KR20210147971A - Recombinant Phage-Based Culture Container For Stem Cell Culture and Uses Thereof - Google Patents
Recombinant Phage-Based Culture Container For Stem Cell Culture and Uses Thereof Download PDFInfo
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- KR20210147971A KR20210147971A KR1020210069003A KR20210069003A KR20210147971A KR 20210147971 A KR20210147971 A KR 20210147971A KR 1020210069003 A KR1020210069003 A KR 1020210069003A KR 20210069003 A KR20210069003 A KR 20210069003A KR 20210147971 A KR20210147971 A KR 20210147971A
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Abstract
Description
본 발명은 줄기세포 배양용 재조합 파지-기반 용기 및 이의 용도에 관한 것이다. The present invention relates to a recombinant phage-based container for culturing stem cells and uses thereof.
줄기세포는 미분화 세포로서 지속적인 계대배양을 통해 분열을 할 수 있으며, 특정 환경 내에서 특정 세포로 분화할 수 있는 세포를 말한다. 줄기세포는 기원이 되는 조직이나, 만드는 방법에 따라, 성체 줄기세포(Adult stem cell), 배아 줄기세포(embryonic stem cell), 유도 만능 줄기세포(induced pluripotent stem cell) 등이 있다. 배아 줄기세포나 유도만능 줄기세포의 경우 모든 세포로 분화가 가능하고, 성체줄기세포의 경우 특정 세포로 분화한다는 특징이 있다. 줄기세포를 이용한 연구나 치료의 한계로는 체외배양시 줄기세포의 노화(senescence)로 인하여 기능의 저하, 충분한 세포수의 확보 등의 문제가 대두되고 있다. 따라서 연구나 치료의 목적으로 사용가능한 줄기세포의 수를 확보하는 배양 방법이 요구된다. Stem cells are undifferentiated cells that can divide through continuous subculture and can differentiate into specific cells within a specific environment. Stem cells are a tissue of origin, but depending on the production method, there are adult stem cells, embryonic stem cells, induced pluripotent stem cells, and the like. In the case of embryonic stem cells or induced pluripotent stem cells, they can be differentiated into all cells, and in the case of adult stem cells, they differentiate into specific cells. As a limitation of research or treatment using stem cells, problems such as a decrease in function and securing a sufficient number of cells due to senescence of stem cells during in vitro culture are emerging. Therefore, there is a need for a culture method that secures the number of stem cells that can be used for research or treatment purposes.
한편, 이러한 줄기세포의 기능을 증진시키기 위해서는 성장인자, 합성화합물, 천연물 유래 화합물 등을 처리하여 줄기세포의 기능을 증진시키는 방법들이 존재하나, 약물을 처리하여 체외배양시 세포가 플레이트에 부착할 수 있는 부착능을 증진시키고 세포의 증식능과 기능을 조절시키기에는 한계가 있다. On the other hand, in order to enhance the function of these stem cells, there are methods for enhancing the function of stem cells by treating growth factors, synthetic compounds, compounds derived from natural products, etc. There is a limit to enhancing the adhesion capacity and controlling the proliferation and function of cells.
이러한 문제를 해결하기 위하여, 체외배양시 줄기세포의 부착능과 기능을 증진시킬 수 있는 세포미세환경을 구현하고 이러한 세포미세환경을 모사한 플레이트를 개발하는 것이 중요하게 생각되고 있다. In order to solve this problem, it is considered important to implement a cell microenvironment capable of enhancing the adhesion and function of stem cells during in vitro culture and to develop a plate that mimics the cell microenvironment.
세포 배양기에 세포미세환경을 구현하는 기술로는 여러 가지 방법들이 존재하는데, 그 중 M13 파지 디스플레이는 박테리아에 특이적으로 감염하는 M13 박테리오파지(M13 bacteriophage)를 기반으로 유전자에 다양한 펩타이드(peptide)를 발현하는 재조합 박테리오파지를 이용해 기판위에 배열시키는 기술이다. 이러한 파지 디스플레이 기술의 경우 인체에 무해하며 줄기세포의 기능 증진을 위한 펩타이드 전달 기술을 바탕으로 이미 검증되어 있다.There are several methods for implementing a cell microenvironment in a cell culture medium. Among them, M13 phage display expresses various peptides in genes based on M13 bacteriophage that specifically infects bacteria. It is a technology for arranging on a substrate using recombinant bacteriophages. In the case of such phage display technology, it is harmless to the human body and has already been verified based on peptide delivery technology for enhancing the function of stem cells.
최근 조직재생 및 조직공학분야에서 나오는 연구결과에 따르면, 세포의 증식 및 분화를 조절할 수 있도록 하는 외부적 환경에 해당하는 세포미세환경, 즉,"니쉬(niche)"의 중요성이 대두되고 있는데 이 외부적 니쉬를 모사할 수 있는 생화학적 및 구조적 특징을 가지는 모사환경을 구성하는 것이 중요하게 생각되고 있다.According to recent research results in the field of tissue regeneration and tissue engineering, the importance of the cell microenvironment, that is, the “niche”, which corresponds to the external environment that can control the proliferation and differentiation of cells, is emerging. It is considered important to construct a simulated environment with biochemical and structural characteristics that can simulate the red varnish.
이러한 세포내 환경을 효과적으로 구현하고, 심장 전구 세포의 증식 및 기능을 증진시키는 데파지 디스플레이(Phage display)를 이용할 수 있다.Phage display that effectively implements such an intracellular environment and promotes proliferation and function of cardiac progenitor cells may be used.
단백질과 단백질간의 상호작용(protein-protein interaction)을 탐색하는 기술에는 여러 가지가 있는데, 그 중 파지 디스플레이(phage display)는 박테리아(bacteria)에 특이적으로 감염하는 기생체인 박테리오파지(bacteriophage)의 유전자에 인위적으로 다양한 아미노산 서열을 생산하는 유전자를 도입하여 생산한 재조합 박테리오파지를 이용해 특정 단백질과 결합능력이 있는 미지의 아미노산 서열을 선발하는 기술로써, 항원 결정기의 동정(epitope mapping), 백신 개발, 작용기-수용체 결합능력 확인(ligand-receptor affinity research), 생리활성 펩티드 선발 등 다양한 분야에서 그 활용성이 검증되어 있다. 대표적으로 쓰이는 M13 파지 디스플레이 시스템의 경우에는 M13 박테리오파지의 게놈 중에서 외피 단백질(coat protein)의 일종인 pIII 또는 pVIII를 생산하는 유전자 말단에 7 ~ 15개의 무작위 아미노산 서열의 펩티드가 발현되도록 인위적으로 유전자 서열을 삽입한 후, 대장균(E.coli)에 감염시켜 얻은 수억 종 이상의 서로 다른 펩티드를 발현한 재조합 박테리오파지를 이용해 바이오패닝(biopanning)이라는 일련의 과정을 거쳐 특정 단백질과 강한 결합능력을 보이는 파지를 선발하도록 고안되어 있다. 이렇게 선발된 박테리오파지로부터 게놈 DNA를 추출해 인위적으로 삽입했던 특정 펩티드를 발현하는 DNA 염기서열을 분석하면 목적하는 작용기 펩티드를 얻을 수 있게 된다.There are several techniques for exploring protein-protein interaction, among which phage display is a parasite that specifically infects bacteria, bacteriophage genes. It is a technology that selects an unknown amino acid sequence capable of binding to a specific protein using recombinant bacteriophage produced by artificially introducing a gene that produces various amino acid sequences. Identification of antigenic determinants (epitope mapping), vaccine development, effector-receptor Its utility has been verified in various fields such as ligand-receptor affinity research and selection of bioactive peptides. In the case of the representative M13 phage display system, the gene sequence is artificially expressed so that peptides of 7 to 15 random amino acid sequences are expressed at the end of the gene that produces pIII or pVIII, a kind of coat protein, in the genome of M13 bacteriophage. After insertion, the recombinant bacteriophage expressing more than hundreds of millions of different peptides obtained by infection with E. coli goes through a series of processes called biopanning to select phages showing strong binding ability with specific proteins. has been devised By extracting genomic DNA from the selected bacteriophage and analyzing the DNA sequence expressing the artificially inserted specific peptide, the desired functional peptide can be obtained.
즉, 파지 디스플레이는 파지의 유전자 정보를 이용하여 파지 표면에 특정 외피 단백질이 발현되도록 하고, 상기 파지 표면의 외피 단백질을 이용하여 세포와 인식하게 하는 표면 단백질을 통해 세포의 혈관으로의 증식 및 분화를 유도 및 조절할 수 있게 할 수 있을 뿐만 아니라, 파지의 나노섬유성 구조체의 형성에 의해 생성되는 물리적 및 기계적 니쉬환경을 제공할 수 있게 한다.That is, the phage display uses the genetic information of the phage to express a specific envelope protein on the surface of the phage, and uses the surface protein of the phage surface to recognize the cell and the cell proliferation and differentiation into blood vessels through the surface protein. In addition to being able to induce and control, it is possible to provide a physical and mechanical varnish environment created by the formation of nanofibrous structures of phages.
따라서, 물리적 및 기계적 니쉬 환경을 제공하는 특정 기질 및 재조합 파지가 코팅된 플레이트를 이용하여 목적 줄기세포를 배양함으로써, 특성을 유지하는 줄기세포를 세포치료제로서 대량 공급할 수 있을 것이다.Therefore, by culturing the target stem cells using a plate coated with a specific substrate and recombinant phage that provides a physical and mechanical nish environment, it will be possible to supply a large amount of stem cells maintaining characteristics as a cell therapy agent.
본 발명자는 목적 줄기세포의 생산 효율을 증가시키는 방법을 개발하기 위하여 예의 노력하였다. 그 결과, 인체조직에 무독한 M13 파지의 구조적 특징을 이용하여 재조합 유전자 공학 기법으로 표면에 세포 전달 시그널 펩티드를 발현하는 M13 파지와 젤라틴을 배양 플레이트에 코팅하여 줄기세포를 배양하였을 때, 상기 젤라틴-파지 기반 플레이트에 의해 배양된 줄기세포가 줄기세포 본래의 고유한 성질은 유지하면서 부착능, 증식능, 이동능 및 혈관생성능이 탁월하게 증진되었음을 규명함으로써 본 발명을 완성하였다.The present inventors made diligent efforts to develop a method for increasing the production efficiency of target stem cells. As a result, when stem cells were cultured by coating a culture plate with M13 phage expressing cell transduction signal peptides and gelatin on the surface by recombinant genetic engineering techniques using the structural features of M13 phage that are non-toxic to human tissues, the gelatin- The present invention was completed by identifying that stem cells cultured by a phage-based plate had excellent adhesion, proliferation, migration and angiogenesis while maintaining the original properties of stem cells.
따라서, 본 발명의 목적은 젤라틴; 및 외피 단백질(coat protein)에 세포 전달 펩티드를 디스플레이(display)한 재조합 파지(Recombinant Phage);가 코팅된 줄기세포 배양용 재조합 파지-기반 용기를 제공하는 데 있다.Accordingly, an object of the present invention is gelatin; And to provide a recombinant phage-based container for culturing stem cells coated with a recombinant phage in which a cell delivery peptide is displayed on a coat protein.
또한, 본 발명의 다른 목적은 상기 줄기세포 배양용 재조합 파지-기반 용기의 제작 방법을 제공하는 데 있다.In addition, another object of the present invention is to provide a method for manufacturing a recombinant phage-based vessel for culturing the stem cells.
또한, 본 발명의 또 다른 목적은 상기 줄기세포 배양용 재조합 파지-기반 용기에 줄기세포를 배양하는 단계를 포함하는, 줄기세포의 부착능, 증식능, 이동능 및 혈관생성능을 향상시키는 방법을 제공하는 데 있다.In addition, another object of the present invention is to provide a method for improving the adhesion, proliferation, migration and angiogenesis of stem cells, comprising the step of culturing the stem cells in the recombinant phage-based vessel for culturing the stem cells. there is
또한, 본 발명의 또 다른 목적은 상기 줄기세포 배양용 재조합 파지-기반 용기에 줄기세포를 배양하는 단계를 포함하는, 줄기세포 생산 방법을 제공하는 데 있다.In addition, another object of the present invention is to provide a method for producing stem cells, including the step of culturing the stem cells in a recombinant phage-based vessel for culturing the stem cells.
본 명세서에서 사용한 용어는 단지 설명을 목적으로 사용된 것으로, 한정하려는 의도로 해석되어서는 안된다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.The terminology used herein is used for the purpose of description only, and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to designate that a feature, number, step, operation, component, part, or a combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 실시예가 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not
이하, 본 발명에 대하여 보다 상세히 설명한다. Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태에 따르면, 본 발명은 젤라틴(Gelatin); 및 외피 단백질(coat protein)에 세포 전달 펩티드를 디스플레이(display)한 재조합 파지(Recombinant Phage);가 코팅된, 줄기세포 배양용 재조합 파지-기반 용기를 제공한다.According to one aspect of the present invention, the present invention is gelatin (Gelatin); And it provides a recombinant phage (Recombinant Phage) for displaying a cell delivery peptide on the coat protein; a recombinant phage-based container for culturing stem cells is coated.
본 발명의 줄기세포 배양용 재조합 파지-기반 용기의 젤라틴은 용기 표면에 코팅되어 목적 줄기세포의 체외배양시 기능을 증진시킬 수 있는 세포미세환경을 구현하는 데 이용된다.The gelatin of the recombinant phage-based container for stem cell culture of the present invention is coated on the surface of the container and used to implement a cell microenvironment capable of enhancing the function of the target stem cell in vitro.
본 발명에서 상기 젤라틴(Gelatin) 농도는, 본 발명에서 목적 효과를 달성하는 한, 제한되지 않으나, 바람직하게는 0.01 내지 10 중량%이고, 보다 바람직하게는 0.1 내지 5 중량%이며, 가장 바람직하게는 1 중량%이다.In the present invention, the concentration of gelatin is not limited as long as the desired effect is achieved in the present invention, but is preferably 0.01 to 10% by weight, more preferably 0.1 to 5% by weight, and most preferably 1% by weight.
본 발명에서 사용되는 용어 "줄기세포 배양용 재조합 파지-기반 용기"는 젤라틴(Gelatin)을 코팅한 후, 외피 단백질(coat protein)에 세포 전달 펩티드를 디스플레이(display)한 재조합 파지(Recombinant Phage)를 코팅시킨 세포 배양 용기를 의미하며, 본 명세서에서 "파지-기반 배양(용) 플레이트"와 혼용하여 사용된다.The term "recombinant phage-based container for stem cell culture" used in the present invention refers to a recombinant phage in which a cell delivery peptide is displayed on a coat protein after coating with gelatin. It refers to a coated cell culture vessel, and is used interchangeably with “phage-based culture (for) plate” herein.
상기 용기는 세포 배양 플레이트, 페트리 디쉬, 배양 플라스크, 챔버 슬라이드, 챔버 또는 튜브 형태일 수 있으며, 이에 제한되지 않으나, 본 발명의 일 실시예에서는 플레이트를 이용하였다.The vessel may be in the form of a cell culture plate, a Petri dish, a culture flask, a chamber slide, a chamber, or a tube, but is not limited thereto, but in an embodiment of the present invention, a plate is used.
또한, 상기 세포 배양 용기는 유리, 폴리스티렌, 폴리카프로락톤 및 폴리프로필렌으로 이루어진 군에서 선택된 1 이상의 소재일 수 있으나, 타겟 세포가 부착되기에 적합한 소재라면 제한없이 사용할 수 있다.In addition, the cell culture vessel may be at least one material selected from the group consisting of glass, polystyrene, polycaprolactone and polypropylene, but any material suitable for attachment of target cells may be used without limitation.
본 발명에서 사용되는 용어 "세포 전달 펩티드"는 "시그널 펩티드" 및 "시그널 서열"과 상호교환적으로 사용되며, 세포부착 모티프에 해당되는 것이다. 따라서, 세포부착 아미노산 서열을 필수 서열로 포함하는 보다 긴 아미노산 서열도 본 발명의 범위에 포함된다.As used herein, the term "cell transduction peptide" is used interchangeably with "signal peptide" and "signal sequence", and corresponds to a cell adhesion motif. Accordingly, longer amino acid sequences including the cell adhesion amino acid sequence as an essential sequence are also included in the scope of the present invention.
본 발명의 바람직한 구현예에 따르면, 본 발명의 세포 전달 펩티드는 RGD(Arg-Gly-Asp), RGDS(Arg-Gly-Asp-Ser), RGDC(Arg-Gly-Asp-Cys), RGDV(Arg-Gly-Asp-Val), RGES(Arg-Gly-Glu-Ser), RGDSPASSKP(Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro), GRGDS(Gly-Arg-Gly-Asp-Ser), GRADSP(Gly-Arg-Ala-Asp-Ser-Pro), KGDS(Lys-Gly-Asp-Ser), GRGDSP(Gly-Arg-Gly-Asp-Ser-Pro), GRGDTP(Gly-Arg-Gly-Asp-Thr-Pro), GRGES(Gly-Arg-Gly-Glu-Ser), GRGDSPC(Gly-Arg-Gly-Asp-Ser-Pro-Cys), GRGESP(Gly-Arg-Gly-Glu-Ser-Pro), SDGR(Ser-Asp-Gly-Arg), YRGDS(Tyr-Arg-Gly-Asp-Ser), GQQHHLGGAKQAGDV (Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val), GPR(Gly-Pro-Arg), GHK(Gly-His-Lys), YIGSR(Tyr-Ile-Gly-Ser-Arg), PDSGR(Pro-Asp-Ser-Gly-Arg), CDPGYIGSR(Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg), LCFR(Leu-Cys-Phe-Arg), EIL(Glu-Ile-Leu), EILDV(Glu-Ile-Leu-Asp-Val), EILDVPST(Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr), EILEVPST(Glu-Ile-Leu-Glu-Val-Pro-Ser-Thr), LDV(Leu-Asp-Val) 및 LDVPS(Leu-Asp-Val-Pro-Ser)로 이루어진 군으로부터 선택되고, 보다 바람직하게는 RGD(Arg-Gly-Asp), RGDS(Arg-Gly-Asp-Ser), RGDC(Arg-Gly-Asp-Cys), RGDV(Arg-Gly-Asp-Val)및 RGES(Arg-Gly-Glu-Ser)로 이루어진 군으로부터 선택되며, 가장 바람직하게는 RGD(Arg-Gly-Asp)이다.According to a preferred embodiment of the present invention, the cell transfer peptide of the present invention is RGD (Arg-Gly-Asp), RGDS (Arg-Gly-Asp-Ser), RGDC (Arg-Gly-Asp-Cys), RGDV (Arg -Gly-Asp-Val), RGES (Arg-Gly-Glu-Ser), RGDSPASSKP (Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro), GRGDS (Gly-Arg-Gly- Asp-Ser), GRADSP (Gly-Arg-Ala-Asp-Ser-Pro), KGDS (Lys-Gly-Asp-Ser), GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), GRGDTP (Gly- Arg-Gly-Asp-Thr-Pro), GRGES (Gly-Arg-Gly-Glu-Ser), GRGDSPC (Gly-Arg-Gly-Asp-Ser-Pro-Cys), GRGESP (Gly-Arg-Gly-Glu) -Ser-Pro), SDGR (Ser-Asp-Gly-Arg), YRGDS (Tyr-Arg-Gly-Asp-Ser), GQQHHLGGAKQAGDV (Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala- Lys-Gln-Ala-Gly-Asp-Val), GPR (Gly-Pro-Arg), GHK (Gly-His-Lys), YIGSR (Tyr-Ile-Gly-Ser-Arg), PDSGR (Pro-Asp- Ser-Gly-Arg), CDPGYIGSR (Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg), LCFR (Leu-Cys-Phe-Arg), EIL (Glu-Ile-Leu), EILDV ( Glu-Ile-Leu-Asp-Val), EILDVPST (Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr), EILEVPST (Glu-Ile-Leu-Glu-Val-Pro-Ser-Thr), LDV (Leu-Asp-Val) and LDVPS (Leu-Asp-Val-Pro-Ser), more preferably RGD (Arg-Gly-Asp), RGDS (Arg-Gly-Asp-Ser) , RGDC(Arg-G ly-Asp-Cys), RGDV (Arg-Gly-Asp-Val) and RGES (Arg-Gly-Glu-Ser), most preferably RGD (Arg-Gly-Asp).
상기 "Arg-Gly-Asp 펩티드" 또는 "RGD 펩티드"는 "수용체의 Arg-Gly-Asp 군", 예를 들면, 인테그린의 수용체에 대한 결합 부위로서 기능할 수 있는 서열을 함유하는 하나 이상의 Arg-Gly-Asp를 갖는 펩티드를 지칭하는 것으로 된다. 또한, RGD 펩티드는 동일한 RGD 수용체와 상호 작용하는 경우 RGD 펩티드의 기능성 동등물인 아미노산을 갖는 펩티드를 포함한다.Said "Arg-Gly-Asp peptide" or "RGD peptide" refers to one or more Arg- containing sequences capable of serving as a binding site for the "Arg-Gly-Asp family of receptors", eg, integrins, for receptors. It is intended to refer to a peptide having Gly-Asp. RGD peptides also include peptides having amino acids that are functional equivalents of RGD peptides when interacting with the same RGD receptor.
본 발명의 RGD 서열을 함유하는 펩티드는 당해 분야에서 익히 공지된 수단에 의해 아미노산으로부터 합성될 수 있다.Peptides containing the RGD sequence of the present invention can be synthesized from amino acids by means well known in the art.
본 명세서에서 사용되는 용어 "파지(phage)" 또는 "박테리오파지(bacteriophage)"는 세균을 감염시키는 바이러스의 일종으로 파지로 부르기도 한다. 파지는 핵산으로 이루어진 유전물질 중심부를 단백질 외피가 싸고 있는 단순한 구조의 유기체이며 핵산은 단일 사슬이거나 이중 사슬인 DNA 또는 RNA로 되어있다. 이 핵산을 단백질 외피가 싸고 있는 단순한 구조로 20면체 머리에 꼬리가 있는 형태, 20면체 머리에 꼬리가 없는 형태, 및 필라멘트형의 3가지 기본형 구조로 나뉜다. 가장 흔한 형태인 20면체 머리에 꼬리가 있는 형태의 박테리오파지는 꼬리 특성에 따라 수축성 꼬리를 가지는 미오오비리데(Myoviridae), 길고 수축성이 없는 꼬리를 갖는 시포비리데(Siphoviridae), 및 짧은 꼬리를 갖는 포도비리데(Podoviridae)로 세분화될 수 있다. 20면체 머리에 꼬리가 없는 박테리오파지는 머리의 형태, 머리의 구성성분 및 외피의 유무에 따라 세분화된다. 마지막으로, 필라멘트 형태의 박테리오파지는 크기, 모양, 외피 및 필라멘트 구성성분에 따라 세분화된다.As used herein, the term “phage” or “bacteriophage” is a kind of virus that infects bacteria and is also called phage. A phage is an organism with a simple structure in which a protein coat surrounds the center of a genetic material composed of nucleic acids. Nucleic acids are composed of single-stranded or double-stranded DNA or RNA. This nucleic acid has a simple structure surrounded by a protein coat, and is divided into three basic structures: an icosahedral head with a tail, an icosahedral head without a tail, and a filamentous type. The most common icosahedral head-tailed bacteriophages are Myoviridae with a contractile tail, Siphoviridae with a long, non-contractile tail, and grapes with a short tail depending on the tail characteristics. It can be subdivided into Podoviridae. Bacteriophages without an icosahedral head and no tail are subdivided according to the shape of the head, the composition of the head, and the presence or absence of an integument. Finally, filamentous bacteriophages are subdivided according to size, shape, integument and filament composition.
상기 파지는 그 종류를 제한하지 않으며, T1,T2, T4, T6 또는 λ(람다), μ(뮤), M13 등일 수 있고, 본 발명에서는 M13을 이용한다.The type of the phage is not limited, and may be T1, T2, T4, T6 or λ (lambda), μ (mu), M13, and the like, and M13 is used in the present invention.
본 발명에서 이용하는 재조합 파지는 M13 파지의 게놈 중에서 외피 단백질을 생산하는 유전자에 목적하는 3 내지 15개의 아미노산 서열의 펩티드가 발현되도록 인위적으로 유전자 서열을 삽입하여 목적의 펩티드를 발현한다.The recombinant phage used in the present invention expresses the target peptide by artificially inserting the gene sequence so that the target peptide of 3 to 15 amino acid sequence is expressed in the gene for producing the envelope protein in the genome of the M13 phage.
상기 파지는 다양한 외피 단백질(coat protein)로 구성되어 있으며, 예를 들면 P3(PIII), P6(PVI), P7(VII), P8(VIII), P9(IX) 등으로 이루어질 수 있다.The phage is composed of various coat proteins, and may be composed of, for example, P3(PIII), P6(PVI), P7(VII), P8(VIII), P9(IX), and the like.
본 발명의 바람직한 구현예에 따르면, 상기 외피 단백질은 P3, P6, P7, P8 및 P9로 이루어진 군에서 선택된 하나 이상이다.According to a preferred embodiment of the present invention, the envelope protein is at least one selected from the group consisting of P3, P6, P7, P8 and P9.
본 발명에서, 상기 재조합 파지는 P3, P8 및 P9로 이루어진 군으로부터 1종이상 선택된 단백질에 펩티드를 포함한다.In the present invention, the recombinant phage includes a peptide in one or more proteins selected from the group consisting of P3, P8 and P9.
또한, 본 발명에서 이용되는 재조합 파지는 주외피 단백질(major coat protein)인 P8에 RGD를 포함하는 5 내지 10 개의 아미노산 서열로 이루어진 세포 전달 펩티드를 포함할 수 있으며, 가장 바람직하게는 RGD를 포함한다.In addition, the recombinant phage used in the present invention may include a cell delivery peptide consisting of 5 to 10 amino acid sequences including RGD in P8, a major coat protein, and most preferably includes RGD. .
즉, 상기 재조합 파지는 세포친화성을 가지는 RGD가 주외피 단백질(major coat protein) P8에서 발현되며, 서열번호 1의 염기서열로 구성되는 유전체를 갖는다. That is, in the recombinant phage, RGD having cell affinity is expressed in the major coat protein P8, and has a genome composed of the nucleotide sequence of SEQ ID NO: 1.
본 발명에서 상기 재조합 파지(Recombinant Phage)의 농도는, 본 발명에서 목적 효과를 달성하는 한, 제한되지 않으나, 바람직하게는 0.001 mg/ml 내지 10 mg/ml이고, 보다 바람직하게는 0.05 mg/ml 내지 0.5 mg/ml이며, 가장 바람직하게는 0.1 mg/ml 농도이다.In the present invention, the concentration of the recombinant phage (Recombinant Phage) is not limited as long as it achieves the desired effect in the present invention, but is preferably 0.001 mg/ml to 10 mg/ml, more preferably 0.05 mg/ml to 0.5 mg/ml, most preferably 0.1 mg/ml concentration.
또한, 본 발명의 바람직한 구현예에 따르면, 본 발명의 줄기세포 배양용 플레이트에 코팅된 재조합 파지에 의해 배양되는 줄기세포는 본래의 고유한 성질은 유지하면서 부착능, 증식능, 이동능 및 혈관생성능(혈관형성능)이 탁월하게 향상된다.In addition, according to a preferred embodiment of the present invention, the stem cells cultured by the recombinant phage coated on the plate for stem cell culture of the present invention maintain adhesion, proliferation, migration and angiogenesis ( angiogenesis) is significantly improved.
바람직하게는, 상기 줄기세포는 EPCs(Endothelial Progenitor Cells), 심장 전구 세포(cardiac progenitor cell, CPC), ECFCs(Endothelial Colony Forming Cells), VPCs(Vasculogenic Progenitor Cells), 중간엽 줄기세포(Mesenchymal Stem Cells), 배아줄기세포(Embryonic Stem Cells) 및 근모세포(Myoblasts)로 이루어진 군으로부터 선택되며, 보다 바람직하게는 CPC (cardiac progenitor cell), EPCs(Endothelial Progenitor Cells), ECFCs(Endothelial Colony Forming Cells) 및 VPCs(Vasculogenic Progenitor Cells)로 이루어진 군으로부터 선택되고, 가장 바람직하게는 EPCs(Endothelial Progenitor Cells)이다.Preferably, the stem cells are EPCs (Endothelial Progenitor Cells), cardiac progenitor cells (CPC), ECFCs (Endothelial Colony Forming Cells), VPCs (Vasculogenic Progenitor Cells), mesenchymal stem cells (Mesenchymal Stem Cells) , is selected from the group consisting of embryonic stem cells (Embryonic Stem Cells) and myoblasts (Myoblasts), more preferably CPC (cardiac progenitor cell), EPCs (Endothelial Progenitor Cells), ECFCs (Endothelial Colony Forming Cells) and VPCs ( Vasculogenic Progenitor Cells), most preferably EPCs (Endothelial Progenitor Cells).
상기 줄기세포 배양용 재조합 파지-기반 용기는 피더세포(feeder cell)를 포함하지 않는 것을 특징으로 한다.The recombinant phage-based container for stem cell culture is characterized in that it does not contain feeder cells.
즉, 본 발명의 줄기세포 배양용 재조합 파지-기반 용기는, 피더세포(feeder cell) 없이, 젤라틴을 코팅하고, 파지 표면에 특정 외피 단백질이 발현되도록 재조합한 파지를 이용함으로써, 상기 파지 표면의 외피 단백질을 이용하여 타겟 세포인 줄기세포(바람직하게는, EPC)와 인식하게 하는 표면 단백질을 통해 세포의 기능을 향상시킬 수 있도록 할 뿐만 아니라, 파지의 나노섬유성 구조체의 형성에 의해 생성되는 물리적 및 기계적 니쉬 환경을 제공하여 혈관내피 전구세포의 줄기세포 본래의 고유한 성질은 유지하면서 부착능, 증식능, 이동능 및 혈관생성능을 촉진시킬 수 있도록 한다.That is, the recombinant phage-based container for stem cell culture of the present invention uses phage recombined so that a specific envelope protein is expressed on the surface of the phage, coated with gelatin, without a feeder cell. By using the protein, not only can the function of the cell be improved through the surface protein that recognizes the target cell, the stem cell (preferably, EPC), but also the physical and By providing a mechanical varnish environment, it is possible to promote adhesion, proliferation, migration and angiogenesis while maintaining the intrinsic properties of stem cells of endothelial progenitors.
따라서, 본 발명은 체외에서 경제적인 비용으로 상술한 목적 줄기세포의 생리학적 활성을 증가시킬 뿐만 아니라, 물리적인 성질을 부여함으로써 목적 줄기세포의 생산 효율을 증가시킴으로써 대량의 줄기세포를 배양할 수 있는 줄기세포 배양용 플레이트라는 이점을 제공한다.Therefore, the present invention can culture a large amount of stem cells by increasing the production efficiency of the target stem cells by giving physical properties as well as increasing the physiological activity of the above-described target stem cells at an economical cost in vitro. It provides the advantage of being a plate for stem cell culture.
또한, 본 발명의 다른 양태에 따르면, 본 발명은 젤라틴(Gelatin); 및 외피 단백질(coat protein)에 세포 전달 펩티드를 디스플레이(display)한 재조합 파지(Recombinant Phage);를, 세포 배양 용기에 코팅시키는 단계를 포함하는 줄기세포 배양용 재조합 파지-기반 용기의 제작 방법을 제공한다.In addition, according to another aspect of the present invention, the present invention is gelatin (Gelatin); and a recombinant phage displaying a cell delivery peptide on a coat protein; and coating a cell culture vessel with a recombinant phage-based vessel for stem cell culture. do.
본 발명의 방법은 상술한 젤라틴 및 재조합 파지를 이용하여 줄기세포 배양용 재조합 파지-기반 용기를 제작하는 것이므로, 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.Since the method of the present invention prepares a recombinant phage-based container for stem cell culture using the above-described gelatin and recombinant phage, the description of common content is omitted to avoid excessive complexity of the present specification due to repeated description.
또한, 본 발명은 상술한 줄기세포 배양용 재조합 파지-기반 용기를 이용하여 줄기세포를 배양하는 방법을 제공한다.In addition, the present invention provides a method for culturing stem cells using the above-described recombinant phage-based vessel for culturing stem cells.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은 상기 줄기세포 배양용 재조합 파지-기반 용기에 줄기세포를 배양하는 단계를 포함하는, 줄기세포의 부착능, 증식능, 이동능 및 혈관생성능을 향상시키는 방법을 제공한다.In addition, according to another aspect of the present invention, the present invention improves the adhesion, proliferation, migration and angiogenesis of stem cells, comprising the step of culturing the stem cells in the recombinant phage-based vessel for culturing the stem cells. provides a way to do it.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은 상기 줄기세포 배양용 재조합 파지-기반 용기에 줄기세포를 배양하는 단계를 포함하는, 줄기세포 생산 방법을 제공하며, 상기 줄기세포는 부착능, 증식능, 이동능 및 혈관생성능이 향상된 줄기세포인 것을 특징으로 한다.In addition, according to another aspect of the present invention, the present invention provides a method for producing stem cells, comprising the step of culturing the stem cells in a recombinant phage-based vessel for culturing the stem cells, wherein the stem cells are capable of adhesion, It is characterized in that it is a stem cell with improved proliferation, migration and angiogenesis.
상기 배양된 줄기세포, 바람직하게는 혈관내피 전구세포는, 세포 본래의 고유한 성질은 유지하면서 부착능, 증식능, 이동능 및 혈관생성능이 향상되므로, 이를 목적 질환의 치료 용도로 적용할 수 있다.The cultured stem cells, preferably vascular endothelial progenitor cells, have improved adhesion, proliferation, migration and angiogenesis while maintaining the intrinsic properties of the cells, and thus can be applied for treatment of target diseases.
본 발명의 줄기세포 배양용 재조합 파지-기반 용기는 파지의 나노섬유성 구조체의 형성에 의해 생성되는 물리적 및 기계적 니쉬 환경을 제공하여 타겟 세포의 생리학적 활성을 증진시키므로, 궁극적으로 특정 조직 재생을 도와 탁월한 대상 질환의 치료제를 제조하는 데 응용할 수 있고, 이를 통해 기능이 향상된 줄기세포 치료제로서 효과적으로 생산할 수 있다.The recombinant phage-based container for stem cell culture of the present invention provides a physical and mechanical nish environment generated by the formation of a nanofibrous structure of phage to enhance the physiological activity of target cells, and thus ultimately helps specific tissue regeneration It can be applied to the manufacture of a therapeutic agent for an excellent target disease, and through this, it can be effectively produced as a stem cell therapeutic agent with improved function.
도 1은 야생형 파지(Wild type M13)(상단); 및 파지의 외피부분인 P8 부위에 RGD 서열이 포함된 재조합 파지(RGD-M13)(하단)의 시퀀싱(sequencing) 결과를 나타낸다.
도 2a는 본 발명의 파지-기반 배양용 플레이트의 제작 과정을 개략적으로 보여준다. 도 2b는 본 발명의 파지-기반 배양용 플레이트에 적용하기 위한 농도별 RGD-M13 처리시 세포 배양 결과를 보여준다.
도 3a은 본 발명의 파지-기반 배양용 플레이트에서 배양한 혈관내피 전구세포(EPC)의 증식능을 확인한 CCK8 assay 결과를 보여준다. 도 3b는 본 발명의 파지-기반 배양용 플레이트에서 배양한 혈관내피 전구세포(EPC)의 형태를 보여준다.
도 4a는 본 발명의 파지-기반 배양용 플레이트에서 배양한 혈관내피 전구세포(EPC)의 이동능 및 혈관형성능을 확인한 migration assay 결과를 보여준다. 도 4b는 상기 결과를 정량화하여 나타낸 그래프이다.
도 5a는 본 발명의 파지-기반 배양용 플레이트에서 배양한 혈관내피 전구세포(EPC)의 혈관형성능을 확인한 tube-formation assay 결과를 보여준다. 도 5b는 상기 결과를 정량화한 그래프이다.1 is a wild type phage (Wild type M13) (top); and a sequencing result of a recombinant phage (RGD-M13) (bottom) in which the RGD sequence was included in the P8 region, which is the outer part of the phage.
Figure 2a schematically shows the manufacturing process of the plate for phage-based culture of the present invention. Figure 2b shows the cell culture results upon treatment with RGD-M13 for each concentration for application to the phage-based culture plate of the present invention.
Figure 3a shows the results of CCK8 assay confirming the proliferative ability of vascular endothelial progenitor cells (EPC) cultured in the phage-based culture plate of the present invention. Figure 3b shows the morphology of vascular endothelial progenitor cells (EPC) cultured in the phage-based culture plate of the present invention.
Figure 4a shows the migration assay results confirming the migration and angiogenesis of vascular endothelial progenitor cells (EPC) cultured in the phage-based culture plate of the present invention. 4B is a graph showing the quantification of the above results.
5a shows the results of a tube-formation assay confirming the angiogenesis performance of vascular endothelial progenitor cells (EPC) cultured in the phage-based culture plate of the present invention. 5B is a graph quantifying the results.
이하, 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the examples are only for explaining the present invention in more detail, and those of ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these examples according to the gist of the present invention It will be self-evident for
실시예 1. 파지-기반 배양 플레이트의 제작 및 배양Example 1. Fabrication and Culture of Phage-Based Culture Plates
본 발명자들은 혈관 질환 치료에 이용할 수 있는 생리학적 활성이 향상된 줄기세포를 획득 및 제공하기 위해, 기능적인 M13 파지(phage)-기반 줄기세포 배양용 플레이트를 하기와 같이 제작하였다.The present inventors prepared a functional M13 phage-based stem cell culture plate as follows to obtain and provide stem cells with improved physiological activity that can be used for the treatment of vascular diseases.
간략하게는, 도 2a에 나타낸 바와 같이, 젤라틴(Gelatine/1g)을 PBS 100ml에 녹인 후 고압멸균(autoclave)하여 제조한 1% 젤라틴 용액을 사용하였다. 1% 젤라틴을 1 시간 동안 1차 코팅하고 PBS로 세척한 후, 특정 농도로 PBS에 현탁한 M13 박테리오파지로 2차 코팅하여 파지-기반 배양용 플레이트를 제작하였다. 상기 젤라틴-파지 기반 플레이트에 EPC를 배양하여 실험을 진행하였다.Briefly, as shown in FIG. 2a, a 1% gelatin solution prepared by dissolving gelatin (Gelatine/1g) in 100ml of PBS and then autoclaving was used. 1% gelatin was first coated for 1 hour, washed with PBS, and then secondary coated with M13 bacteriophage suspended in PBS at a specific concentration to prepare a phage-based culture plate. The experiment was carried out by culturing EPC on the gelatin-phage-based plate.
이때, M13 파지(New England Biolabs)는 유전자 재조합 기법을 바탕으로 주외피 단백질인 p8 부위에 세포친화성을 가지는 RGD를 발현시킨 RGD- M13 파지(서열번호 1; MLSFFAGGRGDSDDYDPAK) 및 야생형 파지(서열번호 2; MLSFAAEGDDPAKAAFN)를 이용하였다(도 1).At this time, M13 phage (New England Biolabs) is RGD-M13 phage (SEQ ID NO: 1; MLSFFAGGRGDSDDYDPAK) and wild-type phage (SEQ ID NO: 2) expressing RGD having cell affinity in the p8 region, the main envelope protein, based on the genetic recombination technique ; MLSFAAEGDDPAKAAFN) was used (FIG. 1).
또한, 가장 최적의 RGD-M13 파지 농도를 확인하기 위하여, 농도별(0.05 mg/ml, 0.1 mg/ml, 0.25 mg/ml 및 0.5 mg/ml)로 파지만을 코팅한 플레이트에 EPC를 배양하여 세포 증식 수준을 관찰하였다.In addition, in order to determine the most optimal RGD-M13 phage concentration, EPC was cultured on a plate coated with only phage at each concentration (0.05 mg/ml, 0.1 mg/ml, 0.25 mg/ml and 0.5 mg/ml). The level of cell proliferation was observed.
그 결과, 도 2b에 나타낸 바와 같이, 0.1 mg/ml 내지 0.25 mg/ml 농도일 때가 가장 효과가 좋은 것으로 나타났다. As a result, as shown in Figure 2b, it was found that the most effective when 0.1 mg / ml to 0.25 mg / ml concentration.
이에, RGD-M13 파지의 유효 농도로서 0.1 mg/ml을 지정하여 하기 실시예들에 적용하였다.Accordingly, 0.1 mg/ml was specified as an effective concentration of RGD-M13 phage and applied to the following Examples.
실시예 2. 본 발명의 재조합 파지-기반 플레이트에 의한 줄기세포 활성 증진 확인Example 2. Recombinant phage-based plate of the present invention to confirm the enhancement of stem cell activity
본 발명자들은 상기 실시예 1에서 구축된 재조합 파지-기반 플레이트 배양 시스템에 의해 획득된 줄기세포의 생리학적 기능 활성 수준을 in vitro에서 확인하였다. The present inventors confirmed in vitro the physiological and functional activity level of stem cells obtained by the recombinant phage-based plate culture system constructed in Example 1 above.
2-1. 본 발명의 재조합 파지-기반 플레이트에 의한 줄기세포의 증식능 증가2-1. Increased proliferative capacity of stem cells by the recombinant phage-based plate of the present invention
본 발명의 재조합 파지-기반 플레이트 배양 시스템에 의해 향상된 줄기세포의 기능 평가를 위해, CCK8 assay 를 실시하여, 재조합 파지-기반 플레이트 배양 시스템에 의해 배양된, 혈관내피 전구세포(EPC; Endothelial Progenitor Cells)의 증식능을 확인하였다. In order to evaluate the function of stem cells improved by the recombinant phage-based plate culture system of the present invention, CCK8 assay was performed, and the recombinant phage-based plate culture system cultured vascular endothelial progenitor cells (EPC; Endothelial Progenitor Cells) The proliferation ability was confirmed.
간략하게는, 상기 실시예 1에서 제작한 재조합 파지-기반플레이트에, 5,000개의 혈관내피 전구세포를 37℃, 5% CO2 환경에서 하루 동안 배양하고, CCK8 kit 용액과 세포배양액이 1:10 비율로 섞인 배지로 교환하였다. 37℃, 5% CO2 환경에서 1시간 반응 시킨 후 450nm에서 흡광도를 측정하여 CCK의 활성을 측정하였고, 아무것도 처리하지 않은 대조군(Control)에 대비한 세포 증식능력을 확인하였다. Briefly, on the recombinant phage-based plate prepared in Example 1, 5,000 vascular endothelial progenitor cells were cultured for one day at 37° C. and 5% CO 2 environment, and the CCK8 kit solution and the cell culture medium were mixed in a 1:10 ratio. was replaced with a medium mixed with After reacting for 1 hour at 37 ° C., 5% CO 2 environment, the absorbance was measured at 450 nm to measure the activity of CCK, and the cell proliferation ability compared to the untreated control (Control) was confirmed.
이때, 파지 농도는 0.1 mg/ml로 이용하였고, 대조군으로는 아무것도 코팅하지 않은 플레이트(Non coating), 젤라틴만을 단독 코팅한 플레이트(Gelatin), 야생형 파지만을 단독 코팅한 플레이트(Wild type), 젤라틴 및 야생형 파지를 코팅한 플레이트(Gelatin+ Wild) 및 RGD-M13 재조합 파지만을 단독 코팅한 플레이트(RGD)에 배양하여 획득한 세포를 이용하여 비교하였다.At this time, the phage concentration was used as 0.1 mg/ml, and as a control, a plate coated with nothing (Non coating), a plate coated with only gelatin (Gelatin), a plate coated with only wild-type phage (Wild type), and gelatin and cells obtained by culturing a plate coated with wild-type phage (Gelatin+ Wild) and a plate coated with only RGD-M13 recombinant phage (RGD) were used for comparison.
그 결과, 도 3a 및 3b에 나타낸 바와 같이, 대조군들과 비교하였을 때, 젤라틴 및 RGD-M13 재조합 파지가 코팅된 플레이트(Gelatin+RGD)에서 배양한 EPC의 증식능이 유의하게 증가하는 것을 확인하였다. As a result, as shown in FIGS. 3A and 3B , it was confirmed that the proliferative capacity of EPCs cultured on a plate (Gelatin+RGD) coated with gelatin and RGD-M13 recombinant phage was significantly increased compared to the control group.
2-2. 본 발명의 재조합 파지-기반 플레이트에 의한 줄기세포의 이동능 증가2-2. Recombinant phage-based plate of the present invention to increase the mobility of stem cells
본 발명의 재조합 파지-기반 플레이트 배양 시스템에 의해 향상된 줄기세포의 기능 평가를 위해, transwell migration assay를 실시하여, 재조합 파지-기반 플레이트 배양 시스템에 의해 배양된 혈관내피 전구세포(EPC)의 이동능을 확인하였다. In order to evaluate the function of stem cells improved by the recombinant phage-based plate culture system of the present invention, transwell migration assay was performed to determine the migration ability of vascular endothelial progenitor cells (EPC) cultured by the recombinant phage-based plate culture system. Confirmed.
간략하게는, 상기 실시예 1에서 제작한 재조합 파지-기반 플레이트에, 5,000개의 혈관내피 전구세포를 37℃, 5% CO2 환경에서 24 시간 동안 배양한 후, 이동능을 분석하였다. 재조합 파지-기반 플레이트 배양시스템에서 배양한 혈관내피 전구세포를 trans-well 위에 안착시킨 후 이동하는 세포의 수를 확인하였다. Briefly, on the recombinant phage-based plate prepared in Example 1, 5,000 vascular endothelial progenitor cells were cultured at 37° C., 5% CO 2 environment for 24 hours, and then their migration ability was analyzed. After the vascular endothelial progenitor cells cultured in the recombinant phage-based plate culture system were seated on the trans-well, the number of migrating cells was confirmed.
이때, 파지 농도는 0.1 mg/ml로 이용하였고, 대조군으로는 아무것도 코팅하지 않은 플레이트(Non coating), 젤라틴만을 단독 코팅한 플레이트(Gelatin), 야생형 파지만을 단독 코팅한 플레이트(Wild type), 젤라틴 및 야생형 파지를 코팅한 플레이트(Gelatin+ Wild) 및 RGD-M13 재조합 파지만을 단독 코팅한 플레이트(RGD)에 배양하여 획득한 세포를 이용하여 비교하였다.At this time, the phage concentration was used as 0.1 mg/ml, and as a control, a plate coated with nothing (Non coating), a plate coated with only gelatin (Gelatin), a plate coated with only wild-type phage (Wild type), and gelatin and cells obtained by culturing a plate coated with wild-type phage (Gelatin+ Wild) and a plate coated with only RGD-M13 recombinant phage (RGD) were used for comparison.
그 결과, 도 4a 및 도 4b에 나타낸 바와 같이, 대조군들과 비교하였을 때, 젤라틴 및 RGD-M13 재조합 파지가 코팅된 플레이트(Gelatin+RGD)에서 배양한 EPC의 이동능이 유의하게 증진되는 것을 확인하였다. As a result, as shown in FIGS. 4a and 4b, it was confirmed that the migration ability of EPC cultured on a plate (Gelatin+RGD) coated with gelatin and RGD-M13 recombinant phage was significantly improved compared to the control group. .
2-3. 본 발명의 재조합 파지-기반 플레이트에 의한 줄기세포의 혈관형성능 증가2-3. Increased angiogenic ability of stem cells by the recombinant phage-based plate of the present invention
본 발명의 재조합 파지-기반 플레이트 배양 시스템에 의해 향상된 줄기세포의 기능 평가를 위해 tube-formation assay를 실시하여, 재조합 파지-기반 플레이트배양 시스템에 의해 배양된 혈관내피 전구세포(EPC)의 혈관형성능을 확인하였다. The tube-formation assay was performed to evaluate the function of stem cells improved by the recombinant phage-based plate culture system of the present invention, and the angiogenesis performance of vascular endothelial progenitor cells (EPC) cultured by the recombinant phage-based plate culture system was evaluated. Confirmed.
간략하게는, 상기 실시예 1에서 제작한 재조합 파지-기반 플레이트에, 5,000개의 혈관내피 전구세포를 37℃, 5% CO2 환경에서 24 시간 동안 배양한 후, 96well plate에 각 well당 Matrigel 60ul를 분주하고 1 시간 뒤, 세포를 6x103 cell/well로 분주(seeding)하여 혈관형성능력을 비교하였다. Briefly, in the recombinant phage-based plate prepared in Example 1, 5,000 vascular endothelial progenitor cells were cultured for 24 hours at 37°C, 5% CO 2 environment, and then 60ul of Matrigel per well was added to a 96-well plate. One hour after dispensing, the cells were seeded at 6x10 3 cells/well to compare the angiogenic ability.
이때, 파지 농도는 0.1 mg/ml로 이용하였고, 대조군으로는 아무것도 코팅하지 않은 플레이트(Non coating), 젤라틴만을 단독 코팅한 플레이트(Gelatin), 야생형 파지만을 단독 코팅한 플레이트(Wild type), 젤라틴 및 야생형 파지를 코팅한 플레이트(Gelatin+ Wild) 및 RGD-M13 재조합 파지만을 단독 코팅한 플레이트(RGD)에 배양하여 획득한 세포를 이용하여 비교하였다.At this time, the phage concentration was used as 0.1 mg/ml, and as a control, a plate coated with nothing (Non coating), a plate coated with only gelatin (Gelatin), a plate coated with only wild-type phage (Wild type), and gelatin and cells obtained by culturing a plate coated with wild-type phage (Gelatin+ Wild) and a plate coated with only RGD-M13 recombinant phage (RGD) were used for comparison.
그 결과, 도 5a 및 도 5b에 나타낸 바와 같이, 대조군들과 비교하였을 때, 젤라틴 및 RGD-M13 재조합 파지가 코팅된 플레이트(Gelatin+RGD)에서 배양한 EPC의 혈관형성능이 유의하게 증진되는 것을 확인하였다. As a result, as shown in FIGS. 5A and 5B , it was confirmed that the angiogenic performance of EPC cultured on a plate (Gelatin+RGD) coated with gelatin and RGD-M13 recombinant phage was significantly improved when compared to the control group. did
결론적으로, 본 발명의 젤라틴 및 RGD 재조합 파지를 코팅한 파지-기반 배양용 플레이트를 이용한 줄기세포의 배양 방법은, 야생형 파지를 이용한 경우와 비교하여, 줄기세포의 효능을 증가시키는 데 최적화된 배양 방법을 제공한다.In conclusion, the method for culturing stem cells using a phage-based culture plate coated with gelatin and RGD recombinant phage of the present invention is optimized to increase the efficacy of stem cells compared to the case of using wild-type phage. provides
이러한 파지-기반 배양 플레이트를 이용한 줄기세포의 배양은 경제적으로 매우 유용할 뿐만 아니라 대규모 배양에도 매우 적합하다는 것을 알 수 있다.It can be seen that the culture of stem cells using such a phage-based culture plate is not only economically very useful, but also very suitable for large-scale culture.
<110> Pusan National University Industry-University Cooperation Foundation
<120> Recombinant Phage-Based Culture Container For Stem Cell Culture
and Uses Thereof
<130> PNU1-409p-1
<160> 2
<170> KoPatentIn 3.0
<210> 1
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> M13-RGD phage
<400> 1
Met Leu Ser Phe Phe Ala Gly Gly Arg Gly Asp Ser Asp Asp Tyr Asp
1 5 10 15
Pro Ala Lys
<210> 2
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> M13 phage
<400> 2
Met Leu Ser Phe Ala Ala Glu Gly Asp Asp Pro Ala Lys Ala Ala Phe
1 5 10 15
Asn
<110> Pusan National University Industry-University Cooperation Foundation
<120> Recombinant Phage-Based Culture Container For Stem Cell Culture
and Uses Thereof
<130> PNU1-409p-1
<160> 2
<170> KoPatentIn 3.0
<210> 1
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> M13-RGD phage
<400> 1
Met Leu Ser Phe Phe Ala Gly Gly Arg Gly Asp Ser Asp
Claims (11)
Gelatin; And Recombinant Phage (Recombinant Phage) for displaying the cell delivery peptide on the coat protein (coat protein); Recombinant phage-based container for stem cell culture is coated.
상기 줄기세포 배양용 재조합 파지-기반 용기는 배양 플레이트, 페트리 디쉬, 배양 플라스크, 챔버 슬라이드, 챔버 및 튜브 형태로 이루어진 군으로부터 선택되는 것을 특징으로 하는, 줄기세포 배양용 재조합 파지-기반 용기.
According to claim 1,
The recombinant phage-based vessel for stem cell culture is characterized in that selected from the group consisting of a culture plate, a Petri dish, a culture flask, a chamber slide, a chamber and a tube form, a recombinant phage for stem cell culture-based vessel.
상기 재조합 파지의 세포 전달 펩티드는 RGD(Arg-Gly-Asp), RGDS(Arg-Gly-Asp-Ser), RGDC(Arg-Gly-Asp-Cys), RGDV(Arg-Gly-Asp-Val), RGES(Arg-Gly-Glu-Ser), RGDSPASSKP(Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro), GRGDS(Gly-Arg-Gly-Asp-Ser), GRADSP(Gly-Arg-Ala-Asp-Ser-Pro), KGDS(Lys-Gly-Asp-Ser),GRGDSP(Gly-Arg-Gly-Asp-Ser-Pro), GRGDTP(Gly-Arg-Gly-Asp-Thr-Pro), GRGES(Gly-Arg-Gly-Glu-Ser),GRGDSPC(Gly-Arg-Gly-Asp-Ser-Pro-Cys), GRGESP(Gly-Arg-Gly-Glu-Ser-Pro), SDGR(Ser-Asp-Gly-Arg),YRGDS(Tyr-Arg-Gly-Asp-Ser), GQQHHLGGAKQAGDV (Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val), GPR(Gly-Pro-Arg), GHK(Gly-His-Lys), YIGSR(Tyr-Ile-Gly-Ser-Arg), PDSGR(Pro-Asp-Ser-Gly-Arg),CDPGYIGSR(Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg), LCFR(Leu-Cys-Phe-Arg), EIL(Glu-Ile-Leu), EILDV(Glu-Ile-Leu-Asp-Val), EILDVPST(Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr), EILEVPST(Glu-Ile-Leu-Glu-Val-Pro-Ser-Thr), LDV(Leu-Asp-Val) 및 LDVPS(Leu-Asp-Val-Pro-Ser)로 이루어진 군으로부터 선택되는 것을 특징으로 하는,
줄기세포 배양용 재조합 파지-기반 용기.
According to claim 1,
The cell delivery peptide of the recombinant phage is RGD (Arg-Gly-Asp), RGDS (Arg-Gly-Asp-Ser), RGDC (Arg-Gly-Asp-Cys), RGDV (Arg-Gly-Asp-Val), RGES (Arg-Gly-Glu-Ser), RGDSPASSKP (Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro), GRGDS (Gly-Arg-Gly-Asp-Ser), GRADSP (Gly -Arg-Ala-Asp-Ser-Pro), KGDS (Lys-Gly-Asp-Ser), GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), GRGDTP (Gly-Arg-Gly-Asp-Thr- Pro), GRGES (Gly-Arg-Gly-Glu-Ser), GRGDSPC (Gly-Arg-Gly-Asp-Ser-Pro-Cys), GRGESP (Gly-Arg-Gly-Glu-Ser-Pro), SDGR ( Ser-Asp-Gly-Arg),YRGDS(Tyr-Arg-Gly-Asp-Ser), GQQHHLGGAKQAGDV (Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly- Asp-Val), GPR (Gly-Pro-Arg), GHK (Gly-His-Lys), YIGSR (Tyr-Ile-Gly-Ser-Arg), PDSGR (Pro-Asp-Ser-Gly-Arg), CDPGYIGSR (Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg), LCFR (Leu-Cys-Phe-Arg), EIL (Glu-Ile-Leu), EILDV (Glu-Ile-Leu-Asp- Val), EILDVPST (Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr), EILEVPST (Glu-Ile-Leu-Glu-Val-Pro-Ser-Thr), LDV (Leu-Asp-Val) and LDVPS (Leu-Asp-Val-Pro-Ser) characterized in that selected from the group consisting of,
Recombinant phage-based vessels for stem cell culture.
상기 재조합 파지는 서열번호 1의 염기서열로 구성되는 유전체를 갖는 것을 특징으로 하는,
줄기세포 배양용 재조합 파지-기반 용기.
According to claim 1,
The recombinant phage is characterized in that it has a genome consisting of the nucleotide sequence of SEQ ID NO: 1,
Recombinant phage-based vessels for stem cell culture.
상기 줄기세포 배양용 재조합 파지-기반 용기에 의해 줄기세포의 부착능, 증식능, 이동능 및 혈관생성능이 향상되는 것을 특징으로 하는,
줄기세포 배양용 재조합 파지-기반 용기.
According to claim 1,
Recombinant phage-based vessel for stem cell culture, characterized in that the adhesion, proliferation, migration and angiogenesis of stem cells are improved,
Recombinant phage-based vessels for stem cell culture.
상기 줄기세포는 EPCs(Endothelial Progenitor Cells), 심장 전구 세포(cardiac progenitor cell, CPC), ECFCs(Endothelial Colony Forming Cells), VPCs(Vasculogenic Progenitor Cells), 중간엽 줄기세포(Mesenchymal Stem Cells), 배아줄기세포(Embryonic Stem Cells) 및 근모세포(Myoblasts)로 이루어진 군으로부터 선택되는 것을 특징으로 하는,
줄기세포 배양용 재조합 파지-기반 용기.
According to claim 1,
The stem cells include Endothelial Progenitor Cells (EPCs), cardiac progenitor cells (CPCs), Endothelial Colony Forming Cells (ECFCs), Vasculogenic Progenitor Cells (VPCs), Mesenchymal Stem Cells, and embryonic stem cells. (Embryonic Stem Cells) and myoblasts (Myoblasts) characterized in that selected from the group consisting of,
Recombinant phage-based vessels for stem cell culture.
상기 줄기세포 배양용 재조합 파지-기반 용기는 피더세포(feeder cell)를 포함하지 않는 것을 특징으로 하는,
줄기세포 배양용 재조합 파지-기반 용기.
According to claim 1,
The recombinant phage-based container for stem cell culture is characterized in that it does not contain a feeder cell,
Recombinant phage-based vessels for stem cell culture.
상기 젤라틴은 0.01 내지 10 중량%이고; 상기 재조합 파지(Recombinant Phage)의 농도는 0.001 mg/ml 내지 10 mg/ml 인 것을 특징으로 하는,
줄기세포 배양용 재조합 파지-기반 용기
According to claim 1,
the gelatin is 0.01 to 10% by weight; The concentration of the recombinant phage (Recombinant Phage) is characterized in that 0.001 mg / ml to 10 mg / ml,
Recombinant phage-based vessel for stem cell culture
Gelatin; And Recombinant phage (Recombinant Phage) displaying a cell delivery peptide on a coat protein; A method of manufacturing a recombinant phage-based vessel for stem cell culture comprising the step of coating the cell culture vessel.
According to any one of claims 1 to 8, wherein any one of the recombinant phage for stem cell culture-based vessel, comprising the step of culturing the stem cells, a method of improving the adhesion, proliferation, migration and angiogenesis of stem cells.
상기 줄기세포는 부착능, 증식능, 이동능 및 혈관생성능이 향상된 줄기세포인 것을 특징으로 하는 방법.
Claims 1 to 8 as a stem cell production method comprising the step of culturing the stem cells in a recombinant phage-based vessel for stem cell culture of any one of claims 1 to 8,
The stem cell is a method, characterized in that the adhesion, proliferation, migration and angiogenesis are improved stem cells.
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