribosome Production

특히 Sirt1 및 Sirt2은 NAD+-의존성 디아세틸라제로 작용하여 p53, Ku70, FOXO 등과 같은 세포의 생존과 사멸에 작용을 하는 여러 단백질을 탈아세틸레이션 시켜 그 활성도를 조절함으로써 세포사멸을 억제하거나 세포가 계속 생존할 수 있게 하는 작용을 한다.44-48 더욱이 여러 종류의 암세포에서도 특이적으로 과발현 되며, 화학물을 이용하여 Sirt1 및 Sirt2를 억제시킴으로써 p53의 활성화로 인한 암세포의 사멸을 유도된다는 것을 논문조사를 통해 알 수 있었다.49-62 본 연구는 이와 같은 내용을 토대로 전능성줄기세포에서 Sirt1 및 Sirt2가 p53의 활성화를 조절하여 세포생존 및 사멸에 기여하는지 확인하고 그것을 억제시킴으로써 세포사멸을 유도 할 수 있는지 연구하였다(도 1). In particular, Sirt1 and Sirt2 act as NAD + -dependent deacetylases to deactivate cell death by deacetylating several proteins that act on the survival and death of cells such as p53, Ku70, FOXO, etc. It works to keep them alive. 44-48 Furthermore, it was found that the overexpression of various types of cancer cells, and that the inhibition of Sirt1 and Sirt2 using chemicals induces the death of cancer cells due to the activation of p53. 49-62 This study investigated whether Sirt1 and Sirt2 contribute to cell survival and death by regulating p53 activation in pluripotent stem cells and induce apoptosis by inhibiting it (Fig. 1). .

Sirt1 및 Sirt2를 통해 유도하는 전능성줄기세포 특이적 세포사멸 연구는 분화세포에서 세포 생존 및 분화에 영향이 없음을 확인하고, 이를 통해 이미 분화시킨 세포들 사이에서 미분화 세포를 효과적으로 제거 할 수 있는지를 연구하였다.Pluripotent stem cell-specific apoptosis studies induced by Sirt1 and Sirt2 confirm that cell viability and differentiation are unaffected in differentiated cells and whether they can effectively remove undifferentiated cells among already differentiated cells. It was.

본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

본 발명자들은 줄기세포의 분화 후에 잔재하는 미분화 전능성 줄기세포를 효과적으로 제거할 수 있는 방법을 개발하고자 노력하였다. 그 결과, 시르투인(Sirtuin)의 억제제를 처리하여 세포를 제거하는 경우에 미분화 전능성 줄기세포를 매우 효과적으로 제거할 수 있음을 확인함으로써, 본 발명을 완성하게 되었다.The present inventors have tried to develop a method capable of effectively removing undifferentiated omnipotent stem cells remaining after the differentiation of stem cells. As a result, the present invention was completed by confirming that undifferentiated omnipotent stem cells can be removed very effectively when the cells are removed by treatment with an inhibitor of sirtuin.

따라서 본 발명의 목적은 미분화 전능성 줄기세포(pluripotent stem cells)의 제거방법을 제공하는 데 있다.Accordingly, an object of the present invention is to provide a method for removing undifferentiated pluripotent stem cells.

본 발명의 다른 목적은 본 발명의 제조방법에 의해 미분화 전능성 줄기세포가 제거된 세포 조성물을 제공하는 데 있다.Another object of the present invention to provide a cell composition in which undifferentiated omnipotent stem cells are removed by the production method of the present invention.

본 발명의 또 다른 목적은 시르투인 억제제를 포함하는 미분화 전능성 줄기세포의 제거용 조성물을 제공하는 데 있다.
Another object of the present invention to provide a composition for removing undifferentiated pluripotent stem cells comprising a sirtuin inhibitor.

본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

본 발명의 일 양태에 따르면, 본 발명은 다음 단계를 포함하는 미분화 전능성 줄기세포(pluripotent stem cells)의 제거방법을 제공한다:According to one aspect of the present invention, the present invention provides a method for removing undifferentiated pluripotent stem cells, comprising the following steps:

(a) 미분화 전능성 줄기세포 및 분화 세포(differentiated cells)를 포함하는 세포 시료를 준비(preparation)하는 단계; 및(a) preparing a cell sample comprising undifferentiated pluripotent stem cells and differentiated cells; And

(b) 상기 세포 시료에 시르투인(Sirtuin) 억제제를 처리하여 미분화 전능성 줄기세포를 선택적으로 사멸시키는 단계.
(b) treating the cell sample with a sirtuin inhibitor to selectively kill undifferentiated pluripotent stem cells.

본 발명자들은 줄기세포의 분화 후에 잔재하는 미분화 전능성 줄기세포를 효과적으로 제거할 수 있는 방법을 개발하고자 노력하였다. 그 결과, 시르투인(Sirtuin)의 억제제를 처리하여 세포를 제거하는 경우에 미분화 전능성 줄기세포를 매우 효과적으로 제거할 수 있음을 확인하였다.
The present inventors have tried to develop a method capable of effectively removing undifferentiated omnipotent stem cells remaining after the differentiation of stem cells. As a result, it was confirmed that undifferentiated omnipotent stem cells can be removed very effectively when the cells are removed by treatment with an inhibitor of sirtuin.

본 발명의 미분화 전능성 줄기세포의 제거방법을 각각의 단계로 나누어 상세하게 설명하면 다음과 같다:The method for removing undifferentiated omnipotent stem cells of the present invention is described in detail by dividing each step as follows:

단계 (a): 미분화 전능성 줄기세포 및 분화 세포를 포함하는 세포 시료의 준비 Step (a): Preparation of Cell Samples Comprising Undifferentiated Omnipotent Stem Cells and Differentiated Cells

본 발명에 따르면, 미분화 전능성 줄기세포 및 분화 세포를 포함하는 세포 시료를 준비한다.According to the present invention, a cell sample comprising undifferentiated pluripotent stem cells and differentiated cells is prepared.

상기 세포 시료는 다양하게 준비될 수 있다. 예를 들어, 미분화 전능성 줄기세포(예컨대, 배아줄기세포 및 유도 전능줄기세포)를 분화시킨(예컨대, 신경 세포로 분화) 결과물이 상기 세포 시료이다. 미분화 전능성 줄기세포를 분화시킨 결과물에는 일반적으로 모든 세포들이 분화세포를 포함하지 않으며, 미분화 전능성 줄기세포가 소량 잔존 해 있다. 따라서, 분화시킨 결과물을 세포 치료제로 이용하기 위해서는 미분화 전능성 줄기세포의 완전한 제거가 필요하다.The cell sample may be prepared in various ways. For example, the resultant cell that differentiates (eg, differentiates into neural cells) undifferentiated pluripotent stem cells (eg embryonic stem cells and induced pluripotent stem cells). In the result of differentiation of undifferentiated pluripotent stem cells, generally all cells do not contain differentiated cells, and a small amount of undifferentiated pluripotent stem cells remain. Therefore, in order to use the differentiated result as a cell therapy, complete removal of undifferentiated omnipotent stem cells is required.

본 발명의 바람직한 구현예에 따르면, 상기 미분화 전능성 줄기세포는 배아줄기세포(embryonic stem cells), 배아생식세포(embryonic germ cells), 배아종양세포(embryonic carcinoma cells) 또는 유도전능줄기세포(induced pluripotent stem cells: iPSCs)이다.According to a preferred embodiment of the invention, the undifferentiated pluripotent stem cells are embryonic stem cells (embryonic stem cells), embryonic germ cells (embryonic germ cells), embryonic tumor cells (embryonic carcinoma cells) or induced pluripotent stem (induced pluripotent stem) cells: iPSCs).

바람직하게는, 미분화 전능성 줄기세포는 배아줄기세포이다. Preferably, the undifferentiated pluripotent stem cells are embryonic stem cells.

보다 바람직하게는, 본 발명에서 이용되는 미분화 전능성 줄기세포는 인간 전능성 줄기세포이다.More preferably, the undifferentiated pluripotent stem cells used in the present invention are human pluripotent stem cells.

본 명세서의 용어 ‘분화 세포’는 줄기세포가 특정 분화 자극에 의해 특정 세포로 분화가 진행된 세포이다.
As used herein, the term 'differentiated cell' refers to a cell in which stem cells are differentiated into specific cells by a specific differentiation stimulus.

단계 (b): 세포 시료에 시르투인 ( Sirtuin ) 억제제를 처리하여 미분화 전능성 줄기세포를 선택적으로 사멸 Step (b): Selective killing the undifferentiated omnipotence stem cell processes the unsealing projection of (Sirtuin) inhibitor in the sample cell

미분화 전능성 줄기세포를 선택적으로 제거하기 위해 세포 시료에 시르투인 억제제를 처리한다. Cell samples are treated with a sirtuin inhibitor to selectively remove undifferentiated omnipotent stem cells.

바람직하게는, 상기 시르투인 억제제는 Sirt1 억제제 또는 Sirt2 억제제이며, 보다 바람직하게는, Sirt1 및 Sirt2에 모두 작용하는 억제제, 또는 Sirt1 억제제와 Sirt2 억제제의 복합(combination)이다. Preferably, the sirtuin inhibitor is a Sirt1 inhibitor or Sirt2 inhibitor, more preferably an inhibitor that acts on both Sirt1 and Sirt2, or a combination of Sirt1 inhibitor and Sirt2 inhibitor.

본 발명에서 이용되는 시르투인 억제제는 시르투인 단백질 활성 억제제 또는 시르투인 유전자 발현 억제제이다.Sirtuin inhibitors used in the present invention are sirtuin protein activity inhibitors or sirtuin gene expression inhibitors.

바람직한 구현예에 따르면, 상기 시르투인 단백질 활성 억제제는 시르투인 단백질에 대한 항체, 니코티나마이드(nicotinamide), 스플리토마이신(splitomycin), 스플리토마이신 유도체(예컨대, 8-브로모-α-페닐스플리토마이신, 8-브로모-β-페닐스플리토마이신 및 디하이드로스플리토마이신), 갬비놀(gambinol), 테노빈-6(Tenovin-6), 시르티놀(Sirtinol), 세일러마이드(salermide), EX527(6-클로로-2,3,4,9-테트라하이드로-1H-카르바졸-1-카르복사마이드) 및 AGK2(2-시아노-3-[5-(2,5-디클로로페닐)-2-퍼릴]-N-5-퀴노리닐아크릴아마이드)로 구성된 군으로부터 선택되는 최소 1종의 억제제이다.According to a preferred embodiment, the inhibitor of sirtuin protein activity is an antibody against a sirtuin protein, nicotinamide, splittomycin, a splittomycin derivative (eg 8-bromo-α- Phenylsplitomycin, 8-Bromo-β-Phenylsplitomycin and Dihydrosplitomycin), Gambibinol, Tenenovin-6, Sirtinol, Sairmide ), EX527 (6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) and AGK2 (2-cyano-3- [5- (2,5-dichlorophenyl ) -2-peryl] -N-5-quinolinylacrylamide).

시르투인 단백질 활성 억제로서 본 발명에서 이용될 수 있는 항체는 시르투인 단백질에 특이적으로 결합하여 활성을 억제하는 폴리클로날 또는 모노클로날 항체이며, 바람직하게는 모노클로날 항체이다. 시르투인 단백질에 대한 항체는 당업계에서 통상적으로 실시되는 방법들, 예를 들어, 융합 방법(Kohler and Milstein, European Journal of Immunology, 6:511-519(1976)), 재조합 DNA 방법(미국 특허 제4,816,56호) 또는 파아지 항체 라이브러리 방법(Clackson et al, Nature, 352:624-628(1991) 및 Marks et al, J. Mol . Biol ., 222:58, 1-597(1991))에 의해 제조될 수 있다. 항체 제조에 대한 일반적인 과정은 Harlow, E. and Lane, D., Using Antibodies : A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies : A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; 및 Coligan , CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY, 1991에 상세하게 기재되어 있으며, 상기 문헌들은 본 명세서에 참조로서 삽입된다. 예를 들어, 단일클론 항체를 생산하는 하이브리도마 세포의 제조는 불사멸화 세포주를 항체-생산 림프구와 융합시켜 이루어지며, 이 과정에 필요한 기술은 당업자에게 잘 알려져 있으며 용이하게 실시할 수 있다. 폴리클로날 항체는 시르투인 단백질 항원을 적합한 동물에게 주사하고, 이 동물로부터 항혈청을 수집한 다음, 공지의 친화성(affinity) 기술을 이용하여 항혈청으로부터 항체를 분리하여 얻을 수 있다.Antibodies that can be used in the present invention as inhibition of sirtuin protein activity are polyclonal or monoclonal antibodies that specifically bind to and inhibit activity of a sirtuin protein, and are preferably monoclonal antibodies. Antibodies to sirtuin proteins may be prepared by methods commonly practiced in the art, for example, by fusion methods (Kohler and Milstein, European). Journal of (Clackson et al., Nature , 352: 624-628 (1991) and Marks et al., J. Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) or phage antibody library methods . Mol Biol, 222:.. 58, can be prepared by 1-597 (1991)). The general process for manufacturing antibodies is Harlow, E. and Lane, D., Using Antibodies : A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies : A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. The polyclonal antibody can be obtained by injecting a suitable animal with a sirtuin protein antigen, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

바람직하게는, 시르투인 유전자의 발현 억제제는 시르투인 유전자에 특이적으로 결합하는 안티센스 올리고뉴클레오타이드 또는 siRNA 올리고뉴클레오타이드이다.Preferably, the inhibitor of expression of a sirtuin gene is an antisense oligonucleotide or siRNA oligonucleotide that specifically binds to a sirtuin gene.

본 명세서에서 용어 "안티센스 올리고뉴클레오타이드”란 특정 mRNA의 서열에 상보적인 핵산 서열을 함유하고 있는 DNA 또는 RNA 또는 이들의 유도체를 의미하고, mRNA내의 상보적인 서열에 결합하여 mRNA의 단백질로의 번역을 저해하는 작용을 한다. 안티센스 서열은 시르투인 mRNA에 상보적이고 시르투인 mRNA에 결합할 수 있는 DNA 또는 RNA 서열을 의미하고, 시르투인 mRNA의 번역, 세포질내로의 전위(translocation), 성숙(maturation) 또는 다른 모든 전체적인 생물학적 기능에 대한 필수적인 활성을 저해할 수 있다. 안티센스 핵산의 길이는 6 내지 100 염기이고, 바람직하게는 8 내지 60 염기이고, 보다 바람직하게는 10 내지 40 염기이다.As used herein, the term "antisense oligonucleotide" refers to DNA or RNA or derivatives thereof that contain a nucleic acid sequence complementary to a sequence of a particular mRNA and binds to the complementary sequence within the mRNA to inhibit translation of the mRNA into a protein. An antisense sequence refers to a DNA or RNA sequence that is complementary to a sirtuin mRNA and capable of binding to a sirtuin mRNA, and includes translation of the sirtuin mRNA, translocation into the cytoplasm, and maturation. ) Or any other overall biological function, the length of the antisense nucleic acid is from 6 to 100 bases, preferably from 8 to 60 bases, more preferably from 10 to 40 bases.

본 발명에서 이용될 수 있는 안티센스 올리고뉴클레오타이드의 디자인은 당업계에 공지된 방법에 따라 쉽게 제작할 수 있다(Weiss, B. (ed.): Antisense Oligodeoxynucleotides and Antisense RNA : Novel Pharmacological and Therapeutic Agents, CRC Press, Boca Raton, FL, 1997; Weiss, B., et al., Antisense RNA gene therapy for studying and modulating biological processes. Cell. Mol . Life Sci ., 55:334-358(1999).The design of antisense oligonucleotides that can be used in the present invention can be readily prepared according to methods known in the art (Weiss, B. (ed.): Antisense Oligodeoxynucleotides and Antisense RNA: Novel Pharmacological and Therapeutic Agents, CRC Press, Boca Raton, FL, 1997;. ... Weiss, B., et al, Antisense RNA gene therapy for studying and modulating biological processes Cell Mol Life Sci . , 55: 334-358 (1999).

본 명세서에서 용어 "siRNA”는 RNA 방해 또는 유전자 사일런싱을 매개할 수 있는 핵산 분자를 의미한다(참조: WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409 및 WO 00/44914). siRNA는 표적 유전자의 발현을 억제할 수 있기 때문에 효율적인 유전자 넉다운 방법으로서 또는 유전자치료 방법으로 제공된다. siRNA는 식물, 벌레, 초파리 및 기생충에서 처음으로 발견되었으나, 최근에 siRNA를 개발/이용하여 포유류 세포 연구에 응용되었다.As used herein, the term “siRNA” refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99 / 07409 and WO 00/44914) siRNAs are provided as an efficient gene knockdown method or as a gene therapy method because they can inhibit the expression of target genes siRNA was first discovered in plants, worms, fruit flies and parasites. siRNA was developed and used for mammalian cell research.

본 발명에서 siRNA 분자가 이용되는 경우, 센스 가닥(시르투인 mRNA 서열에 상응하는 (corresponding) 서열)과 안티센스 가닥(시르투인 mRNA 서열에 상보적인 서열)이 서로 반대쪽에 위치하여 이중쇄를 이루는 구조를 가질 수 있으며, 또는 자기-상보성(self-complementary) 센스 및 안티센스 가닥을 가지는 단일쇄 구조를 가질 수 있다.When siRNA molecules are used in the present invention, the sense strand (corresponding sequence corresponding to a sirtuin mRNA sequence) and the antisense strand (sequence complementary to a sirtuin mRNA sequence) are positioned opposite to each other to form a double strand. It may have a structure, or it may have a single chain structure with self-complementary sense and antisense strands.

바람직하게는, 상기 시르투인 활성 억제제 또는 시르투인 발현 억제제는 Sirt1 또는 Sirt2의 발현 또는 활성을 억제하여 p53, Ku70, MyoD(Myogenic regulatory factor), PPAR(Peroxisome proliferator-activated Receptor)γ, PGC1α(Peroxisome proliferator-activated receptor gamma coactivator 1-α), NFκB(Nuclear factor κ-light-chain-enhancer of activated B cells), FOXO1(Forkhead box protein 01), Zyxin, Hsf(Heat shock factor protein), STAT(Signal transducer and activator of transcription)-3, α-튜불린, 히스톤 H4, FOXO3a 또는 HNF(Hepatocyte nuclear factor)4α를 활성화시킨다. Preferably, the sirtuin activity inhibitor or sirtuin expression inhibitor inhibits the expression or activity of Sirt1 or Sirt2, thereby inhibiting p53, Ku70, MyoD (Myogenic regulatory factor), PPAR (Peroxisome proliferator-activated Receptor) γ, PGC1α ( Peroxisome proliferator-activated receptor gamma coactivator 1-α, Nuclear factor κ-light-chain-enhancer of activated B cells, Forkhead box protein 01 (FOXO1), Zyxin, Heat shock factor protein (STAT), and Signal (STAT) Activates transducer and activator of transcription (3) -3, α-tubulin, histone H4, FOXO3a or Hepatocyte nuclear factor (HNF) 4α.

본 발명의 바람직한 구현예에 따르면, 상기 시르투인 활성 억제제는 p53을 활성화 시켜 세포 사멸하게 된다.
According to a preferred embodiment of the present invention, the sirtuin activity inhibitor activates p53 to kill cells.

본 발명의 다른 양태에 따르면, 본 발명은 상기 본 발명의 방법에 의해 미분화 전능성 줄기세포가 제거된 세포 조성물을 제공한다.
According to another aspect of the present invention, the present invention provides a cell composition in which undifferentiated pluripotent stem cells are removed by the method of the present invention.

본 발명의 또 다른 양태에 따르면, 시르투인(Sirtuin) 억제제를 포함하는 미분화 전능성 줄기세포의 제거용 조성물이다.According to another aspect of the present invention, a composition for removing undifferentiated omnipotent stem cells comprising a sirtuin inhibitor.

본 발명의 조성물은 상기 시르투인 억제제를 이용하기 때문에, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.
Since the composition of the present invention utilizes the sirtuin inhibitor, the common content between the two is omitted in order to avoid excessive complexity of the present specification.

본 발명의 특징 및 이점을 요약하면 다음과 같다: The features and advantages of the present invention are summarized as follows:

(a) 전능성 줄기세포의 분화과정 중 미분화로 남아있는 줄기세포를 효율적으로 제거하여 세포 분화 초기 미분화 세포에 의한 분화효율 저해 및 세포 간 분화 정도 차이를 감소시킬 수 있다.(a) By effectively removing stem cells remaining undifferentiated during the differentiation process of pluripotent stem cells, it is possible to reduce the efficiency of differentiation by the undifferentiated cells and to differentiate the degree of differentiation between cells.

(b) 분화과정 중 이식 후 종양생성의 가능성을 지닌 미분화 세포를 선택적으로 제거하여 전능성줄기세포의 임상적용에 있어 안전성 확보가 기대된다.
(b) It is expected to secure safety in clinical application of pluripotent stem cells by selectively removing undifferentiated cells with the possibility of tumor formation after transplantation during differentiation.

도 1은 Sirt1 및 Sirt2 억제제를 통한 전능성줄기세포 특이적 세포사멸을 보여준다. Sirt1 및 Sirt2 억제제를 처리하면 p53이 활성화되어 세포사멸이 유도된다는 것을 설명하는 모식도이다.
도 2는 Sirt1 및 Sirt2 억제제를 처리하여 배아줄기세포의 세포사멸을 확인한 결과이다. 활성화된 카스파아제 3(caspase 3)를 면역형광염색법으로 확인한 결과를 보여준다.
도 3은 Sirt1 및 Sirt2 억제제인 테노빈-6을 처리하여 세포사멸을 확인한 결과를 보여준다. Sirt1 및 Sirt2 억제제를 처리한 세포시료에서 세포사멸 인자인 아세틸레이션된(acetylated) p53 및 활성-캐스패이즈 3를 확인한 웨스턴블럿 결과이다.
도 4는 배아체로의 분화유도 후 Sirt1 및 Sirt2 억제제를 처리하여 배아줄기세포만의 세포사멸을 유도한 현미경사진을 보여준다
도 5는 배아체의 분화유도 후 Sirt1 및 Sirt2 억제제를 처리하여 미분화 세포의 감소를 Oct3/4 발현으로 확인한 실시간 PCR 결과이다. 1 shows pluripotent stem cell specific apoptosis through Sirt1 and Sirt2 inhibitors. Schematic diagram illustrating that treatment of Sirt1 and Sirt2 inhibitors activates p53 to induce apoptosis.
Figure 2 shows the results of apoptosis of embryonic stem cells by treatment with Sirt1 and Sirt2 inhibitors. Activated caspase 3 is shown by immunofluorescence staining.
Figure 3 shows the results of confirming apoptosis by treatment with Sirt1 and Sirt2 inhibitor tennobin-6. Western blot results confirming apoptosis factors acetylated p53 and activating caspase 3 in cells treated with Sirt1 and Sirt2 inhibitors.
Figure 4 shows the micrographs inducing apoptosis of only embryonic stem cells by treatment with Sirt1 and Sirt2 inhibitors after induction of differentiation into embryos
5 is a real-time PCR result confirming Oct3 / 4 expression of reduction of undifferentiated cells by treatment with Sirt1 and Sirt2 inhibitors after induction of differentiation of embryos.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

실시예Example

실시예 1: Sirt1, Sirt2 억제제의 세포사멸 유도Example 1 Induction of Apoptosis of Sirt1 and Sirt2 Inhibitors

생쥐에서 mSir2로 처음 알려져 있는 시르투인 유전자는 주로 영양제한 및 활성산소가 과다한 상태에서 과발현 되는 NAD+-의존성 디아세틸라제이며, 세포사멸을 억제하는 유전자로 많이 알려져 있다. 특히 Sirt1은 여러 종류의 암세포에서 과발현 되어 있고 주역할로는 활성산소 등에 의한 스트레스 상황에서 세포의 수명을 연장하거나 p53을 탈아세틸화함으로써 세포사멸을 억제하는 것으로 알려져 있다.44-48 최근에는 Sirt1은 배아줄기세포에서 스트레스 상황이 아닌 보통의 배양환경에서도 분화된 다른 세포들과 비교하여 많이 발현이 되어있음이 확인되었다. 하지만 전능성줄기세포의 세포사멸에 있어서 구체적인 기작연구는 아직 이루어 지지 않고 있다.The sirtuin gene, first known as mSir2 in mice, is a NAD + -dependent deacetylase that is overexpressed mainly in the state of nutrient restriction and free radicals, and is known as a gene that inhibits apoptosis. In particular, Sirt1 is overexpressed in various types of cancer cells, and its main role is known to inhibit cell death by prolonging cell life or deacetylating p53 under stress caused by active oxygen. 44-48 Sirt1 was recently confirmed that this is a lot of expression compared with the other cell differentiation in normal culture environment rather than a stressful situation in embryonic stem cells. However, specific mechanisms for the cell death of pluripotent stem cells have not been studied yet.

전능성줄기세포에서 Sirt1의 과발현이 암세포에서와 유사한 역할을 할 것이라는 가설을 가지고 사전실험을 통해 Sirt1 및 Sirt2 억제제가 세포사멸을 유도 하는지 확인하였다. 사전실험 결과 Sirt1 및 Sirt2 억제제가 전능성줄기세포 특이적인 세포사멸을 보이는 것을 확인할 수 있었으며, 이를 바탕으로 시르투인이 전능성줄기세포 내에서 어떠한 기전을 통해서 세포 사멸이 이루어지는지 알아보고자 하였다. 또한 분화과정을 통해서 분화된 세포군내에서 미분화 세포가 Sirt1 억제제 및 Sirt2 억제제를 처리 하였을 때 특이적으로 세포사멸이 유도되는지 확인하였다. With the hypothesis that overexpression of Sirt1 in pluripotent stem cells would play a role similar to that in cancer cells, previous experiments confirmed whether Sirt1 and Sirt2 inhibitors induce apoptosis. As a result of the preliminary experiments, it was confirmed that Sirt1 and Sirt2 inhibitors showed pluripotent stem cell-specific apoptosis. Based on this, we tried to investigate the mechanism by which sirtuin kills cells in pluripotent stem cells. In addition, it was confirmed that apoptosis was induced specifically when undifferentiated cells were treated with Sirt1 and Sirt2 inhibitors in the differentiated cell population.

시르투인의 세포 내 기작을 알아보기 위해서 시르투인의 활성도의 조절이 필요한 화학물를 통해 Sirt1 및 Sirt2를 억제하여 음성대조군 및 억제제 처리군에서의 비교실험을 하였다. 이때 Sirt1 및 Sirt2 모두 억제하는 화학물질로 테노빈-6(Tenovin-6, Santa Cruz, 미국), 시르티놀(Sirtinol, Santa Cruz, 미국) 및 세일러마이드(Salermide, Santa Cruz, 미국)를 사용하였다.In order to investigate the intracellular mechanism of sirtuin, sirt1 and Sirt2 were inhibited by chemicals that required the regulation of sirtuin activity and compared in the negative control group and inhibitor treatment group. Tennovin-6 (Tenovin-6, Santa Cruz, USA), sirtinol (Sirtinol, Santa Cruz, USA) and sailamide (Salermide, Santa Cruz, USA) were used as chemicals that inhibit both Sirt1 and Sirt2.

본 실험에서 미분화된 전능성 줄기세포로 H9(위스콘신 세포주) 배아줄기세포와 Y2-3(하버드대학 세포주) 및 J10(질병 세포주/본 실험실 제작)의 두 종류의 유도만능 줄기세포를 사용하였다. 위의 세포주들은 DMEM F12(Gibco, 미국) 기본 배양액 및 KSR(Knockout Serum Replacement, Gibco, 미국)을 8:2로 혼합하여 배양액을 만들었다. 세포배양시 bFGF(basic fibroblast growth factor)를 4 ng/㎖ 첨가하여, 젤라틴(Gelatin) 코팅된 세포배양 접시에서 STO(fibroblast, ATCC)라는 지지세포위에서 공배양 하거나, mTeSR(STEMCELL Technologies, 미국)배양액을 이용하여 매트리겔(Matrigel)로 코팅 된 세포배양 접시에서 단독 배양하였다. 상기 언급한 전능성 줄기세포 세포주들은 20-50 계대의 세포주를 사용하였고, 시르투인 억제제의 효능을 테스트 하기 위해 계대 후 72시간 후 억제제를 처리하였다. 각각의 억제제는 이전 논문보고에 따라 농도를 조절하여 농도별로 테스트 하였으며, 각각의 억제제의 농도는 시르티놀(Sirtinol, 10-100 uM), 세일러마이드(Salermide, 10-100 uM), 테노빈-6(Tenovin-6, 0.1-10 uM) 이다.In this experiment, two types of induced pluripotent stem cells were H9 (Wisconsin cell line) embryonic stem cells, Y2-3 (Harvard cell line), and J10 (disease cell line / manufactured). The above cell lines were made by mixing DMEM F12 (Gibco, USA) base culture and KSR (Knockout Serum Replacement, Gibco, USA) at 8: 2. 4 ng / ml of basic fibroblast growth factor (bFGF) was added during cell culture and co-cultured on support cells called STO (fibroblast, ATCC) in gelatin-coated cell culture dishes, or mTeSR (STEMCELL Technologies, USA) culture solution. It was cultured alone in a cell culture dish coated with Matrigel (Matrigel). The omnipotent stem cell lines mentioned above used cell lines of 20-50 passages and were treated with inhibitors 72 hours after passage to test the efficacy of sirtuin inhibitors. Each inhibitor was tested for each concentration by adjusting the concentration according to previous paper reports. The concentrations of each inhibitor were sirtinol (10-100 uM), sailamide (10-100 uM), and tenobin-6. (Tenovin-6, 0.1-10 uM).

각각의 억제제의 처리 후 효과를 비교해본 결과 시르티놀 및 세일러마이드는 20 uM부터 효과를 보이기 시작하며, 50 uM의 농도에서 12시간에서 24시간 사이에 대부분의 전능성 줄기세포가 세포사멸과정을 거쳐 세포가 죽는 것을 확인하였다. 또한 테노빈-6의 경우 저농도에서도 상기 두 억제제와 같은 효과를 보았으며, 효과를 보이는 농도는 1 uM부터이고, 5 uM에서 최적의 효과를 보이며, 그 이상의 농도에서 5 uM과 같은 효과를 보이거나 억제제의 독성에 의한 사멸을 보이는 것을 확인하였다.
Comparing the effects of each inhibitor after treatment, sirtinol and sailamide began to be effective at 20 uM, with most omnipotent stem cells undergoing apoptosis between 12 and 24 hours at a concentration of 50 uM. Confirmed dying. In addition, tenobin-6 showed the same effect as the two inhibitors at low concentrations, and the effective concentration was from 1 uM, and showed the optimal effect at 5 uM, and at 5 or more concentrations. It was confirmed that death by the toxicity of the inhibitor.

세포 사멸 확인Apoptosis confirmation

시르투인을 효과적으로 억제시킴으로 예상되는 결과는 세포 사멸이며 이것을 효과적으로 보여주기 위해 세포 사멸 상황에서 발현되는 활성화된 형태의 캐스페이즈 3(cleaved Caspase 3, Cell signaling, 미국) 및 전능성 줄기세포 마커인 SSEA4(stage-specific embryonic antigen-4, Millipore, 미국)의 면역형광 염색법을 통해 세포사멸이 일어나는 세포가 전능성줄기세포임을 확인하였다(도 2). The results expected to effectively inhibit sirtuins are cell death, and activated forms of caspase 3 (Cell signaling, USA) and SSEA4, an omnipotent stem cell marker, expressed in apoptosis situations to demonstrate this effectively. Stage-specific embryonic antigen-4, Millipore, USA) immunofluorescence staining was confirmed that the cells in which apoptosis occurs pluripotent stem cells (Fig. 2).

전능성 줄기세포에서 세포사멸과정 중 세포가 완전히 죽기 전 상태의 단백질 발현을 확인 하기위해 세포에 억제제와 대조군인 DMSO(Dimethyl sulfoxide)를 처리 후, 6시간을 배양하고 고정액(파라포름알데하이드)를 이용하여 세포를 고정한 후 면역현광염색법을 실시하였다. 실시방법을 열거하면 다음과 같다: In order to confirm the expression of the protein before the cell dies completely in the process of apoptosis in pluripotent stem cells, the cells were treated with DMSO (dimethyl sulfoxide) as an inhibitor and a control group, and then cultured for 6 hours using a fixed solution (paraformaldehyde). After fixing the cells, immunofluorescence staining was performed. The implementation method is as follows:

세포에 고정액을 처리하고 PBS(phospate buffered saline) 3회 세포세척한 후에, 5% NDS(Normal donkey serum)을 이용하여 블로킹하였다. 2시간(상온) 또는 12시간(4℃)동안 SSEA4 및 활성-캐스페이즈3 항체를 5% NDS에1:100으로 희석하여 동시 처리한다. 형광 발현을 보기위해 각각 항체에 대한 이차 형광항체를 30분동안 처리 후 슬라이드에 마운팅하여 현관현미경을 통해 관찰하였다. The cells were treated with fixative solution and washed three times with PBS (phospate buffered saline), and then blocked using 5% normal donkey serum (NDS). SSEA4 and active-Casephase3 antibodies are diluted 1: 100 in 5% NDS for 2 hours (room temperature) or 12 hours (4 ° C) and co-treated. In order to see the fluorescence expression, the secondary fluorescent antibody for each antibody was treated for 30 minutes and then mounted on a slide and observed through a vestibule microscope.

도 2와 같이 전능성 줄기세포에 DMSO를 처리 한 군에서는 활성-캐스페이즈3가 전능성 줄기세포 마커인 SSEA4와 함께 발현되는 부분이 거의 보이지 않는 반면 실험군인 테노빈-6를 처리한 세포에서는 대부분 SSEA4가 발현되는 세포에서 활성-캐스페이즈3가 함께 발현되는 것을 관찰 할 수 있었다. SSEA4가 염색되는 않는 부분은 대조군인 DMSO 처리군과 테노빈-6 처리한 실험군에서 차이를 보이지 못하였다. 이는 Salermide 및 시르티놀 억제제에서도 같은 효과를 보여 Sirt1 및 Sirt2 억제제의 공통적인 효과를 확인하였다.
As shown in FIG. 2, in the group treated with DMSO to pluripotent stem cells, the portion of active-caspase 3 expressed with SSEA4, a pluripotent stem cell marker, was hardly seen, whereas in the group treated with tennobin-6, SSEA4 was mostly In the cells to be expressed, the activity-Casephase3 was co-expressed. SSEA4 stained portion did not show a difference between the control group DMSO treatment group and tenobin-6 treated group. The same effect was observed for Salermide and sirtinol inhibitors, confirming the common effects of Sirt1 and Sirt2 inhibitors.

Sirt1Sirt1 및  And Sirt2Sirt2 의 영향 분석Impact analysis

세포사멸 및 세포주기 변화에서 나타난 실험결과를 분석하기 위해 종래 보고된 Sirt1 및 Sirt2가 영향을 주는 단백질을 분석하였다. Sirt1이 세포사멸과 관련되어 영향을 주는 p53은 사포사멸에 관여하는 중요한 단백질로 알려져 있다. p53은 Sirt1 및 Sirt2에 의해 탈아세틸레이션 되며 Sirt1 및 Sirt2를 억제함으로써 p53의 아세틸레이션과 동시에 활성이 높아짐을 웨스턴 블럿으로 확인하였고, p53의 영향을 받아 세포사멸과정을 일으키는 활성-카스파아제 3(Cleaved-caspase 3)를 정량하여 세포사멸에 영향을 주는 것을 확인하였다. 이를 위해 Sirt1 및 Sirt2 에 의한 탈아세틸레이션 정도에 따른 활성 정도를 항-아세틸레이티드(acetylated) p53 항체(Cell signaling, 미국) 및 항-디아세틸레이티드(deacetylated) p53 항체(Cell signaling, 미국)로 웨스턴 블럿을 수행하여 그 정도를 측정하여 각각의 연관성과 세포사멸 및 생존에 있어서 어떠한 기전을 가지고 있는지 확인하였다(도 3).In order to analyze the experimental results in apoptosis and cell cycle changes, the proteins reported by Sirt1 and Sirt2 were analyzed. P53, which affects apoptosis by Sirt1, is known to be an important protein involved in apoptosis. p53 was deacetylated by Sirt1 and Sirt2, and Western blot confirmed that the activity was increased simultaneously with the acetylation of p53 by inhibiting Sirt1 and Sirt2, and activated-caspase 3 (Cleaved) caused the apoptosis process under the influence of p53. -caspase 3) was quantified to confirm that it affects apoptosis. To this end, the degree of activity according to the degree of deacetylation by Sirt1 and Sirt2 was determined by anti-acetylated p53 antibody (Cell signaling, USA) and anti-deacetylated p53 antibody (Cell signaling, USA). The western blot was performed to determine the extent of each mechanism, and the mechanism of each association and apoptosis and survival (Fig. 3).

웨스턴블럿을 위해 억제제와 DMSO를 6시간동안 처리한 세포를 트립신/EDTA(T/E)를 이용하여 단일세포로 떼어내어 세포를 모아 용해 버퍼(Lysis buffer)을 이용하여 세포를 13000 rpm으로 15분간 원심분리하여 상층액에 있는 단백질을 수득하였다. SDS-PAGE 전기연동 후, 단백질을 막(membrand)에 전송하여 단백질을 분리하였다. 분리된 단백질은 항체염색을 위해 5% 소 혈청 알부민(Bovin serum albumin:BSA)으로 1시간 동안 블로킹을 실시하였으며 이후 Sirt1, p53, Acetylated p53 및 활성-캐스페이즈3에 대한 항체를 4℃에서 12시간동안 각각 처리하였다. TBST을 이용하여 세척 후 각각의 항체에 대한 HRP(horse radish peroxidase) 반응을 위한 처리 후 나타나는 각각 단백질에 대한 발광밴드를 x-ray필름을 통해 찍어 낸 후 나타난 밴드를 분석하였다. 도 3과 같이 대조군인 DMSO 처리군과 실험군인 테노빈-6 처리군에서는 전체 p53에 대한 양의 차이는 없었으나, Sirt1 및 Sirt2 억제제에 의해 Acetyl-p53의 양이 현저하게 증가하여 있는 것을 확인 하였고 그 결과 p53이 안정되어, 원래 크기의 p53이 더 많이 유지되고 있음을 확인하였다. 53 kD보다 위의 밴드는 전능성 줄기세포에서 p53은 유비퀴티네이션에 의해 계속적인 분해가 일어나기 때문에 분해 과정 중의 결과물이다. 또한 이렇게 활성화된 p53(Acetylated)에 의해 발현된 활성-캐스페이즈3를 확인할 수 있었으며 이것의 발현이 대조군에 비하여 현저하게 증가로 세포사멸을 확인할 수 있었다. 이로써 Sirt1 및 Sirt2 억제제를 통하여 p53의 활성을 통해 캐스페이즈3를 활성화 시켜 전능성 줄기세포 특이적인 세포사멸을 유도한다는 결론을 얻어낼 수 있었다.
For Western blotting, cells treated with inhibitor and DMSO for 6 hours were separated into single cells using trypsin / EDTA (T / E), and the cells were collected. The cells were lysed using Lysis buffer for 15 minutes at 13000 rpm. Centrifugation gave the protein in the supernatant. After SDS-PAGE electrophoresis, proteins were transferred to membranes to separate proteins. The isolated protein was blocked with 5% bovin serum albumin (BSA) for 1 hour for antibody staining, and then the antibody against Sirt1, p53, Acetylated p53 and active-Casephase 3 was 12 hours at 4 ° C. Each treatment. After washing with TBST, the luminescence band for each protein after treatment for HRP (horse radish peroxidase) reaction for each antibody was photographed through an x-ray film, and then the band appeared. As shown in FIG. 3, there was no difference in the amount of total p53 in the DMSO-treated group and the tenobin-6-treated group, but it was confirmed that the amount of Acetyl-p53 was significantly increased by Sirt1 and Sirt2 inhibitors. As a result, it was confirmed that p53 was stabilized and more p53 of the original size was maintained. The band above 53 kD is the result of the degradation process in pluripotent stem cells because p53 is continuously degraded by ubiquitination. In addition, it was possible to confirm the activity-Casephase 3 expressed by the p53 (Acetylated) thus activated, and its expression was markedly increased compared to the control group and confirmed apoptosis. Thus, it was concluded that p53 activation via Sirt1 and Sirt2 inhibitors induces caspase 3 to induce pluripotent stem cell-specific apoptosis.

미분화 세포의 제거Removal of undifferentiated cells

Sirt1 및 Sirt2 억제제의 전능성줄기세포 특이적 세포사멸 작용을 응용하여, 줄기세포 분화과정에서 미분화 세포를 제거하는데 이용하였다. 이를 확인하기 위해 전능성줄기세포로부터 분화를 유도하여 배아체를 형성하게 하고 아무런 처리를 하지 않은 상태의 미분화 세포가 남아있는 비율을 FACS분석(Facscaliber, Becton Dickinson, 미국) 및 실시간 유전자 발현분석(Realtime RT-PCR(Bio-Rad, 미국))을 통해 확인하였다. 배아체의 형성은 배양중의 전능성줄기세포를 콜라게나제(collagenase, Warthington biochemical Corporation, 미국)를 이용하여 배양접시에서 떨어뜨린 후, 7일 동안 구형의 배아체를 유지하였다. 이때 배양액은 전능성줄기세포 배양액에서 줄기세포 유지인자인 bFGF를 제거한 배양액(DMEM F12 80%, KSR 20%)을 사용하게 된다. 미분화 인자 유전자 분석을 위해 Oct3/4의 프라이머(정방향 서열; 5'- TGG GCT CGA GAA GGA TGT G -3', 역방향 서열; 5'- GCA TAG TCG CTG CTT GAT CG -3')를 이용하여 실시간 PCR을 통해 발현을 확인하였다. 상기 방법으로 배아체를 4-7일 동안 Sirt1 및 Sirt2 억제제를 처리하여 미분화세포가 얼마나 많이 제거되었는지 확인하였다(도 5). 실험결과 Oct4의 발현양이 처리군에서 대조군에 비해 약 70%정도의 감소를 확인 할 수 있었으며, 테노빈-6의 농도 1 uM 및 5 uM에서 발현이 줄어드는 것을 보아 농도의존적 효과를 확인하였다. 또한 남아있는 20-30%의 Oct4는 분화된 세포에서 남아있는 미량의 발현이라 판단되며, 이를 면역형광염색법을 통해 확인하였다(도 2). 결과적으로, Sirt1 및 Sirt2 억제제가 미분화세포 제거에 효과가 있음을 확인하였으며, 미분화 세포로 인한 종양생성억제 등의 안전성을 확보할 수 있음을 보여준다.Pluripotent stem cell specific apoptosis of Sirt1 and Sirt2 inhibitors was applied to remove undifferentiated cells during stem cell differentiation. To confirm this, induced differentiation from pluripotent stem cells to form embryoid bodies, and the percentage of undifferentiated cells remaining untreated was analyzed by FACS analysis (Facscaliber, Becton Dickinson, USA) and real-time gene expression analysis (Realtime RT). -PCR (Bio-Rad, USA). Embryonic formation was carried out by dropping the pluripotent stem cells in culture using a collagenase (collagenase, Warthington biochemical Corporation, USA) in a culture dish, and then maintained a globular embryonic body for 7 days. At this time, the culture medium is to use a culture medium (b. DMEM F12 80%, KSR 20%) from the pluripotent stem cell culture medium removed bFGF. Real time using primers of Oct3 / 4 (forward sequence; 5'- TGG GCT CGA GAA GGA TGT G -3 ', reverse sequence; 5'- GCA TAG TCG CTG CTT GAT CG -3') for undifferentiated factor gene analysis Expression was confirmed by PCR. Embryos were treated with Sirt1 and Sirt2 inhibitors for 4-7 days in this manner to determine how much undifferentiated cells were removed (FIG. 5). As a result, it was confirmed that the amount of Oct4 expression in the treated group was reduced by about 70% compared to the control group, and the concentration-dependent effect was confirmed by decreasing the expression at the concentrations of 1 uM and 5 uM of tenobin-6. In addition, the remaining 20-30% of Oct4 was determined to be a trace expression remaining in the differentiated cells, it was confirmed by immunofluorescence staining (Fig. 2). As a result, it was confirmed that Sirt1 and Sirt2 inhibitors are effective in removing undifferentiated cells, and it can be seen that it is possible to secure safety such as tumor suppression caused by undifferentiated cells.

이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

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Claims (14)

다음 단계를 포함하는 미분화 전능성 줄기세포의 제거방법:
(a) 미분화 전능성 줄기세포 및 분화 세포(differentiated cells)를 포함하는 세포 시료를 준비(preparation)하는 단계; 및
(b) 상기 세포 시료에 시르투인(Sirtuin) 억제제를 처리하여 미분화 전능성 줄기세포를 선택적으로 사멸시키는 단계.
A method for removing undifferentiated omnipotent stem cells comprising the following steps:
(a) preparing a cell sample comprising undifferentiated pluripotent stem cells and differentiated cells; And
(b) treating the cell sample with a sirtuin inhibitor to selectively kill undifferentiated pluripotent stem cells.
제 1 항에 있어서, 상기 미분화 전능성 줄기세포는 배아줄기세포(embryonic stem cells), 배아생식세포(embryonic germ cells), 배아종양세포(embryonic carcinoma cells) 또는 iPSCs(induced pluripotent stem cells)인 것을 특징으로 하는 방법.
The method of claim 1, wherein the undifferentiated pluripotent stem cells are embryonic stem cells (embryonic stem cells), embryonic germ cells (embryonic germ cells), embryonic tumor cells (embryonic carcinoma cells) or iPSCs (induced pluripotent stem cells) characterized in that How to.
제 2 항에 있어서, 상기 미분화 전능성 줄기세포는 배아줄기세포(embryonic stem cells)인 것을 특징으로 하는 방법.
3. The method of claim 2, wherein said undifferentiated pluripotent stem cells are embryonic stem cells.
제 1 항에 있어서, 상기 미분화 전능성 줄기세포는 인간으로부터 유래된 것을 특징으로 하는 방법.
2. The method of claim 1, wherein said undifferentiated pluripotent stem cells are derived from humans.
제 1 항에 있어서, 상기 시르투인 억제제는 Sirt1 및 Sirt2에 모두 작용하는 억제제, 또는 Sirt1 억제제와 Sirt2 억제제의 복합(combination)인 것을 특징으로 하는 방법.
The method of claim 1, wherein the sirtuin inhibitor is an inhibitor that acts on both Sirt1 and Sirt2, or a combination of Sirt1 inhibitor and Sirt2 inhibitor.
제 1 항에 있어서, 상기 시르투인 억제제는 시르투인 단백질 활성 억제제 또는 시르투인 유전자 발현 억제제인 것을 특징으로 하는 방법.
The method of claim 1, wherein the sirtuin inhibitor is a sirtuin protein activity inhibitor or a sirtuin gene expression inhibitor.
제 6 항에 있어서, 상기 시르투인 억제제는 시르투인 단백질에 대한 항체, 니코티나마이드(nicotinamide), 스플리토마이신(splitomycin), 스플리토마이신 유도체, 갬비놀(gambinol), 테노빈-6(Tenovin-6), 시르티놀(Sirtinol), 세일러마이드(salermide), EX527(6-클로로-2,3,4,9-테트라하이드로-1H-카르바졸-1-카르복사마이드) 및 AGK2(2-시아노-3-[5-(2,5-디클로로페닐)-2-퍼릴]-N-5-퀴노리닐아크릴아마이드)로 구성된 군으로부터 선택되는 최소 1종의 억제제인 것을 특징으로 하는 방법.
The method of claim 6, wherein the sirtuin inhibitor is an antibody against a sirtuin protein, nicotinamide, splitomycin, splitomycin derivatives, gambinol, tenobin-6 ( Tenovin-6), Sirtinol, salermide, EX527 (6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) and AGK2 (2- At least one inhibitor selected from the group consisting of cyano-3- [5- (2,5-dichlorophenyl) -2-furyl] -N-5-quinolinylacrylamide).
제 7 항에 있어서, 상기 억제제는 Sirt1 또는 Sirt2를 억제하여 p53, Ku70, FoxO1, NF-κB, PPARγ 또는 HNF4α를 활성화시키는 것을 특징으로 하는 방법.
8. The method of claim 7, wherein the inhibitor activates p53, Ku70, FoxOl, NF-κB, PPARγ or HNF4α by inhibiting Sirt1 or Sirt2.
삭제delete 시르투인(Sirtuin) 억제제를 포함하는 미분화 전능성 줄기세포의 선택적 제거용 조성물.
Composition for selective removal of undifferentiated omnipotent omnipotent stem cells comprising a sirtuin inhibitor.
제 10 항에 있어서, 상기 시르투인 억제제는 Sirt1 및 Sirt2에 모두 작용하는 억제제, 또는 Sirt1 억제제와 Sirt2 억제제의 복합(combination)인 것을 특징으로 하는 조성물.
The composition of claim 10, wherein the sirtuin inhibitor is an inhibitor that acts on both Sirt1 and Sirt2, or a combination of Sirt1 inhibitor and Sirt2 inhibitor.
제 10 항에 있어서, 상기 시르투인 억제제는 시르투인 단백질 활성 억제제 또는 시르투인 유전자 발현 억제제인 것을 특징으로 하는 조성물.
The composition of claim 10, wherein the sirtuin inhibitor is a sirtuin protein activity inhibitor or a sirtuin gene expression inhibitor.
제 12 항에 있어서, 상기 시르투인 억제제는 시르투인 단백질에 대한 항체, 니코티나마이드(nicotinamide), 스플리토마이신(splitomycin), 스플리토마이신 유도체, 갬비놀(gambinol), 테노빈-6(Tenovin-6), 시르티놀(Sirtinol), 세일러마이드(salermide), EX527(6-클로로-2,3,4,9-테트라하이드로-1H-카르바졸-1-카르복사마이드) 및 AGK2(2-시아노-3-[5-(2,5-디클로로페닐)-2-퍼릴]-N-5-퀴노리닐아크릴아마이드)로 구성된 군으로부터 선택되는 최소 1종의 억제제인 것을 특징으로 하는 조성물.
The method according to claim 12, wherein the sirtuin inhibitor is an antibody against a sirtuin protein, nicotinamide, splictomycin, a splitomycin derivative, a gambinol, tenobin-6 ( Tenovin-6), Sirtinol, salermide, EX527 (6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) and AGK2 (2- At least one inhibitor selected from the group consisting of cyano-3- [5- (2,5-dichlorophenyl) -2-furyl] -N-5-quinolinylacrylamide).
제 11 항에 있어서, 상기 억제제는 Sirt1 및 Sirt2를 억제하여 p53, Ku70, Myo(Myogenic regulatory factor)D, PPAR(Peroxisome proliferator-activated Receptor)γ, PGC1α(Peroxisome proliferator-activated receptor gamma coactivator 1-α), NFκB(Nuclear factor κ-light-chain-enhancer of activated B cells), FOX(Forkhead box protein)O1, Zyxin, Hsf(Heat shock factor protein)1, STAT(Signal transducer and activator of transcription)-3, α-튜불린, 히스톤 H4, FOXO3a 또는 HNF(Hepatocyte nuclear factor)4α를 활성화시키는 것을 특징으로 하는 조성물.
The method of claim 11, wherein the inhibitor inhibits Sirt1 and Sirt2, p53, Ku70, Myogenic regulatory factor (Myo) D, Peroxisome proliferator-activated Receptor (PPAR) γ, PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-α) , NFκB (Nuclear factor κ-light-chain-enhancer of activated B cells), FOX (Forkhead box protein) O1, Zyxin, Hsf (Heat shock factor protein) 1, Signal transducer and activator of transcription (STAT) -3, α A composition characterized by activating tubulin, histone H4, FOXO3a or HNF (Hepatocyte nuclear factor) 4α.
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