KR102181316B1 - Composition for inducing selective apoptosis undifferentiated human induced plurioptent stem cells comprising bee venom and the method for inducing selective apoptosis using the same - Google Patents
Composition for inducing selective apoptosis undifferentiated human induced plurioptent stem cells comprising bee venom and the method for inducing selective apoptosis using the same Download PDFInfo
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Abstract
Description
본 발명은 봉독을 유효성분으로 포함하는 미분화 유도만능 줄기세포의 선택적 세포사멸 유도용 조성물에 관한 것으로서, 구체적으로는 봉독을 유효성분으로 포함하는 미분화 유도만능 줄기세포의 선택적 세포사멸 유도용 조성물, 선택적 제거용 조성물, 미분화 유도만능 줄기세포의 제거방법, 및 상기 방법으로 미분화 유도만능 줄기세포가 제거된 세포 조성물에 관한 것이다.The present invention relates to a composition for inducing selective apoptosis of undifferentiated induced pluripotent stem cells containing bee venom as an active ingredient, and specifically, a composition for inducing selective apoptosis of undifferentiated induced pluripotent stem cells containing bee venom as an active ingredient, selective It relates to a composition for removal, a method of removing undifferentiated induced pluripotent stem cells, and a cell composition from which undifferentiated induced pluripotent stem cells are removed by the method.
유도만능 줄기세포는 만능분화능(pluripotency)을 가지고 있지 않던 분화된 세포들이 인위적인 역분화 과정을 통해 만능분화능을 가지도록 유도된 세포들을 일컫는 말로서, ① 세포의 모양(둥근 모양, 큰 핵과 인, 적은 세포질)과 자라는 속도(배아줄기세포의 분열시간: 17 hr)가 배아줄기세포와 유사하며, ② 유전자 발현(gene expression)과 염색체 변형(chromatin modification) 패턴이 배아줄기세포와 유사하며, ③ 만능분화능을 가지고, ④ 면역 결핍 생쥐에서 테라토마를 형성 할 수 있으며, ⑤ 생쥐의 배반포(blastocyst)에 삽입시켰을 때 키메라(chimera) 생쥐를 형성하며, ⑥ 유전자의 생식선 전이(germ line transmission)가 가능한 특성을 가진다.Induced pluripotent stem cells refer to cells in which differentiated cells that did not have pluripotency are induced to have pluripotency through an artificial dedifferentiation process. ① Cell shape (round shape, large nucleus and phosphorus, small cytoplasm ) And growth rate (dividing time of embryonic stem cells: 17 hr) are similar to embryonic stem cells, ② gene expression and chromatin modification patterns are similar to embryonic stem cells, and ③ pluripotency. It has, ④ can form teratoma in immunodeficient mice, ⑤ forms chimera mice when inserted into the blastocyst of mice, and ⑥ has the property of being capable of germ line transmission of genes.
2006년 세계 최초로 일본 교토대 야마나카 교수팀에 의해 다분화능(Pluripotency)에 핵심적인 역할을 하는 리프로그래밍 유도 전사인자들(Oct4,Sox-2, Klf4, cMyc)의 복합적인 과발현을 통해 체세포로부터 유도만능줄기세포를 생산할 수 있는 역분화 기술이 개발됨에 따라(Cell, 126: 663-676, 2006), 상기 기술을 바탕으로 세포치료제 및 신약개발을 주도하기 위한 세계 각국의 기술 개발 경쟁이 치열하게 진행되고 있다.In 2006, for the first time in the world by Professor Yamanaka of Kyoto University in Japan, induced pluripotency from somatic cells through complex overexpression of reprogramming-inducing transcription factors (Oct4, Sox-2, Klf4, cMyc) that play a key role in pluripotency. With the development of dedifferentiation technology capable of producing stem cells (Cell, 126: 663-676, 2006), competition for technology development around the world to lead the development of cell therapy products and new drugs based on the technology is fierce. have.
이러한 역분화 기술은 환자에게 신체적 손상 및 불편함이 거의 없는 비교적 손쉬운 방법으로 자가-체세포를 확보하고, 이로부터 인간배아줄기 세포와 같은 특성의 유도만능줄기세포를 제조하는 것을 가능케 함으로써, 환자-체세포로부터 맞춤형 자가 전분화능 줄기세포주를 확립하는 전략을 획기적으로 진화시켰으며, 인간 배아줄기세포 이용 시 야기될 수 있는 생명윤리 논란 및 면역 적합성 문제를 극복 할 수 있는 최적의 해법으로 인식되면서 미래 재생의료 기술 개발 분야에 무한한 가능성을 제시하고 있다. 또한, 이러한 역분화 만능줄기세포는 환자 면역 적합형 세포 치료제 개발을 가능케 하며, 세포를 얻기 어려운 기관인 뇌나 심장 등의 세포로 분화 등을 가져올 수 있어 뇌질환이나 심장질환의 원인 규명과 치료 방법을 규명할 수 있는 이점을 가진다. 하지만, 역분화 기술의 우수성에도 불구하고, 이를 실질적으로 세포치료제 및 신약 개발 단계에 활용하기 위해서는 낮은 역분화 효율, 역분화 과정에 소요되는 긴 시간 프레임 등의 극복되어야 하는 문제점들이 아직 남아 있는 실정이어서, 유도만능줄기세포의 제조 효율을 증진시킬 수 있는 기술의 개발이 요구되어 왔다.This dedifferentiation technology makes it possible to secure auto-somatic cells in a relatively easy way with little physical damage and discomfort to the patient, and to produce induced pluripotent stem cells of the same characteristics as human embryonic stem cells, thereby making it possible to produce patient-somatic cells. The strategy to establish a customized autologous pluripotent stem cell line has been remarkably evolved, and the future regenerative medical technology is recognized as the optimal solution to overcome the bioethical controversy and immune compatibility issues that may arise when using human embryonic stem cells. It presents infinite possibilities in the field of development. In addition, these dedifferentiated pluripotent stem cells enable the development of patient immune-adaptive cell therapy, and can lead to differentiation into cells such as the brain or heart, which are difficult to obtain cells, to identify the cause of brain disease or heart disease and to investigate the treatment method. It has the advantage of being able to do it. However, despite the excellence of dedifferentiation technology, problems that must be overcome such as low dedifferentiation efficiency and a long time frame required for the dedifferentiation process still remain in order to use it in the development stage of cell therapy and new drugs. In addition, there has been a demand for the development of technology that can improve the production efficiency of induced pluripotent stem cells.
미분화 유도만능 줄기세포에서 유래된 분화세포가 종양을 형성하는지는 알려져 있지 않지만, 세포치료제의 성분 내에 포함된 미분화 세포에 의한 오염이 이소성 이식에서 인 비보(in vivo) 테라토마를 야기할 수 있다는 큰 우려가 있으며 (Lee AS, Tang C, Rao MS et al. Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies (2013). Nat Med 2013;19:998-1004; Miura K, et al. Variation in the safety of induced pluripotent stem cell lines (2009). Nat Biotechnol 27(8):743-745), 실험적으로 이 우려가 마우스 배아줄기세포에서는 증명된 바 있다. 미미하지만, 테라토마나 다른 비정상적인 세포 성장 (종양)의 형태는 상당한 건강상의 위험성을 갖고 있으며, 이는 이식한 세포나 조직의 기능의 손실을 야기할 수 있다. 실제로, 최근 보고에서 SCID 마우스에 이식한 인간 태아 조직 내에 주입된 인간배아줄기세포(hESCs)가 악성종양으로 발전된 결과가 있다. 이러한 종양 형성 가능성 때문에, 재생의학의 세포치료에 미분화 유도만능 줄기세포, 구체적으로는 미분화 유도만능줄기세포를 임상적으로 적용하는 것을 어렵게 한다.It is not known whether differentiated cells derived from undifferentiated induced pluripotent stem cells form tumors, but there is a great concern that contamination by undifferentiated cells contained in components of cell therapy products may cause in vivo teratoma in ectopic transplantation. (Lee AS, Tang C, Rao MS et al. Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies (2013).Nat Med 2013;19:998-1004; Miura K, et al. Variation in the safety of induced pluripotent stem cell lines (2009).Nat Biotechnol 27(8):743-745), experimentally, this concern has been demonstrated in mouse embryonic stem cells. Although minor, teratomas or other abnormal forms of cell growth (tumor) pose significant health risks, which can lead to loss of function of the transplanted cells or tissues. In fact, in a recent report, there is a result that human embryonic stem cells (hESCs) injected into human fetal tissue transplanted into SCID mice developed into malignant tumors. Due to the possibility of tumor formation, it is difficult to clinically apply undifferentiated induced pluripotent stem cells, specifically, undifferentiated induced pluripotent stem cells, to cell therapy in regenerative medicine.
이러한 불확실성 때문에, 미분화 유도만능 줄기세포-유래 분화세포의 임상적 적용은, 이 세포가 종양형성의 위험성으로부터 자유롭다는 것에 대한 확실한 보장을 요구한다. 이러한 문제를 해결하기 위하여, 다양한 접근방식들이 제안되고 있는 추세이기는 하나, 여전히 이러한 접근에 대한 문제점은 한계가 있다는 문제점도 존재한다.Because of this uncertainty, the clinical application of undifferentiated induced pluripotent stem cell-derived differentiated cells requires a certain guarantee that these cells are free from the risk of tumorigenesis. In order to solve this problem, various approaches are being proposed, but there is still a problem that the problem with this approach is limited.
한편, 봉독(bee venom)은 꿀벌의 복부에서 생성되는 산성 및 염기성 분비물의 혼합물로써 무색의 쓴 액체의 형태를 띄며, 그 주된 성분은 펩타이드인 멜리틴(melittin), 아파민(apamin) 및 비만세포 탈과립화(mast cell degranulating; MCD) 펩타이드 및 효소인 포스포리파아제 A2(phospholipase A2; PLA2) 등이며, 이외 여러 가지 미량의 성분을 포함한다. 이는 종양 세포에 대한 세포독성 및 세포 성장을 억제하거나 세포 사멸 및 괴사를 유도하는 능력이 있다고 보고된 바 있으나, 유도만능 줄기세포의 선택적 사멸과 관련하여서는 보고된 바가 없다.On the other hand, bee venom is a mixture of acidic and basic secretions produced in the abdomen of bees and takes the form of a colorless bitter liquid, and its main components are the peptides melittin, apamin, and mast cells. Degranulating (mast cell degranulating; MCD) peptides and enzyme phospholipase A2 (PLA2), etc., and other trace components. It has been reported that it has cytotoxicity to tumor cells and the ability to inhibit cell growth or induce apoptosis and necrosis, but there is no report regarding the selective death of induced pluripotent stem cells.
이러한 배경하에, 본 발명자들은 줄기세포의 분화 후에 잔재하는 미분화 유도만능 줄기세포를 효과적으로 제거할 수 있는 방법을 개발하고자 노력하였고, 그 결과, 봉독을 처리할 경우 미분화된 유도만능 줄기세포의 선택적인 세포사멸을 유도하여 미분화 유도만능 줄기세포를 매우 효과적으로 제거할 수 있음을 확인함으로써, 본 발명을 완성하였다.Under this background, the present inventors have tried to develop a method that can effectively remove undifferentiated induced pluripotent stem cells remaining after differentiation of stem cells, and as a result, when bee venom is treated, selective cells of undifferentiated induced pluripotent stem cells By inducing death and confirming that undifferentiated induced pluripotent stem cells can be very effectively removed, the present invention was completed.
본 발명의 하나의 목적은 봉독을 유효성분으로 포함하는, 미분화 유도만능 줄기세포의 선택적 사멸 유도용 조성물을 제공하는 것이다.One object of the present invention is to provide a composition for inducing selective death of undifferentiated induced pluripotent stem cells, comprising bee venom as an active ingredient.
본 발명의 다른 하나의 목적은 봉독을 유효성분으로 포함하는, 미분화 유도만능 줄기세포의 선택적 제거용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for selectively removing undifferentiated induced pluripotent stem cells comprising bee venom as an active ingredient.
본 발명의 또 다른 하나의 목적은 (a) 미분화 유도만능 줄기세포 (undifferentiated induced pluripotent stem cells) 및 분화세포(differentiated cells)를 포함하는 세포 시료를 준비하는 단계; 및 (b) 상기 세포 시료에 봉독을 처리하여 미분화 유도만능 줄기세포를 선택적으로 사멸시키는 단계;를 포함하는, 미분화 유도만능 줄기세포의 제거방법을 제공하는 것이다.Another object of the present invention is (a) preparing a cell sample comprising undifferentiated induced pluripotent stem cells and differentiated cells; And (b) treating the cell sample with bee venom to selectively kill undifferentiated induced pluripotent stem cells; comprising, a method of removing undifferentiated induced pluripotent stem cells.
본 발명의 또 다른 하나의 목적은 상기 방법으로 미분화 유도만능 줄기세포가 제거된 세포 조성물을 제공하는 것이다.Another object of the present invention is to provide a cell composition from which undifferentiated induced pluripotent stem cells are removed by the above method.
본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.Each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific description described below.
상기 목적을 달성하기 위한 본 발명의 하나의 양태는 봉독을 유효성분으로 포함하는, 미분화 유도만능 줄기세포의 선택적 사멸 유도용 조성물을 제공하는 것이다.One aspect of the present invention for achieving the above object is to provide a composition for inducing selective death of undifferentiated induced pluripotent stem cells comprising bee venom as an active ingredient.
또한, 본 발명의 다른 하나의 양태는 봉독을 유효성분으로 포함하는, 미분화 유도만능 줄기세포의 선택적 제거용 조성물을 제공하는 것이다.In addition, another aspect of the present invention is to provide a composition for selective removal of undifferentiated induced pluripotent stem cells comprising bee venom as an active ingredient.
본 발명의 용어, “봉독”은 꿀벌(Apismellifera)의 복부에서 생성되는 산성 및 염기성 분비물의 혼합물로서 무색의 쓴 액체형태를 띠며, 그 주된 성분은 펩타이드인 멜리틴(melittin), 아파민(apamin) 및 비만세포 탈과립화(mast cell degranulating; MCD) 펩타이드 및 효소인 포스포리파아제 A2(phospholipase A2; PLA2) 등이며, 이외 여러 가지 미량의 성분을 포함한다. 본 발명의 봉독은 꿀벌에서 분리되는 것일 수 있으나 이에 제한되지 않는다.The term "bee venom" of the present invention is a mixture of acidic and basic secretions produced in the abdomen of a bee (Apismellifera) and has a colorless bitter liquid form, and its main components are the peptides melittin and apamin. And mast cell degranulating (MCD) peptides and enzymes, such as phospholipase A2 (PLA2), and other trace components. The bee venom of the present invention may be isolated from bees, but is not limited thereto.
본 발명에서 사용하는 봉독은 정제 봉독, 봉독 정제물 또는 봉독 추출물을 포함하는 것일 수 있으며, 구체적으로는 정제 봉독 또는 봉독 정제물일 수 있으나 이에 제한되지 않는다.The bee venom used in the present invention may include purified bee venom, purified bee venom, or bee venom extract, and specifically, may be purified bee venom or purified bee venom, but is not limited thereto.
본 발명에서는, 이러한 봉독을 미분화 유도만능 줄기세포에 처리하는 경우, 미분화 유도만능 줄기세포의 선택적 세포 사멸을 유도하는 것을 규명하여, 이를 미분화 줄기세포의 제거 용도로 사용할 수 있음을 최초로 규명하였다.In the present invention, when such bee venom is treated on undifferentiated induced pluripotent stem cells, it was identified that it induces selective cell death of undifferentiated induced pluripotent stem cells, and it was first identified that it can be used for removal of undifferentiated stem cells.
본 발명에서의 용어, “유도만능 줄기세포 (iPSC: induced pluripotent stem cells)”는 분화된 세포들로부터 인위적인 역분화 과정을 통해 다능성 분화능을 가지도록 유도된 세포들을 일컫는 말로서 역분화줄기세포 이라고도 한다. 인위적인 역분화 과정은 레트로바이러스 및 렌티바이러스를 이용한 바이러스-매개 또는 비바이러스성 벡터 이용, 단백질 및 세포 추출물 등을 이용하는 비바이러스-매개 역분화인자의 도입에 의해 수행되거나, 줄기세포 추출물, 화합물 등에 의한 역분화 과정을 포함한다. 유도만능 줄기세포는 배아줄기세포와 거의 같은 특성을 가지며, 구체적으로는, 비슷한 세포 모양을 보여준다. 또한, 유전자, 단백질 발현 패턴이 유사하고, in vitro 및 in vivo에서 전분화능을 가지며, 테라토마 (teratoma)를 형성하고, 생쥐의 배반포 (blastocyst)에 삽입시켰을 때, 키메라 (chimera) 생쥐를 형성하고, 유전자의 생식선 전이 (germline transmission)가 가능하다. 본 발명의 유도만능줄기 세포로는 인간, 원숭이, 돼지, 말, 소, 양, 개, 고양이, 생쥐, 토끼 등의 모든 유래의 유도만능 줄기세포를 포함하나, 바람직하게는 인간 유래의 유도만능 줄기세포이다.The term "induced pluripotent stem cells (iPSCs)" in the present invention refers to cells induced to have pluripotent differentiation capacity from differentiated cells through an artificial dedifferentiation process, and is also referred to as dedifferentiated stem cells. . The artificial dedifferentiation process is performed by introduction of a non-viral-mediated dedifferentiation factor using a virus-mediated or non-viral vector using retroviruses and lentiviruses, protein and cell extracts, etc., or by stem cell extracts, compounds, etc. Including the dedifferentiation process. Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, and specifically, show similar cell shapes. In addition, gene and protein expression patterns are similar, have pluripotency in vitro and in vivo, form teratoma, and when inserted into a blastocyst of mice, chimera mice are formed, Germline transmission of genes is possible. The induced pluripotent stem cells of the present invention include induced pluripotent stem cells derived from all humans, monkeys, pigs, horses, cattle, sheep, dogs, cats, mice, rabbits, etc., but preferably human induced pluripotent stem cells It is a cell.
본 발명에서의 용어, “분화”는 세포가 분열 증식하여 성장하는 동안에 서로 구조나 기능이 특수화하는 현상. 즉 생물의 세포, 조직 등이 각각에게 주어진 일을 수행하기 위하여 형태나 기능이 변해가는 것을 의미하며, “분화된 세포”는 줄기세포가 특정 분화 자극에 의해 특정 세포로 분화가 진행된 세포, 구체적으로는 0% 이상 내지 100% 미만의 세포 분화능을 가지는 세포를 의미한다. 이에, 역분화에 의해 전부 또는 일부가 미분화될 수 있는 범주 내의 세포는 모두 분화된 세포로 포함되며, 예를 들어 체세포 또는 전구세포가 포함된다. 상기 0%의 세포 분화능을 가지는 세포는 체세포에 해당될 것이다.The term "differentiation" in the present invention refers to a phenomenon in which structures and functions are specialized to each other during growth by division and proliferation of cells. In other words, means that cells and tissues of an organism change their shape or function in order to perform a task given to each, and “differentiated cells” refers to cells in which stem cells are differentiated into specific cells by specific differentiation stimulation. Refers to a cell having a cell differentiation capacity of 0% or more to less than 100%. Accordingly, all cells within the category in which all or part of the cells can be undifferentiated by dedifferentiation are included as differentiated cells, and, for example, somatic cells or progenitor cells are included. Cells having the cell differentiation ability of 0% will correspond to somatic cells.
또한, “미분화”는 상기 분화와 반대되는 의미를 가진 용어로서, 세포가 분열 증식하기 전의 상태를 의미하며, 미분화 유도만능 줄기세포는 특정 세포로 분화되지 않은 채 원래와 세포와 동일한 성질을 가지는, 즉, 전분화능을 가지는 다능성 줄기세포로 유지되어 있는 세포를 의미한다.In addition, “undifferentiated” is a term having the opposite meaning to the above differentiation, and refers to a state before a cell divides and proliferates, and an undifferentiated induced pluripotent stem cell does not differentiate into a specific cell and has the same properties as the original cell, That is, it refers to cells maintained as pluripotent stem cells having pluripotency.
본 발명에서의 용어, “체세포”는 성체를 구성하는 세포로서 분화능 및 자가생산능이 제한된 세포를 의미한다.In the present invention, the term "somatic cell" refers to a cell constituting an adult body and has limited differentiation and self-producing ability.
본 발명에서의 용어, “전구세포”는 특정 세포의 형태 및 기능을 갖추기 전단계의 특정 세포 계통의 세포로 분화되거나 특정 유형의 조직으로 형성될 수 있는 세포로서, 자가재생산능(self-renewal)은 가지나 극히 제한된 분화능을 갖는 세포를 의미하며, 내배엽 전구세포, 중배엽 전구세포, 및 외배엽 전구세포가 모두 포함된다.In the present invention, the term "progenitor cell" is a cell that can be differentiated into cells of a specific cell lineage or formed into a specific type of tissue prior to having the shape and function of a specific cell, and self-renewal is It refers to a cell having a branched but extremely limited differentiation ability, and includes all endoderm progenitor cells, mesodermal progenitor cells, and ectodermal progenitor cells.
본 발명에서 용어, “세포사멸”은 세포자살 또는 아폽토시스(Apoptosis)와 혼용되어 사용되며, 우리 몸 안에 입력되어 있는 생체 프로그램으로 비정상 세포, 손상된 세포, 노화된 세포가 스스로 자살해 사멸함으로써 전체적인 신체건강을 유지하게 해 주는 메커니즘을 말한다.In the present invention, the term "cell death" is used interchangeably with apoptosis or apoptosis. Abnormal cells, damaged cells, and aging cells kill themselves by committing suicide as a biological program input in our body. It refers to the mechanism that allows you to maintain.
본 발명에서 말하는 “선택적 세포사멸”은 분화된 세포에는 영향, 구체적으로는 세포독성의 영향을 미치지 아니하고, 분화되지 않은 미분화 유도만능 줄기세포를 선택적으로 세포 사멸을 유도하는 것을 말한다.In the present invention, "selective apoptosis" refers to inducing apoptosis by selectively inducing undifferentiated induced pluripotent stem cells that are not differentiated without affecting differentiated cells, specifically cytotoxicity.
본 발명에서 미분화 유도만능 줄기세포를 언급하면서 사용되는 용어 “제거(depletion or removal)”는 세포 시료 즉 비균질 세포 파퓰레이션으로부터 미분화 유도만능 줄기세포를 실질적으로 제거되어 테라토마를 형성하지 않는 세포 조성물을 의미한다. 예를 들어, 용어 제거는 본 발명의 방법이 적용된 세포 시료에서 미분화 유도만능 줄기세포의 양이 1% 미만, 바람직하게는 0.5% 미만, 보다 바람직하게는 0.1% 미만이 되도록 하는 것을 의미한다. 상기 용어 “%”는 세포개수(cellcount)를 기준으로 한 백분율이다.The term “depletion or removal” used in referring to undifferentiated induced pluripotent stem cells in the present invention refers to a cell composition that does not form a teratoma by substantially removing undifferentiated induced pluripotent stem cells from a cell sample, that is, a heterogeneous cell population. . For example, the term removal means that the amount of undifferentiated induced pluripotent stem cells in the cell sample to which the method of the present invention is applied is less than 1%, preferably less than 0.5%, more preferably less than 0.1%. The term "%" is a percentage based on the cell count.
본 발명의 구체적인 일 실시예에서는 봉독을 처리 후 2D 배양조건에서 미분화 유도만능 줄기세포의 세포생존율이 감소하고, 형태학적 변화가 생기는 것을 확인하였고 (도 1A 내지 B), 3D 조건에서는 스페로이드 크기(spheroid size)가 농도 의존적으로 감소되는 것을 확인하였으며 (도 2 내지 3), 웨스턴 블롯을 통해 미분화 유도만능 줄기세포에서 선택적으로 death receptor의 발현이 향상되는 것을 확인하였지만 (도 4), 봉독이 분화 줄기세포에서는 DNA에 손상을 미치지 않음을 확인하였다 (도 5).In a specific embodiment of the present invention, it was confirmed that the cell viability of undifferentiated induced pluripotent stem cells decreased and morphological changes occurred in 2D culture conditions after treatment with bee venom (FIGS. 1A to B), and spheroid size ( spheroid size) was confirmed to decrease in a concentration-dependent manner (FIGS. 2 to 3), and it was confirmed that expression of death receptors was selectively improved in undifferentiated induced pluripotent stem cells through Western blot (FIG. 4), but bee venom stems differentiation. It was confirmed that the cells did not damage DNA (FIG. 5).
본 발명의 또 다른 하나의 양태는 (a) 미분화 유도만능 줄기세포(undifferentiated induced pluripotent stem cells) 및 분화세포(differentiated cells)를 포함하는 세포 시료를 준비하는 단계; 및 (b) 상기 세포 시료에 봉독을 처리하여 미분화 유도만능 줄기세포를 선택적으로 세포사멸시키는 단계;를 포함하는, 미분화 유도만능 줄기세포의 제거방법을 제공한다.Another aspect of the present invention includes the steps of: (a) preparing a cell sample comprising undifferentiated induced pluripotent stem cells and differentiated cells; And (b) selectively killing undifferentiated induced pluripotent stem cells by treating the cell sample with bee venom. It provides a method for removing undifferentiated induced pluripotent stem cells, including.
상기 용어 “미분화”, “유도만능 줄기세포”, “분화”, “봉독”, “선택적 세포사멸” 및 “제거”는 상기에서 설명한 바와 같다.The terms “undifferentiated”, “induced pluripotent stem cell”, “differentiated”, “bee venom”, “selective cell death” and “removal” are as described above.
상기 발명에서 이용되는 미분화 유도만능 줄기세포는 인간, 소, 말, 염소, 양, 개, 고양이, 마우스, 래트 또는 조류로부터 유래된 것이고, 가장 바람직하게는 인간 유도만능 줄기세포이다.The undifferentiated induced pluripotent stem cells used in the present invention are derived from humans, cattle, horses, goats, sheep, dogs, cats, mice, rats or birds, and most preferably human induced pluripotent stem cells.
본 발명의 또 다른 하나의 양태는 상기 본 발명의 제조방법에 의해 미분화 유도만능 줄기세포가 제거된 세포 조성물을 제공한다.Another aspect of the present invention provides a cell composition from which undifferentiated induced pluripotent stem cells are removed by the method of the present invention.
상기 용어 “미분화”, “유도만능 줄기세포” 및 “제거”는 상기에서 설명한 바와 같다.The terms “undifferentiated”, “derived pluripotent stem cells” and “removed” are as described above.
유도만능 줄기세포가 분화하는 과정에서 잔존하는 미분화 줄기세포는 세포치료제로 이식 시 테라토마(기형종)를 형성할 수 있는 문제점을 가지는 상황에서, 본 발명의 봉독을 유효성분으로 포함하는 조성물은 유도만능 줄기세포가 분화하는 과정에서 미분화 상태로 남아있는 줄기세포만을 효과적으로 제거할 수 있으므로, 안전한 줄기세포 치료제 개발에 활용될 수 있다.In a situation where undifferentiated stem cells remaining in the process of differentiation of induced pluripotent stem cells have a problem in that they can form teratoma (teratoma) when transplanted as a cell therapy agent, the composition containing bee venom of the present invention as an active ingredient is induced pluripotent. Since only stem cells remaining in an undifferentiated state can be effectively removed during the process of stem cell differentiation, it can be used to develop a safe stem cell therapeutic agent.
도 1은 봉독이 미분화 유도만능 줄기세포에만 선택적으로 세포사멸을 유도하는 것을 2D 세포 배양 조건에서 확인한 결과를 나타낸 것이다.
도 2는 미분화 유도만능 줄기세포 및 분화 줄기세포를 3D 배양 시 봉독을 함께 처리하여 미분화 유도만능 줄기세포에만 선택적으로 세포사멸을 유도하는 것을 확인한 결과를 나타낸 것이다.
도 3은 미분화 유도만능 줄기세포 및 분화 줄기세포를 3D 배양한 후 봉독을 처리하여 미분화 유도만능 줄기세포에만 선택적으로 세포사멸을 유도하는 것을 확인한 결과를 나타낸 것이다.
도 4는 미분화 유도만능 줄기세포 및 분화 줄기세포에 봉독을 처리하여 Death receptor의 발현 변화를 비교한 것이다.
도 5는 분화된 줄기세포에 봉독을 처리하여 DNA의 손상 유무를 확인한 것이다.1 shows the results of confirming that bee venom selectively induces apoptosis only in undifferentiated induced pluripotent stem cells under 2D cell culture conditions.
FIG. 2 shows the results of confirming that undifferentiated induced pluripotent stem cells and differentiated stem cells are selectively induced apoptosis only in undifferentiated induced pluripotent stem cells by treating bee venom together during 3D culture.
3 shows the results of confirming that undifferentiated induced pluripotent stem cells and differentiated stem cells are cultured in 3D and then treated with bee venom to selectively induce apoptosis only in undifferentiated induced pluripotent stem cells.
4 is a comparison of the expression change of the death receptor by treating bee venom to undifferentiated induced pluripotent stem cells and differentiated stem cells.
5 shows whether or not DNA is damaged by processing bee venom on differentiated stem cells.
이하, 실시예를 통하여 본 발명의 구성 및 효과를 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들 실시예에 의해 한정되는 것은 아니다.Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.
실시예 1: 봉독 준비Example 1: bee venom preparation
봉독은 ㈜청진바이오텍에서 구입하였다. 주성분은 Melittin (45-65%), PLA2 (11.5-13%), Apamin (2-3%)이며, 이 외에도 MCD-Peptide401, Adolapin, protease inhibitor, Secapine, Procamine A, B, Hyaluronidase, Histamine, Dopamine을 함유하고 있다. 이를 실험에 사용하기 위해, 봉독 파우더 100 mg을 증류수 10 ml에 녹인 후, 0.22 μm syringe filter를 이용하여 여과한 후 -20℃에 보관하여 사용하였다.Bee venom was purchased from Chungjin Biotech. The main ingredients are Melittin (45-65%), PLA2 (11.5-13%), and Apamin (2-3%). In addition, MCD-Peptide401, Adolapin, protease inhibitor, Secapine, Procamine A, B, Hyaluronidase, Histamine, Dopamine Contains. To use this in the experiment, 100 mg of bee venom powder was dissolved in 10 ml of distilled water, filtered using a 0.22 μm syringe filter, and stored at -20°C.
실시예 2: 미분화 인간 유도만능 줄기세포 및 분화 인간 유도만능 줄기세포에 대한 봉독의 세포독성 확인 (2D 배양조건)Example 2: Confirmation of cytotoxicity of bee venom against undifferentiated human induced pluripotent stem cells and differentiated human induced pluripotent stem cells (2D culture conditions)
봉독을 농도별로 처리하여 24시간 후 세포생존률을 CCK 어세이로 분석하였다. 이를 위해, Matrigel Matrix (hESC-qualified)가 코팅된 24-웰 배양접시(24-well culture plate)에 미분화 인간 유도만능 줄기세포를 접종(seeding)한 후 mTeSR1 배양배지 (STEMCELL technologies, USA)에서 24시간 배양하였다. 분화 인간 유도만능 줄기세포는 24-웰 배양접시에 접종 후 DMEM 배지 (10% FBS, NEAA, PS, 2-ME 첨가)에서 24시간 배양하였다. 약 50% 정도 세포 컨플루언시(confluency)가 확인되면, 상기 실시 예에서 제조한 봉독을 0.5 ㎍/㎖, 1 ㎍/㎖, 2.5 ㎍/㎖, 5 ㎍/㎖ 및 10 ㎍/㎖ 의 농도로 처리한 후 37℃, 5% CO₂인큐베이터에서 24시간 동안 배양하였다. 그 후 CCK 용액 (Cell Counting Kit-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan)을 첨가하고 1-4시간 경과한 후, SpectraMaxi3 Multi-mode reader (Molecular Devices,LLC, Sunnyvale, CA, USA)를 이용하여 450 nm에서 흡광도를 측정하여 세포 생존율을 확인하였다.Bee venom was treated by concentration, and cell viability after 24 hours was analyzed by CCK assay. To this end, after seeding undifferentiated human induced pluripotent stem cells in a 24-well culture plate coated with Matrigel Matrix (hESC-qualified), mTeSR1 culture medium (STEMCELL technologies, USA) Incubated for hours. Differentiated human induced pluripotent stem cells were inoculated in a 24-well culture dish and cultured in DMEM medium (10% FBS, NEAA, PS, 2-ME added) for 24 hours. When about 50% of cell confluency was confirmed, the bee venom prepared in the above example was at concentrations of 0.5 µg/ml, 1 µg/ml, 2.5 µg/ml, 5 µg/ml, and 10 µg/ml After treatment with, it was incubated for 24 hours in a 5% CO2 incubator at 37°C. Thereafter, CCK solution (Cell Counting Kit-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added and after 1-4 hours elapsed, SpectraMaxi3 Multi-mode reader (Molecular Devices, LLC, Sunnyvale, CA, USA) By measuring the absorbance at 450 nm using, the cell viability was confirmed.
또한, 봉독을 처리하고 24시간 후 세포의 형태학상 변화를 확인하기 위해, 배양 플레이트에 부착되어 있는 세포 모양 및 부착성이 떨어져 세포모양이 둥글게 변화는 등의 세포사멸 형태를 현미경으로 확인하였다.In addition, in order to confirm the change in the morphology of the cells 24 hours after treatment with bee venom, the shape of the cells attached to the culture plate and the shape of apoptosis, such as a rounded change in cell shape due to poor adhesion, were confirmed with a microscope.
그 결과, 도 1에서 볼 수 있듯이, 봉독 0.5 ㎍/㎖ 이상에서 농도의존적으로 미분화 유도만능 줄기세포에서만 세포생존율이 감소됨을 확인하였다 (도 1A 내지 B).As a result, as can be seen in Figure 1, it was confirmed that the cell viability was decreased only in undifferentiated induced pluripotent stem cells in a concentration-dependent manner at 0.5 ㎍/㎖ or more of bee venom (FIGS. 1A to B).
이를 통해, 봉독이 미분화 유도만능 줄기세포에만 선택적으로 세포독성이 있어, 선택적 세포사멸 효능이 있음을 확인하였다.Through this, it was confirmed that bee venom is selectively cytotoxic only to undifferentiated induced pluripotent stem cells, and thus has selective apoptosis effect.
실시예 3: 미분화 인간 유도만능 줄기세포 및 분화 인간 유도만능 줄기세포에 대한 봉독의 세포독성 확인 (3D 배양조건)Example 3: Confirmation of cytotoxicity of bee venom against undifferentiated human induced pluripotent stem cells and differentiated human induced pluripotent stem cells (3D culture conditions)
3D-spheroid culture 조건에서 봉독 처리에 의한 변화를 확인하기 위해, 미분화 인간 유도만능 줄기세포 및 분화 인간 유도만능 줄기세포를 mTeSR1 배양배지 및 DMEM 배양배지에 각각 부유시킨 후 96-well U-bottomed Ultra Low Attachment (ULA) 배양접시에 접종한 후 (200 μl/well), 200 g에서 5분간 원심분리하였다. 부유시키는 단계에서 봉독을 처리하는 co-treatment, 1일동안 스페로이드(spheroid) 형태로 배양시킨 후, 봉독을 처리하는 post-treatment 조건에서 각각 효과를 확인하였다. 봉독을 0.5 ㎍/㎖, 1 ㎍/㎖, 2.5 ㎍/㎖, 5 ㎍/㎖ 및 10 ㎍/㎖의 농도로 처리한 후 48시간 배양하면서, 스페로이드 형태를 관찰하고 스페로이드 지역을 Image J 소프트웨어를 사용하여 측정하였다.In order to confirm the change by bee venom treatment in 3D-spheroid culture conditions, undifferentiated human induced pluripotent stem cells and differentiated human induced pluripotent stem cells were suspended in mTeSR1 culture medium and DMEM culture medium, respectively, and then 96-well U-bottomed Ultra Low. After inoculation on the attachment (ULA) culture dish (200 μl/well), it was centrifuged at 200 g for 5 minutes. The effects were confirmed in the co-treatment treatment of bee venom in the floating step, and in the form of spheroids for 1 day, and post-treatment conditions for treatment of bee venom. Bee venom was treated at concentrations of 0.5 µg/ml, 1 µg/ml, 2.5 µg/ml, 5 µg/ml, and 10 µg/ml, and cultured for 48 hours to observe the spheroid morphology and determine the spheroid area with Image J software. It was measured using.
그 결과, 도 2에서 보듯이, 봉독을 co-treatment 조건으로 처리했을 경우, 0.5 ㎍/㎖ 이상에서 농도의존적으로 미분화 유도만능 줄기세포의 스페로이드 크기가 감소됨을 확인하였다 (도 2A 내지 B).As a result, as shown in FIG. 2, when the bee venom was treated under co-treatment conditions, it was confirmed that the spheroid size of the undifferentiated induced pluripotent stem cells was decreased in a concentration-dependent manner at 0.5 µg/ml or more (FIGS. 2A to B).
또한, 도 3에서 보듯이, 봉독을 post-treatment 조건으로 처리했을 경우, 0.5 ㎍/㎖ 이상에서 농도의존적으로 미분화 유도만능 줄기세포의 스페로이드 크기가 감소됨을 확인하였다 (도 3A 내지 B).In addition, as shown in FIG. 3, when bee venom was treated under post-treatment conditions, it was confirmed that the spheroid size of undifferentiated induced pluripotent stem cells was decreased in a concentration-dependent manner at 0.5 µg/ml or more (FIGS. 3A to B).
이를 통해, 봉독이 미분화 유도만능 줄기세포에만 선택적으로 세포독성이 있어, 선택적 세포사멸 효능이 있음을 확인하였다.Through this, it was confirmed that bee venom is selectively cytotoxic only to undifferentiated induced pluripotent stem cells, and thus has selective apoptosis effect.
실시예 4: 미분화 인간 유도만능 줄기세포에 봉독 처리 시 Death receptor 발현 증가 확인Example 4: Confirmation of increased expression of death receptor upon treatment of bee venom in undifferentiated human induced pluripotent stem cells
미분화 인간 유도만능 줄기세포 및 분화 인간 유도만능 줄기세포에 봉독을 처리한 후 death receptor인 DR4, DR5, FAS 단백질의 발현에 어떠한 영향을 미치는지 확인하기 위하여, 웨스턴 블롯을 수행하였다.Western blot was performed to determine how it affects the expression of the death receptors DR4, DR5, and FAS proteins after treatment with bee venom on undifferentiated human iPS cells and differentiated human iPS cells.
보다 구체적으로, 미분화 인간 유도만능 줄기세포를 Matrigel Matrix (hESC-qualified)가 코팅된 60 mm 배양접시에 접종한 다음, 37℃, 5% CO₂ 인큐베이터에서 24시간 동안 배양하였다. 분화 인간 유도만능 줄기세포는 60 mm 배양접시에 접종한 다음, 상기 서술한 바와 같이 24시간 동안 배양하였다. 배양 후, 봉독을 2.5 ㎍/㎖ 및 5 ㎍/㎖의 농도로 처리하여, 24시간 동안 추가 배양하였다. 추가 배양 후, 배양접시에 부착되지 않은 세포와 부착된 세포를 모두 모은 다음 생리 식염수로 2회 세척하고, 세포의 전체 cell lysate는 MPER Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL)를 사용하여 준비하였다. cell lysate은 BCA 단백질 분석 (BCA(Bicinchoninic Acid) protein assay)으로 단백질 농도를 정량하였고, 동량의 단백질을 SDS-PAGE에 전기영동한 후 Immunobilon PVDF membrane(Millipore, Bedford, MA)에 옮긴 후 Immunoblotting을 수행하였다. 단백질 발현 정도는 ECL 용액과 반응시킨 후 ImageQuant LAS 4000 mini(GE Healthcare, Piscataway, NJ, USA)를 사용하여 가시화하였다. More specifically, undifferentiated human induced pluripotent stem cells were inoculated into a 60 mm culture dish coated with Matrigel Matrix (hESC-qualified), and then cultured in a 5% CO₂ incubator at 37°C for 24 hours. Differentiated human induced pluripotent stem cells were inoculated into a 60 mm culture dish and then cultured for 24 hours as described above. After cultivation, bee venom was treated at concentrations of 2.5 µg/ml and 5 µg/ml, and further cultured for 24 hours. After further cultivation, all cells that are not attached to the culture dish and attached cells are collected and washed twice with physiological saline, and the total cell lysate of the cells is prepared using MPER Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL). I did. Cell lysate was quantified by BCA protein analysis (Bicinchoninic Acid (BCA) protein assay), and the same amount of protein was electrophoresed on SDS-PAGE, transferred to Immunobilon PVDF membrane (Millipore, Bedford, MA), and then subjected to Immunoblotting. I did. Protein expression level was visualized using ImageQuant LAS 4000 mini (GE Healthcare, Piscataway, NJ, USA) after reacting with ECL solution.
그 결과, 도 4에서 보듯이, 봉독의 세포사멸기작을 확인한 결과, 미분화 인간 유도만능 줄기세포의 death receptor (FAS, DR4, DR5) 발현은 증가한 반면, 분화된 인간 유도만능 줄기세포에서는 DR5 발현이 미약하게 증가하였고, FAS 및 DR4 발현은 거의 나타나지 않았다 (도 4).As a result, as shown in FIG. 4, as a result of confirming the apoptosis mechanism of bee venom, expression of death receptors (FAS, DR4, DR5) in undifferentiated human induced pluripotent stem cells increased, whereas DR5 expression in differentiated human induced pluripotent stem cells was increased. It increased slightly, and FAS and DR4 expression were hardly observed (Fig. 4).
이를 통해, 봉독이 미분화 유도만능 줄기세포에만 선택적으로 세포독성이 있어, 선택적 세포사멸 효능이 있음을 확인하였다.Through this, it was confirmed that bee venom is selectively cytotoxic only to undifferentiated induced pluripotent stem cells, and thus has selective apoptosis effect.
실시예 5: 봉독에 의한 분화 인간 유도만능 줄기세포의 DNA 손상 확인Example 5: DNA damage confirmation of human induced pluripotent stem cells differentiated by bee venom
분화 인간 유도만능 줄기세포에 봉독을 처리한 후 DNA 손상 시 증가되는 것으로 알려진 phosphor-ATM 단백질의 발현정도를 웨스턴 블롯을 통해 확인하였다.The expression level of phosphor-ATM protein, which is known to increase when DNA is damaged after treatment with bee venom in differentiated human induced pluripotent stem cells, was confirmed through Western blot.
구체적으로, DNA 손상을 유도하는 양성대조군으로 cisplatin을 사용하였다. 보다 구체적으로, 분화 인간 유도만능 줄기세포를 60 mm 배양접시에 접종한 다음, 37℃, 5%, CO₂ 조건의 인큐베이터에서 24시간 동안 배양하였다. 배양 후, cisplatin을 50 μM과 100 μM, 봉독을 5 ㎍/㎖의 농도로 처리하여, 24시간 동안 추가 배양하였다. 추가 배양 후, 세포를 모두 회수한 다음 생리 식염수로 2회 세척하고, 세포의 전체 cell lysate는 MPER Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL)를 사용하여 준비하였다. cell lysate은 BCA 단백질 분석 (BCA(Bicinchoninic Acid) protein assay)으로 단백질 농도를 정량하였고, 동량의 단백질을 SDS-PAGE에 전기영동한 후 Immunobilon PVDF membrane(Millipore, Bedford, MA)에 옮긴 후 Immunoblotting을 수행하였다. 단백질 발현 정도는 ECL 용액과 반응시킨 후 ImageQuant LAS 4000 mini(GE Healthcare, Piscataway, NJ, USA)를 사용하여 가시화하였다.Specifically, cisplatin was used as a positive control inducing DNA damage. More specifically, differentiated human induced pluripotent stem cells were inoculated into a 60 mm culture dish, and then cultured for 24 hours in an incubator at 37°C, 5%, and CO₂. After incubation, cisplatin was treated at a concentration of 50 μM and 100 μM and bee venom at a concentration of 5 μg/ml, followed by additional culture for 24 hours. After further culture, all the cells were recovered and washed twice with physiological saline, and the total cell lysate of the cells was prepared using MPER Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL). Cell lysate was quantified by BCA protein analysis (Bicinchoninic Acid (BCA) protein assay), and the same amount of protein was electrophoresed on SDS-PAGE, transferred to Immunobilon PVDF membrane (Millipore, Bedford, MA), and then subjected to Immunoblotting. I did. Protein expression level was visualized using ImageQuant LAS 4000 mini (GE Healthcare, Piscataway, NJ, USA) after reacting with ECL solution.
그 결과, 도 5A에서 보듯이, cisplatin을 분화 유도만능 줄기세포에 처리하자, DNA 수리에 관여하는 인산화된 ATM 단백질(p-ATM) 발현이 증가한 반면, 봉독을 처리군에서는 p-ATM 발현이 크게 증가하지 않음을 웨스턴 블롯을 통해 확인하였다 (도 5A).As a result, as shown in Fig. 5A, when cisplatin was treated with pluripotent stem cells for differentiation, the expression of phosphorylated ATM protein (p-ATM) involved in DNA repair increased, whereas p-ATM expression was significantly increased in the bee venom treated group. It was confirmed through Western blot that there was no increase (Fig. 5A).
또한, 분화 인간 유도만능 줄기세포에 봉독을 처리한 후 DNA 손상 시 증가되는 것으로 알려진 γ-H2AX foci 형성정도를 면역형광염색을 통해 확인하였다.In addition, the degree of formation of γ-H2AX foci, which is known to be increased when DNA is damaged after treatment with bee venom on differentiated human induced pluripotent stem cells, was confirmed through immunofluorescence staining.
보다 구체적으로, 분화 인간 유도만능 줄기세포를 35 mm 유리접시 (SPL Lifesciences, 한국)에 접종한 후, 37℃, 5% CO₂ 조건의 인큐베이터에서 24시간 동안 배양하였다. 배양 후, cisplatin을 50 μM과 100 μM, 봉독을 5 ㎍/㎖의 농도로 처리하여, 24시간 동안 추가 배양하였다. 추가 배양 후, 생리 식염수로 2회 세척하고 10% 포르말린 (formalin)을 넣어 30분간 실온에서 고정하였다. 세척한 후 0.1% Triton X-100를 넣고 실온에서 30분간 침투 (permeabilization) 시킨 후 다시 생리 식염수로 2회 세척하였다. 3% BSA가 포함된 생리 식염수를 넣은 후 실온에서 60분간 blocking 시킨 후, 항-γ-H2AX 항체 (1:250 희석)를 넣어 4℃에서 하룻동안 반응시켰다. 생리 식염수로 3회 세척한 후, Alexa-594가 라벨 된 2차 항체 (1:500 희석)를 넣어 실온에서 3시간 반응시켰다. 다시 생리 식염수로 3회 세척한 후, DAPI로 대조 염색하여 형광현미경으로 관찰하였다. More specifically, differentiated human induced pluripotent stem cells were inoculated into a 35 mm glass dish (SPL Lifesciences, Korea) and then cultured for 24 hours in an incubator at 37° C. and 5% CO₂. After incubation, cisplatin was treated at a concentration of 50 μM and 100 μM and bee venom at a concentration of 5 μg/ml, followed by additional culture for 24 hours. After further incubation, the mixture was washed twice with physiological saline, and 10% formalin was added and fixed at room temperature for 30 minutes. After washing, 0.1% Triton X-100 was added, permeabilized at room temperature for 30 minutes, and washed twice with physiological saline. After adding physiological saline containing 3% BSA, blocking was performed at room temperature for 60 minutes, and then anti-γ-H2AX antibody (1:250 dilution) was added and reacted at 4° C. for one day. After washing three times with physiological saline, a secondary antibody labeled with Alexa-594 (1:500 dilution) was added and reacted at room temperature for 3 hours. After washing again with physiological saline three times, counter-staining with DAPI was performed and observed with a fluorescence microscope.
그 결과, 도 5B에서 보듯이, DNA 손상에 의해 생성된 γ-H2AX foci가 cisplatin을 처리한 군에서는 현저하게 증가된 반면, 봉독을 처리한 군에서는 나타나지 않음을 형광염색을 통해 확인하였다 (도 5B).As a result, as shown in FIG. 5B, it was confirmed through fluorescence staining that the γ-H2AX foci generated by DNA damage was significantly increased in the group treated with cisplatin, but did not appear in the group treated with bee venom (FIG. 5B ).
이를 통해, 봉독이 분화 인간 유도만능 줄기세포의 DNA에 손상을 미치지 않음을 확인하였다.Through this, it was confirmed that bee venom does not damage the DNA of differentiated human induced pluripotent stem cells.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.
Claims (5)
A composition for inducing selective apoptosis of undifferentiated induced pluripotent stem cells, comprising bee venom as an active ingredient.
The composition of claim 1, wherein the undifferentiated induced pluripotent stem cells are derived from humans.
A composition for selective removal of undifferentiated induced pluripotent stem cells, comprising bee venom as an active ingredient.
(b) 상기 세포 시료에 봉독을 처리하여 미분화 유도만능 줄기세포를 선택적으로 세포사멸시키는 단계;를 포함하는, 미분화 유도만능 줄기세포의 제거방법.
(a) preparing a cell sample comprising undifferentiated induced pluripotent stem cells and differentiated cells; And
(b) treating the cell sample with bee venom to selectively kill undifferentiated induced pluripotent stem cells; comprising, a method for removing undifferentiated induced pluripotent stem cells.
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