KR101700598B1 - Biomarker composition for isolating fibroblast with capacity of multipotency comprising CD105 and method for isolating fibroblast with capacity of multipotency using the same marker - Google Patents

Biomarker composition for isolating fibroblast with capacity of multipotency comprising CD105 and method for isolating fibroblast with capacity of multipotency using the same marker Download PDF

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KR101700598B1
KR101700598B1 KR1020140156129A KR20140156129A KR101700598B1 KR 101700598 B1 KR101700598 B1 KR 101700598B1 KR 1020140156129 A KR1020140156129 A KR 1020140156129A KR 20140156129 A KR20140156129 A KR 20140156129A KR 101700598 B1 KR101700598 B1 KR 101700598B1
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fibroblasts
cells
stem cell
separating
present
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이승범
박선후
김민정
심세환
이승숙
신혜윤
이선주
장원석
진영우
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한국원자력의학원
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Abstract

본 발명은 CD105를 포함하는 줄기세포능을 가진 섬유아세포 분리용 바이오마커 조성물 및 이를 이용한 줄기세포능을 가진 섬유아세포 분리 방법에 관한 것이다. 줄기세포능을 가진 섬유아세포의 선택적 분리를 평가하는데 유용한 바이오 표면마커로써 CD105 항체를 제공한다. 본 발명에 따르면 마우스 피부 진피세포에서 분리된 섬유아세포는 CD73 발현이 되지 않고 섬유아세포 특이적 마커 발현 확인으로 중간엽줄기세포와는 구별됨을 확인하였다. 또한 지속적인 계대배양시 특이적으로 감소하는 CD105 표면마커 발현에 따른 CD105 양성 및 음성 섬유아세포들을 분리한 후 줄기세포 분화능 비교분석시 CD105 양성 섬유아세포에서 다분화능이 매우 높음을 알 수 있었다. 따라서, 본 발명에서 선별된 CD105를 이용함으로써 줄기세포능 유무가 있는 섬유아세포를 선택적으로 분리할 수 있는 방법 개발에 활용될 수 있을 것이다.The present invention relates to a biomarker composition for the isolation of fibroblasts having stem cell function including CD105, and a method for separating fibroblasts having stem cell function using the same. Provides CD105 antibodies as biosurfactant markers useful for assessing selective isolation of fibroblasts with stem cell potential. According to the present invention, fibroblasts isolated from mouse skin dermis cells are not CD73-expressing, and are confirmed to be differentiated from mesenchymal stem cells by confirmation of fibroblast-specific marker expression. In addition, CD105 positive and negative fibroblasts were separated by CD105 surface marker expression in a continuous subculture, and CD105-positive fibroblasts were found to have a high degree of pluripotency. Therefore, by using the CD105 selected in the present invention, it can be utilized in the development of a method for selectively separating fibroblasts having stem cell function.

Description

CD105를 포함하는 줄기세포능을 가진 섬유아세포 분리용 바이오마커 조성물 및 이를 이용한 줄기세포능을 가진 섬유아세포 분리 방법{Biomarker composition for isolating fibroblast with capacity of multipotency comprising CD105 and method for isolating fibroblast with capacity of multipotency using the same marker}[0001] The present invention relates to a biomarker composition for the isolation of fibroblasts having stem cell capability including CD105 and a method for separating fibroblasts having stem cell potential using the same. [0002] Biomarker composition for isolating fibroblast with capacity of multipotency comprising CD105 and method for isolating fibroblast with capacity of multipotency using the same marker}

본 발명은 CD105를 포함하는 줄기세포능을 가진 섬유아세포 분리용 바이오마커 조성물 및 이를 이용한 줄기세포능을 가진 섬유아세포 분리 방법에 관한 것이다.The present invention relates to a biomarker composition for the isolation of fibroblasts having stem cell function including CD105, and a method for separating fibroblasts having stem cell function using the same.

줄기세포는 미분화된 세포로서 자가 재생 및 인체의 다양한 세포로의 분화능 (줄기세포능)을 가지고 있어 치료제로서 적용하려는 연구가 최근 많이 이루어지고 주목받고 있다.Since stem cells are undifferentiated cells and have self-renewal and the ability to differentiate into various cells of the human body (stem cell function), many studies for applying them as therapeutic agents have recently been attracting attention.

피부진피에는 중간엽줄기세포, 섬유아세포 등 다양한 세포들이 존재한다. 최근에는 섬유아세포가 중간엽줄기세포처럼 다분화능 특성이 있음이 보고되고 있다. 이러한 섬유아세포에는 줄기세포능이 없는 섬유아세포와 줄기세포능이 있는 섬유아세포들이 존재하지만, 이들을 쉽게 분리할 수 있는 방법 또는 이러한 섬유아세포들에서 줄기세포능 유무를 선택적으로 구별할 수 있는 바이오마커는 아직 보고되지 않았다.There are various cells such as mesenchymal stem cells and fibroblasts in the skin dermis. Recently, it has been reported that fibroblasts have differentiation characteristics like mesenchymal stem cells. Although fibroblasts lacking stem cell capability and fibroblasts having stem cell potential exist in such fibroblasts, there is still a method of easily separating them, or a biomarker capable of selectively discriminating the presence or absence of stem cell function in such fibroblasts It was not.

한국특허공개공보 제10-2008-0106686호(2008.12.09)Korean Patent Laid-Open No. 10-2008-0106686 (December, 2008)

본 발명의 목적은 CD105를 포함하는 줄기세포능을 가진 섬유아세포 분리용 바이오마커 조성물 및 이를 이용한 줄기세포능을 가진 섬유아세포 분리방법을 제공하는데 있다.It is an object of the present invention to provide a biomarker composition for the isolation of fibroblasts having stem cell function comprising CD105 and a method for separating fibroblasts having stem cell function using the same.

본 발명은 CD105를 포함하는 줄기세포능을 가진 섬유아세포 분리용 바이오마커 조성물을 제공한다.The present invention provides a biomarker composition for the isolation of fibroblasts having stem cell ability comprising CD105.

또한, 본 발명은 CD105에 특이적으로 결합하는 항체를 포함하는 줄기세포능을 가진 섬유아세포 분리용 키트를 제공한다.In addition, the present invention provides a kit for separating fibroblasts having stem cell function comprising an antibody specifically binding to CD105.

또한, 본 발명은 진피 섬유아세포에서 CD105를 발현하는 CD105 양성세포를 분리하는 단계를 포함하는 줄기세포능을 가진 섬유아세포 분리방법을 제공한다.In addition, the present invention provides a method for separating fibroblast cells having stem cell function, which comprises separating CD105-positive cells expressing CD105 in dermal fibroblasts.

또한, 본 발명은 CD105를 발현하는 피부 진피 섬유아세포를 유효성분으로 포함하는 피부조직재생용 조성물을 제공한다.The present invention also provides a composition for regenerating skin tissue comprising skin dermal fibroblasts expressing CD105 as an active ingredient.

본 발명은 CD105를 포함하는 줄기세포능을 가진 섬유아세포 분리용 바이오마커 조성물 및 이를 이용한 줄기세포능을 가진 섬유아세포 분리 방법에 관한 것이다. 줄기세포능을 가진 섬유아세포의 선택적 분리를 평가하는데 유용한 바이오 표면마커로써 CD105 항체를 제공한다. 본 발명에 따르면 마우스 피부 진피세포에서 분리된 섬유아세포는 CD73 발현이 되지 않고 섬유아세포 특이적 마커 발현 확인으로 중간엽줄기세포와는 구별됨을 확인하였다. 또한 지속적인 계대배양시 특이적으로 감소하는 CD105 표면마커 발현에 따른 CD105 양성 및 음성 섬유아세포들을 분리한 후 줄기세포 분화능 비교분석시 CD105 양성 섬유아세포에서 다분화능이 매우 높음을 알 수 있었다. 따라서, 본 발명에서 선별된 CD105를 이용함으로써 줄기세포능 유무가 있는 섬유아세포를 선택적으로 분리할 수 있는 방법 개발에 활용될 수 있을 것이다.The present invention relates to a biomarker composition for the isolation of fibroblasts having stem cell function including CD105, and a method for separating fibroblasts having stem cell function using the same. Provides CD105 antibodies as biosurfactant markers useful for assessing selective isolation of fibroblasts with stem cell potential. According to the present invention, fibroblasts isolated from mouse skin dermis cells are not CD73-expressing, and are confirmed to be differentiated from mesenchymal stem cells by confirmation of fibroblast-specific marker expression. In addition, CD105 positive and negative fibroblasts were separated by CD105 surface marker expression in a continuous subculture, and CD105-positive fibroblasts were found to have a high degree of pluripotency. Therefore, by using the CD105 selected in the present invention, it can be utilized in the development of a method for selectively separating fibroblasts having stem cell function.

도 1은 마우스 피부에서 분리된 진피 섬유아세포의 특성을 나타낸 결과이다.
도 2는 계대배양에 따른 섬유아세포의 중간엽줄기세포 표면 마커 분석 결과를 나타낸다.
도 3은 CD105 양성, 음성세포 분리 및 CD105 발현 분석 결과를 나타낸다.
도 4는 CD105 양성 섬유아세포에서의 효율적인 다분화능 확인 결과이다.FIG. 1 shows the characteristics of dermal fibroblasts isolated from mouse skin.
Figure 2 shows the results of mesenchymal stem cell surface marker analysis of fibroblasts following subculture.
Figure 3 shows the results of CD105 positive, negative cell sorting and CD105 expression analysis.
Figure 4 shows the results of efficient multi-differentiability assays in CD105-positive fibroblasts.

본 발명은 CD105를 포함하는 줄기세포능을 가진 섬유아세포 분리용 바이오마커 조성물을 제공한다. 상세하게는 상기 섬유아세포는 피부 진피 유래일 수 있지만, 이에 제한되는 것은 아니다.
The present invention provides a biomarker composition for the isolation of fibroblasts having stem cell ability comprising CD105. Specifically, the fibroblasts may be derived from skin dermis, but are not limited thereto.

본 발명의 "CD105"는 세포막에 존재하는 동종 이합 당단백질로서, TGF (transforming growth factor) beta 1 복합체의 일부로 아직 기능적 역할은 명확히 밝혀지지 않았다. 본 발명의 CD105는 사람, 소, 염소, 양, 돼지, 마우스, 토끼 등의 포유류를 포함하는 CD105를 가지는 모든 진핵 생물 유래의 단백질일 수 있으며, 바람직하게는 NCBI accession no. BC029080.1 일 수 있으나, 이에 한정되는 것은 아니다.
The "CD105" of the present invention is a homologous recombinant glycoprotein present in the cell membrane, and its functional role as a part of TGF (transforming growth factor) beta 1 complex has not yet been clarified. The CD105 of the present invention may be any eukaryotic protein having CD105 including mammals such as human, bovine, goat, sheep, pig, mouse, rabbit and the like, preferably NCBI accession no. BC029080.1, but is not limited thereto.

또한, 본 발명의 CD105에 특이적으로 결합하는 항체를 포함하는 줄기세포능을 가진 섬유아세포 분리용 키트를 제공한다.
In addition, there is provided a kit for separating fibroblasts having stem cell function, which comprises an antibody specifically binding to CD105 of the present invention.

본 명세서에서 용어 “항체”란 당해 기술분야에 공지된 용어로서 항원성 부위에 대하여 지시되는 특이적인 면역 글로불린을 의미한다. 본 발명에서의 항체는 본 발명의 줄기세포능을 가진 섬유아세포 분리용 바이오마커인 CD105에 대해 특이적으로 결합하는 항체를 의미하며, 당해 기술분야의 통상적인 방법에 따라 항체를 제조할 수 있다. 상기 항체의 형태는 폴리클로날 항체 또는 모노클로날 항체를 포함하며, 모든 면역글로불린 항체가 포함된다. 상기 항체는 2개의 전체 길이의 경쇄 및 2 개의 전체 길이의 중쇄를 갖는 완전한 형태를 의미한다. 또한, 상기 항체는 인간화 항체 등의 특수 항체도 포함된다.
The term " antibody " as used herein refers to a specific immunoglobulin as indicated in the art and directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to CD105 which is a biomarker for fibroblast separation with stem cell function of the present invention, and the antibody can be produced according to a conventional method in the art. The forms of the antibodies include polyclonal or monoclonal antibodies, including all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes a special antibody such as a humanized antibody.

한편, 본 발명의 항체 대신에 본 발명의 바이오마커에 특이적으로 결합하는 앱타머를 이용할 수도 있으며, 앱타머는 올리고핵산 또는 펩타이드 분자일 수 있다.
Alternatively, an aptamer that specifically binds to the biomarker of the present invention may be used instead of the antibody of the present invention, and the aptamer may be an oligonucleic acid or a peptide molecule.

또한 본 발명의 키트는 마커 성분에 특이적으로 결합하는 항체, 기질과의 반응에 의해서 발색하는 표지체가 접합된 2차 항체 접합체(conjugate), 상기 표지체와 발색 반응할 발색 기질 용액, 세척액 및 효소반응 정지용액 등을 포함할 수 있으며, 사용되는 시약 성분을 포함하는 다수의 별도 패키징 또는 컴파트먼트로 제작될 수 있다.In addition, the kit of the present invention comprises an antibody specifically binding to a marker component, a secondary antibody conjugate conjugated with a label that develops upon reaction with the substrate, a coloring substrate solution to be colored with the label, A reaction stop solution, and the like, and may be manufactured from a number of separate packaging or compartments including the reagent components used.

상기 2차 항체 접합체의 표지체는 발색반응을 하는 통상의 발색제가 바람직하며, HRP(horseradish peroxidase), 염기성 탈인산화효소(alkaline phosphatase), 콜로이드 골드(coloid gold), FITC(poly L-lysine-fluorescein isothiocyanate), RITC(rhodamine-B-isothiocyanate) 등의 형광물질(fluorescein), 및 색소(dye) 등이 사용될 수 있다.
The marker of the secondary antibody conjugate is preferably a conventional coloring agent that undergoes a chromogenic reaction and is preferably selected from the group consisting of HRP (horseradish peroxidase), alkaline phosphatase, colloid gold, poly L-lysine-fluorescein fluorescein such as isothiocyanate and rhodamine-B-isothiocyanate, and dye may be used.

또한, 본 발명은 피부에서 진피 섬유아세포를 분리하는 단계; 및 상기 분리된 진피 섬유아세포에서 CD105를 발현하는 CD105 양성세포를 분리하는 단계를 포함하는 줄기세포능을 가진 섬유아세포 분리방법을 제공한다.The present invention also relates to a method for the treatment of dermal fibroblasts, comprising: separating dermal fibroblasts from the skin; And separating the CD105-positive cells expressing CD105 from the separated dermal fibroblasts.

바람직하게는 상기 CD105 양성세포 분리하는 단계는 자석 활성 세포 분류법(Magnetic activated cell sorting; MACS) 또는 유세포 분석(Flow cytometry Analysis)을 통해 분리될 수 있으나, 이에 한정되는 것은 아니다.
Preferably, the step of separating the CD105-positive cells may be separated by magnetic activated cell sorting (MACS) or flow cytometry analysis, but is not limited thereto.

또한, 본 발명은 CD105를 발현하는 피부 진피 섬유아세포를 유효성분으로 포함하는 피부조직재생용 조성물을 제공한다.
The present invention also provides a composition for regenerating skin tissue comprising skin dermal fibroblasts expressing CD105 as an active ingredient.

본 발명에서 사용되는 피부조직재생용 조성물은 증상이나 피부손상의 정도에 따라서 다를 수 있으나 일반적으로 0.01mg/ml～10mg/ml의 농도로 사용될 수 있다.The composition for regenerating skin tissue used in the present invention may be used in a concentration of 0.01 mg / ml to 10 mg / ml, though it may vary depending on symptoms and degree of skin damage.

피부결손조직에는 통증, 염증이 수반되거나 결손부위가 피부 내 조직에 복합적으로 초래될수 도 있어, 본 발명의 CD105를 발현하는 피부 진피 섬유아세포 이외에 타 성장인자나 세포외기질 성분과 소염진통제, 항균제, 부신피질호르몬에서 선택된 약물을 1종 이상 더 함유시킬 수도 있다. 이러한 약물로서 케토프로펜(ketoprofen), 플루비프로펜(flurbiprofen), 페노프로펜(fenoprofen), 이부프로펜(ibuprofen)을 포함하는 페닐프로피온산 유도체계열의 엔세이드류; 피록시캄(piroxicam), 테녹시캄(tenoxicam), 멜록시캄(meloxicam)을 포함하는 옥시캄 유도체계열의 엔세이드류(NSAIDs); 디클로페낙(diclofenac); 및 인도메타신(indomethacin) 중에서 선택된 소염진통제, metronidazole, Amoxicillin, Amoxicillin-clavulanate, Ampicillin-sulbactam, Ampicillin, Piperacillin, Benzathine penicillin, Cephalosporin, Cefazolin, Other cephalosporin, Lincosamide (Clindamycin), Macrolide (Erythromycin), 테트라사이클린 하이드로클로라이드, 세틸피리디늄 클로라이드, 클로르헥시딘 하이드로클로라이드에서 선택된 항균제, 메틸프레드니솔론, 하이드로코르티손 아세테이트에서 선택된 부신피질호르몬제 약물을 함유시킬 수 있다.
In addition to the skin dermal fibroblasts expressing CD105 of the present invention, other skin growth factors, extracellular matrix components, anti-inflammatory agents, antimicrobial agents, anti-inflammatory agents, One or more drugs selected from adrenocortical hormones may be further contained. Such drugs include, but are not limited to, enantiomers of phenylpropionic acid derivatives including ketoprofen, flurbiprofen, fenoprofen, ibuprofen; (NSAIDs) based on oxycam derivatives, including piroxicam, tenoxicam, and meloxicam; Diclofenac; Amoxicillin-clavulanate, Ampicillin-sulbactam, Ampicillin, Piperacillin, Benzathine penicillin, Cephalosporin, Cefazolin, Other cephalosporin, Clincamycin, Macrolide An antimicrobial selected from hydrochloride, cetylpyridinium chloride, chlorhexidine hydrochloride, methylprednisolone, hydrocortisone acetate, and the like.

또한, 본 발명의 피부조직재생용 조성물은 CD105를 발현하는 피부 진피 섬유아세포의 유효량 및 약제학적으로 허용되는 담체나 부형제를 포함한다. 본 발명에서 사용될 수 있는 담체 및 부형제는 덱스트로스 락토스, 수크로스, 솔비톨, 전분, 아카시아 고무, 인산칼슘, 소디움 알지네이트, 젤라틴, 소디움 CMC, 전분, 인산칼슘, 폴리비닐피롤리돈(PVP), 물, 알코올, 글리세린, 식물유등을 포함할 수 있다. 본 발명에서는 이들 성분이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제등을 더 함유할 수 있다. 이러한 성분은 의약분야, 화장품 분야 등에서 통상적으로 사용되는 성분들이며, 이들 이외에 이 분야에서 통상으로 사용되는 성분들을 더 함유할 수도 있다.
In addition, the composition for regenerating skin tissue of the present invention includes an effective amount of dermal fibroblasts expressing CD105 and a pharmaceutically acceptable carrier or excipient. Carriers and excipients that can be used in the present invention include, but are not limited to, dextrose lactose, sucrose, sorbitol, starch, acacia rubber, calcium phosphate, sodium alginate, gelatin, sodium CMC, starch, calcium phosphate, polyvinylpyrrolidone , Alcohols, glycerin, vegetable oils, and the like. In the present invention, in addition to these components, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative and the like may be further contained. These ingredients are commonly used in the fields of medicine, cosmetics and the like, and may further contain other ingredients commonly used in this field.

본 발명의 제제형태는 액제, 현탁제, 유화제, 엑스제, 분말제, 과립제, 정제 또는 캡슐제의 행태로 제형화 할 수 있으며, 피부용제로서는 액제, 현탁제, 유착제, 페이스트, 겔제, 크림제, 로션, 파우더, 계면활성제 함유 크린싱제, 오일제, 스프레이제 등으로 제형화 할 수 있다.
The form of the preparation of the present invention can be formulated into a form of a liquid, a suspension, an emulsifier, an excipient, a powder, a granule, a tablet or a capsule. Examples of the dermal solvent include a liquid, a suspension, A lotion, a powder, a surfactant-containing cleansing agent, an oil agent, a spraying agent and the like.

이하에서는, 본 발명을 한정하지 않는 실시예에 따라 본 발명을 상세히 설명한다. 본 발명의 하기 실시예는 본 발명을 구체화하기 위한 것일 뿐 본 발명의 권리범위를 제한하거나 한정하는 것이 아님은 물론이다. 따라서, 본 발명의 상세한 설명 및 실시예로부터 본 발명이 속하는 기술분야의 전문가가 용이하게 유추할 수 있는 것은 본 발명의 권리범위에 속하는 것으로 해석된다.
Hereinafter, the present invention will be described in detail with reference to embodiments which do not limit the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

< < 실시예Example >  > 줄기세포능을Stem cell function 가진 섬유아세포의 선택적 분리를 위한  For selective isolation of fibroblasts 바이오마커Biomarker 발굴 excavation

1. 마우스 피부에서 진피세포 분리1. Dermal cell separation from mouse skin

실험에 사용한 동물은 중앙실험동물에서 공급하는 C57/B6 mice에서 태어난 생후 하루된 마우스 피부에서 세포를 분리하였다. 얻어진 피부에서 지방층을 제거한 후 2-4 mm × 2-4 mm 크기의 절편으로 조각 낸 뒤, Liberase (0.1% W/V in phosphate buffered saline, Gibco BRL, USA)용액을 첨가하고 4℃에서 O/N 정치하였다. 이후 핀셋을 이용하여 표피와 진피를 분리하고 진피를 칼로 잘게 조각낸 후 type I collagenase 1시간 정치배양하였다. 효소처리에 의해 분리된 진피세포를 실험에 사용하였다.
The animals used in the experiments were isolated from the mouse skin of the day after birth born in C57 / B6 mice fed from the central laboratory animals. After removing the fat layer from the obtained skin, it was sliced into 2-4 mm × 2-4 mm sections, and then added with Liberase (0.1% W / V phosphate buffered saline, Gibco BRL, USA) N politically. Subsequently, the epidermis and dermis were separated using tweezers, the dermis was finely cut with a knife, and then type I collagenase was cultured for 1 hour. Dermis cells isolated by enzyme treatment were used for the experiments.

2. 세포배양2. Cell culture

세포는 일반 배지 DMEM에, 0.48 mg/mL 글루타민(glutamine), 100 IU/mL 페니실린(penicillin), 50 ㎍/mL 스트렙토마이신(streptomycin), 10% 우태아혈청(fetal bovine serum), 10ng/ml of bFGF를 첨가한 후 37℃, 5% CO2 조건하에서 배양하고 2번 계대배양 이후의 세포를 실험에 사용하였다.
Cells were cultured in normal DMEM medium supplemented with 0.48 mg / mL glutamine, 100 IU / mL penicillin, 50 μg / mL streptomycin, 10% fetal bovine serum, 10 ng / ml of bFGF was added, and the cells were cultured under the condition of 37 ° C and 5% CO 2 , and the cells after the second subculture were used for the experiment.

3. 유세포 분석기 (Flow cytometry Analysis)를 통한 간엽성 줄기세포 표지자 발현 분석3. Analysis of hepatic stem cell marker expression by flow cytometry analysis

유세포 분석을 이용하여 피부로부터 분리 배양된 진피세포에서 간엽성 줄기세포 표지자 kit (R&D system, Minneapolis, MN, USA)를 이용하여 간엽성 줄기세포의 표지자인 Sca-1, CD106, CD105, CD73, CD54, CD44, CD29 와 내피세포 표지자(endothelial cell marker)인 CD11b, 골수조혈모세포 표면 표지자 (CD45)의 발현양상을 관찰하였다. 표면 표지자 발현을 관찰하기 위하여 간엽성 줄기세포 표지자 또는 내피세포 표지자 일차 항체를 5 μg/1×106 cells 농도로 4℃에서 30분 동안 반응시켰다. 염색된 세포를 유세포 분석 염색 완충액(flow cytometry staining buffer)으로 세척 후 이차 항체로서 R-피코에리트린(R-phycoerythrin; R-PE) 또는 플루오레세인 이소티오시아네이트(fluorescein isothiocyanate; FITC)가 결합된 goat anti-rat IgM (eBioscience, USA)을 4℃에서 30분 동안 반응시켰다. 대조군은 일차 항체를 첨가하지 않고 진행하였다. 염색된 세포들은 FACSCantoFlow Cytometer(BD Biosciences)을 사용하여 세포의 형광량 측정 및 발현 양상을 분석하였다.
CD106, CD105, CD73, and CD54, which are markers of mesenchymal stem cells, were obtained from dermal cells isolated from skin using flow cytometry using a mesenchymal stem cell marker kit (R & D system, Minneapolis, MN, USA) , CD44 and CD29, and endothelial cell marker CD11b and bone marrow hematopoietic cell surface marker (CD45). To observe surface marker expression, the mesenchymal stem cell marker or endothelial cell marker primary antibody was reacted at a concentration of 5 μg / 1 × 10 6 cells for 30 minutes at 4 ° C. After the stained cells were washed with a flow cytometry staining buffer, R-phycoerythrin (R-PE) or fluorescein isothiocyanate (FITC) was bound as a secondary antibody (Goat anti-rat IgM, eBioscience, USA) was reacted at 4 ° C for 30 minutes. The control group proceeded without addition of the primary antibody. The stained cells were analyzed for fluorescence intensity and expression pattern using FACSCantoFlow Cytometer (BD Biosciences).

4. MACS 방법 또는 유세포 분석기(Flow cytometry Analysis)을 이용한 CD105 양성 및 음성 세포분리 4. CD105 positive and negative cell separation using MACS method or flow cytometry analysis

CD105 발현세포를 순수분리하기위해서 계대배양된 진피세포를 R-피코에리트린(R-phycoerythrin; R-PE)이 결합된 CD105에 대한 항체를 이용하여 MACS 방법으로 분리하였다. CD105-PE 항체를 MACS buffer (0.5% BSA, 2mM EDTA in PBS)에 1:100로 희석하여 30분간 냉장 상태로 처리하고 PBS 1회 세척하였다. MACS bead가 결합된 PE 항체로 냉장에서 30분 처리한 후 제조회사 매뉴얼 (Miltenyi Biotec, Germany)에 따라 양성세포와 음성세포를 분리하였다.To isolate CD105 expressing cells, subcultured dermal cells were separated by MACS method using an antibody against R-phycoerythrin (R-PE) bound CD105. The CD105-PE antibody was diluted 1: 100 in MACS buffer (0.5% BSA, 2 mM EDTA in PBS), refrigerated for 30 minutes, and washed once with PBS. MACS bead-conjugated PE antibody was treated for 30 minutes in the refrigerator, and then positive cells and negative cells were separated according to the manufacturer's manual (Miltenyi Biotec, Germany).

또는, 배양된 진피세포를 플루오레세인 이소티오시아네이트(fluorescein isothiocyanate; FITC)가 결합된 CD105 (eBioscience, USA)를 FACS buffer (0.5% BSA in PBS)에 희석하고 제조회사의 매뉴얼에 따라 처리 후 4℃에서 30분 동안 반응시켰다. 염색된 세포들을 FACSArial Flow Cytometer(BD Biosciences)을 사용하여 발현 양상을 분석하고 양성세포와 음성세포를 순수분리하였다.
Alternatively, the cultured dermal cells were diluted in FACS buffer (0.5% BSA in PBS) with CD105 (eBioscience, USA) with fluorescein isothiocyanate (FITC) conjugated thereto and treated according to the manufacturer's manual And reacted at 4 DEG C for 30 minutes. The stained cells were analyzed by FACSArial Flow Cytometer (BD Biosciences) for expression and positive cells and negative cells were separated.

5. RT-PCR을 통한 MACS 분리 배양세포의 CD105 발현 및 다분화능 비교 분석 5. Comparison of CD105 expression and differentiation potential of cultured MACS cells by RT-PCR

MACS 분리 배양세포의 CD105 (NCBI accession no. BC029080.1) 발현 양상 확인과 지방세포 및 연골세포로의 분화 정도를 확인하기 위하여 역전사 중합효소 연쇄반응 (RT-PCR)을 통하여 지방세포 또는 연골세포 특이적 유전자 발현을 측정하였다. 각 세포들의 total RNA를 RNeasy mini kit (Qiagen, CA, USA)로 추출하였다. 추출된 RNA 1㎍을 역전사 (reverse transcription) 반응으로 cDNA를 합성하여 각각의 유전자에 대한 primer set로 PCR을 수행하였다. 각각의 PCR은 변성(denaturation)은 95℃에서 5분, 결합(anealing) cycle은 94℃에서 30초, 각 결합(anealing) 온도에서 30초, 72℃에서 30초간 각 primer의 적정 cycle 수에 맞추어 실험한 후, 중합(polymerization)은 72℃에서 5분 동안 반응하여 결과를 관찰하였다.
In order to confirm the expression pattern of CD105 (NCBI accession No. BC029080.1) in MACS-isolated cultured cells and to determine the degree of differentiation into adipocytes and chondrocytes, RT-PCR was performed to determine the adipocyte or chondrocyte specificity Gene expression was measured. Total RNA of each cell was extracted with RNeasy mini kit (Qiagen, CA, USA). CDNA was synthesized by reverse transcription of 1 추출 of the extracted RNA and PCR was performed with a primer set for each gene. Each PCR was denaturation at 95 ° C for 5 minutes, anealing cycle at 94 ° C for 30 seconds, 30 seconds at each anealing temperature, and 30 seconds at 72 ° C. After the experiment, the polymerization was carried out at 72 ° C for 5 minutes, and the result was observed.

6. 지방세포, 뼈세포로의 분화능 분석6. Analysis of differentiation into adipocytes and bone cells

지방세포로의 분화능력을 확인하기 위하여 MACS 분리 배양세포를 12 또는 6 well 배양접시에 1×104 cells/cm2 농도로 분양한 다음 세포가 거의 차게 될때 분화 배지로 교환하였다. 지방세포로의 분화 유도는 DMEM 배지에 10% FBS, 1% 항생제(antibiotics), 1 μM 덱사메타손(dexamethasone) (Sigma), 10 μg/ml 인간 인슐린(human insulin) (Sigma), 100 μM 인도메타신(indomethacin) (Sigma), 500 μM 3-이소부틸-메틸잔틴(3-isobutyll-methylxanthine; IBMX, Sigma)를 첨가한 배지로 사용하였으며 일주일에 두 번씩 배지 교환하며 3주 동안 정치배양하였다. 분화 3주 후 지방세포로의 분화능을 알아보기 위해 오일 레드 O(Oil red O) 염색을 시행하였다. 염색을 위해 세포를 4% 파라포름알데히드(paraformaldehyde)를 사용하여 상온에서 30분 동안 고정한 후 0.5% 오일 레드 O(Oil red O) (Sigma) 용액을 상온에서 10분간 적용시켜 염색하였으며, 증류수로 세척한 후에 광학현미경으로 세포의 분화 양상을 관찰하였다. 뼈세포로의 분화능력을 확인하기 위하여 DMEM 배지에 10% FBS, 1% 항생제(antibiotics), 1 μM 덱사메타손(dexamethasone) (Sigma), 2mM β-글리세롤포스페이트(β-glycerolphosphate) (Sigma) 및 50 μM 아스코르빅산 2-포스페이트(ascorbic acid 2-phosphate) (Sigma)를 첨가한 연골분화용 배지를 처리하여 5주간 세포를 분화시켰다. 뼈세포로의 분화능을 알아보기 위해 알리자린 레드 S(Alizarin Red S) 염색을 시행하였다. 염색을 위해 세포를 4% 파라포름알데히드(paraformaldehyde) 사용하여 상온에서 30분 동안 고정한 후 2% 알리자린 레드 S(Alizarin Red S) (pH=4.2; Sigma) 용액을 상온에서 5분간 적용시켜 염색하였으며, 증류수로 세척한 후에 광학현미경으로 세포의 분화 양상을 관찰하였다.
To confirm the differentiation ability of adipocytes, MACS-isolated cultured cells were pre-cultured at a concentration of 1 × 10 4 cells / cm 2 in a 12 or 6-well culture dish, and were then replaced with differentiation medium when the cells became almost full. Differentiation induction in adipocytes was induced by adding 10% FBS, 1% antibiotics, 1 μM dexamethasone (Sigma), 10 μg / ml human insulin (Sigma), 100 μM indomethacin indomethacin (Sigma) and 500 μM 3-isobutyll-methylxanthine (IBMX, Sigma). The cells were cultured for 3 weeks. Three weeks after the differentiation, oil red O staining was performed to evaluate the differentiation potential of adipocytes. For staining, the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, stained with 0.5% Oil red O (Sigma) solution at room temperature for 10 minutes, washed with distilled water And then the differentiation pattern of the cells was observed with an optical microscope. To confirm the ability to differentiate into bone cells, DMEM medium was supplemented with 10% FBS, 1% antibiotics, 1 μM dexamethasone (Sigma), 2 mM β-glycerolphosphate (Sigma) and 50 μM Cells were differentiated for 5 weeks by treating the medium for cartilage differentiation supplemented with ascorbic acid 2-phosphate (Sigma). Alizarin Red S staining was performed to examine the differentiation potential into bone cells. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 2% Alizarin Red S (pH = 4.2; Sigma) solution for 5 min at room temperature. After washing with distilled water, the differentiation pattern of cells was observed with an optical microscope.

7. 결과7. Results

마우스 피부에서 분리된 진피 섬유아세포의 특성을 확인하였다. 마우스 피부에서 분리된 진피세포는 중간엽줄기세포 대표적 표면 마커인 CD73 발현이 안되는 것을 유세포분석(flowcytometry)으로 확인하였으며(도 1A), 면역염색을 통해 섬유아세포의 특이적 마커인 피브로넥틴(Fibronectin), alpha-SMA, 비멘틴(Vimentin) 발현을 확인할 수 있었다(도 1B). The characteristics of dermal fibroblasts isolated from mouse skin were confirmed. The dermis cells isolated from mouse skin were confirmed by flowcytometry that CD73 expression, which is a representative surface marker of mesenchymal stem cells, was not observed (Fig. 1A). Fibronectin, which is a specific marker of fibroblast, alpha-SMA, and vimentin (Fig. 1B).

계대배양에 따른 섬유아세포의 중간엽줄기세포 표면 마커 분석한 결과, 지속적인 계대배양시 줄기세포 표면 마커 중 하나인 CD105 발현이 특이적으로 감소함을 확인할 수 있었으며, 중간엽줄기세포 대표적 표면 마커인 CD73 발현은 여전히 안되는 것을 유세포분석(flowcytometry)으로 확인하였다(도 2). As a result of analysis of mesenchymal stem cell surface markers of fibroblasts by subculture, it was confirmed that CD105 expression, which is one of the stem cell surface markers, was specifically decreased in continuous passage culture, and CD17 Flowcytometry was used to confirm that expression was still not observed (Fig. 2).

CD105 양성, 음성세포 분리 및 CD105 발현 분석 결과, R-피코에리트린(R-phycoerythrin; R-PE)이 결합된 CD105-PE 항체를 이용하여 MACS 방법으로 분리된 CD105 양성 및 음성 섬유아세포의 순도가 96% 이상임을 유세포분석(flowcytometry)으로 확인하였으며(도 3A), 여섯번의 계대배양 후에도 CD105 발현 양상이 MACS로 분리된 두 세포들과 일치함을 RT-PCR로 확인할 수 있었다(도 3B).The purity of CD105 positive and negative fibroblasts isolated by MACS method using CD105-PE antibody conjugated with R-phycoerythrin (R-PE) was analyzed by CD105-positive, negative cell- (FIG. 3A), and that the CD105 expression pattern coincided with the two cells separated by MACS even after six times of passage, by RT-PCR (FIG. 3B).

CD105 양성 섬유아세포에서의 효율적인 다분화능을 확인하였다. CD105는 중간엽줄기세포의 줄기세포능 조절에 관여하므로 MACS 분리 배양된 섬유아세포들을 중간엽줄기세포의 다분화능 방향인 지방세포(adipogenesis)와 골세포 (Osteogenesis)로 분화 유도하여 그 특성을 비교분석하였다. CD105 양성 섬유아세포들이 지방세포(도 4A)와 뼈세포(도 4C)로의 분화 양상이 매우 높음을 오일 레드 O(Oil red O) 염색과 알리자린 레드 S(Alizarin Red S) 염색으로 확인할 수 있었으며, 각각의 지방세포 특이적 유전자들(PPARr, LPL)과 뼈세포 특이적 유전자들(ALP, Runx2) 발현도 또한 높음을 RT-PCR로 확인할 수 있었다(도 4B 및 도 4D). 대조적으로 CD105 음성 섬유아세포들에서의 다분화능은 매우 미약함을 확인하였다.
CD105-positive fibroblasts. &Lt; / RTI &gt; CD105 is involved in the regulation of stem cell function of mesenchymal stem cells. Therefore, the fibroblasts isolated from MACS are induced to differentiate into adipogenesis and osteogenesis, which are the multipotential direction of mesenchymal stem cells. Respectively. The high degree of differentiation of CD105-positive fibroblasts into adipocytes (Fig. 4A) and bone cells (Fig. 4C) was confirmed by oil red O staining and Alizarin Red S staining, (PPARr, LPL) and bone cell specific genes (ALP, Runx2) were also highly expressed by RT-PCR (Fig. 4B and Fig. 4D). In contrast, it was confirmed that the pluripotency of CD105 negative fibroblasts was very weak.

Claims (7)

삭제delete 삭제delete CD105에 특이적으로 결합하는 항체 및 CD73에 특이적으로 결합하는 항체를 포함하며, 지방세포 및 골세포로 분화가 가능한 줄기세포능을 가진 피부 진피 섬유아세포 분리용 키트.A kit for separating skin dermis fibroblasts, comprising an antibody specifically binding to CD105 and an antibody specifically binding to CD73, and having stem cell potential capable of differentiating into adipocytes and bone cells. 제3항에 있어서, 상기 항체는 모노클로날 항체 또는 폴리클로날 항체인 것을 특징으로 하는 지방세포 및 골세포로 분화가 가능한 줄기세포능을 가진 피부 진피 섬유아세포 분리용 키트.The kit for separating dermal fibroblasts according to claim 3, wherein the antibody is a monoclonal antibody or polyclonal antibody, and has stem cell potential capable of differentiating into adipocytes and bone cells. 피부에서 진피 섬유아세포를 분리하는 단계; 및
상기 분리된 진피 섬유아세포에서 CD105를 발현하는 CD105 양성세포이면서, CD73을 발현하지 않는 CD73 음성세포를 분리하는 단계를 포함하는 지방세포 및 골세포로 분화가 가능한 줄기세포능을 가진 피부 진피 섬유아세포 분리방법.Separating dermal fibroblasts from the skin; And
And separating CD73-negative cells that do not express CD73 while being CD105-positive cells expressing CD105 in the isolated dermal fibroblasts, and dividing the dermal fibroblast cells having stem cell potential capable of differentiating into adipocytes and osteoblasts Way. 제5항에 있어서, 상기 CD105를 발현하는 CD105 양성세포이면서, CD73을 발현하지 않는 CD73 음성세포를 분리하는 단계는 자석 활성 세포 분류법(Magnetic activated cell sorting; MACS) 또는 유세포 분석(Flow cytometry Analysis)을 통해 분리하는 것을 특징으로 하는 지방세포 및 골세포로 분화가 가능한 줄기세포능을 가진 피부 진피 섬유아세포 분리방법.6. The method according to claim 5, wherein the step of isolating CD73-negative cells that are CD105-positive cells expressing CD105 but not CD73 is performed by Magnetic Activated Cell Sorting (MACS) or Flow cytometry Analysis The present invention relates to a method for separating dermal fibroblasts from a dermal fibroblast. CD105를 발현하면서, CD73은 발현하지 않고, 지방세포 및 골세포로 분화가 가능한 줄기세포능을 가진 피부 진피 섬유아세포를 유효성분으로 포함하는 피부조직재생용 조성물. A composition for regenerating a skin tissue comprising CD165 and a skin dermal fibroblast having stem cell potential capable of differentiating into adipocytes and osteocytes without expressing CD73.
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