WO2015088153A1 - Composition containing sagunjatang as active ingredient for promoting reprogramming of cells into induced pluripotent stem cells, and method for preparing induced pluripotent stem cells using same - Google Patents

Composition containing sagunjatang as active ingredient for promoting reprogramming of cells into induced pluripotent stem cells, and method for preparing induced pluripotent stem cells using same Download PDF

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WO2015088153A1
WO2015088153A1 PCT/KR2014/011124 KR2014011124W WO2015088153A1 WO 2015088153 A1 WO2015088153 A1 WO 2015088153A1 KR 2014011124 W KR2014011124 W KR 2014011124W WO 2015088153 A1 WO2015088153 A1 WO 2015088153A1
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cells
induced pluripotent
stem cells
pluripotent stem
reprogramming
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French (fr)
Korean (ko)
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김기모
허덕림
방옥선
김노수
이준
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한국한의학연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/76—Undefined extracts from plants

Definitions

  • composition for promoting reprogramming of cells into induced pluripotent stem cells including Sagunja-tang as an active ingredient, and a method for producing induced pluripotent stem cells using the same
  • the present invention was made by the task number C13040 in support of the Ministry of Science, ICT and Future Planning, the research management specialized organization of the task is the Korea Institute of Oriental Medicine, the research project name is “creative research project”, the research title is “medicinal herbs based pluripotent induction stem cells ( ips) Development of technology to enhance efficiency ", The lead institution is Korea Institute of Oriental Medicine.
  • the period of research is 2013.02.01 ⁇ 2013.11.30.
  • the present invention relates to a composition for promoting reprogramming into induced pluripotent stem cells (iPSc) of differentiated cells, including Sagunja-tang as an active ingredient, and a method for producing induced pluripotent stem cells using the same.
  • iPSc induced pluripotent stem cells
  • Induced pluripotent progenitor cells refer to cells in which differentiated cells that did not have pluripotency were induced to have pluripotent capacity through artificial dedifferentiation process.
  • 1 Cell shape (isometric, large nucleus and phosphorus, small number) Cytoplasm) and growth rate (embryonic stem cell division time: 17 hr) are similar to embryonic stem cells, 2 gene expression and chromatin modification patterns are similar to embryonic stem cells, 3 pluripotent differentiation ability 4 Can form teratoma in immunodeficient mice, 5 form chimera mouse when inserted into blastocyst of mouse, 6 has germ line transmission of gene .
  • This dedifferentiation technique enables the patient-somatic cells to obtain autologous somatic cells in a relatively easy way, with little physical damage and discomfort to the patient, and thereby to produce induced pluripotent stem cells having characteristics such as human embryonic cells.
  • the company has evolved a strategy to establish a customized self-differentiating stem cell line, and it is recognized as an optimal solution to overcome the bioethics controversy and immunocompatibility problems that may occur when using human embryonic stem cells. It presents unlimited possibilities in the field of development.
  • these dedifferentiated pluripotent stem cells enable the development of immune immunosynthetic cell therapeutics for patients, and may cause differentiation into cells such as brain or heart, which are difficult to obtain cells, to identify the causes and treatment methods for brain diseases and heart diseases.
  • Sagunja-tang is a medicinal herb that consists of ginseng (Panax ginseng), bokyeong (Per ia cocos), baekrye (At ractylodi sj aponi ca) and licorice (Glycyrrhi za uralens is).
  • One object of the present invention is to provide a composition for promoting reprogramming into induced pluripotent stem cells (iPSc) of differentiated cells, including Sagunja-tang as an active ingredient.
  • iPSc induced pluripotent stem cells
  • Another object of the present invention is to provide a method for promoting reprogramming of differentiated cells into induced pluripotent stem cells, comprising the step of treating Sagunja-tang to differentiated cells into which reprogramming factors have been introduced.
  • the present invention provides a composition for promoting reprogramming of differentiated cells into induced pluripotent stem cells (iPSc) comprising Sagunja-tang as an active ingredient.
  • the term "Sagunja-tang" means herbal medicines derived from ' Ginseng (Panax ginseng), Bokryeong (Peria cocos), Baekchul (Atractylodis japonica) and licorice (Glycyrrhiza uralensis) as a medicinal herb.
  • Sagunjatang of the present invention is preferably a hot water extract of ginseng, Bokyeong, Baekchul and Licorice.
  • hot water extraction is carried out with 1-2: 1-2: 1—2: 1- 2 weight ratio of ginseng, bokyeong, baekrye and licorice in 5 ⁇ 15 times of water and heated at 90 ⁇ 100 ° C for 1 ⁇ 4 hours. It can be done by.
  • the Sagunjatang may be commonly used with 'SGT-4'.
  • when treating the Sagunja-tang to differentiated cells introduced with a reprogramming induction factor it was found that the reprogramming to induced pluripotent cells can be promoted, and the recombination of the differentiated cells into induced pluripotent stem cells It was first identified that it can be used for promoting programming or reverse differentiation.
  • induced pluri otent stem cell refers to cell differentiation into differentiated somatic cells or cells with limited differentiation capacity. It is a cell that induces pluripotency, such as embryonic stem cells, which is returned to the cell stage before differentiation through injection of related genes and reprogramming. These induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically, show similar cell shapes, gene and protein expression patterns are similar, and have the characteristics of differentiation in and out of the body. These induced pluripotent stem cells have the same pluripotency as human embryonic stem cells, but do not involve ethical problems such as human embryonic stem cells because they are derived from already differentiated somatic cells rather than obtained from female eggs.
  • reprogramming refers to a process by which differentiated cells can be restored or converted into a state having pluripotent.
  • the reprogramming includes any process of returning differentiated cells having a differentiation capacity of 0% to less than 100% to an undifferentiated state, preferably, differentiated cells having a differentiation capacity of 0% or more than 0%. It includes both restoring or converting partially differentiated cells having a differentiation capacity of less than 100% into cells having 100% differentiation ability.
  • Such reprogramming may be performed by a reprogramming factor.
  • reprogramming inducer 1 refers to a substance that, when processed or expressed in differentiated cells, induces iPSc-specific gene expression and induces induced pluripotent stem cells having pluripotency. Induction of reprogramming Factors may be included in the present invention as long as the substance induces reprogramming of differentiated cells, and examples thereof may include 0ct3 / 4, sox-2, c-Myc, and Kl f-4. It can select according to the kind of cell and is not limited to the above example.
  • Differentiated cells into which the reprogramming inducer may be introduced may be cells derived from various animals such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits, preferably humans It may be a cell derived from, but is not limited to the differentiated cells that can be differentiated into induced pluripotent stem cells.
  • Such differentiated cells may be somatic cells.
  • Such a somatic cell is a cell constituting an adult means a cell having limited differentiation and self-producing ability, and the kind thereof is not particularly limited, but may be, for example, fibroblast.
  • 0ct3 / 4, c-Myc, Kl f4 and sox-2 were introduced into human fibroblasts, and oct4, sox-2 and Nanog expression was confirmed in the introduced cells.
  • the results showed that the pluripotent stem cell marker protein was expressed (FIG. 2).
  • four groups of fibroblasts into which four genes were introduced were treated with four-fold ethanol, induced pluripotent stem cell marker genes Nanog, sox-2, and oct4.
  • the present invention provides a method for promoting reprogramming of differentiated cells into induced pluripotent stem cells, comprising the step of treating Sagunja-tang to differentiated cells into which reprogramming factors have been introduced.
  • the reprogramming factor, differentiated cells, Sagunja-tang, induced pluripotent cells and reprogramming are as described above.
  • the method specifically includes the steps of: (a) introducing a reprogramming factor into differentiated cells; And (b) treating Sagunja-tang with the differentiated cells into which the reprogramming factor is introduced.
  • the method of introducing a reprogramming factor into the differentiated cells can be used without limitation the method of providing a substance to the cells commonly used in the art, according to the type of reprogramming inducer, differentiation of the reprogramming inducer
  • the method of administering to the culture of the cells, or the method of directly injecting the reprogramming inducer into the differentiated cells can be used, wherein the reprogramming inducer used is packaged transfected with a viral vector inserted the gene of the factor Viruses obtained from cells, plasmid vectors containing the corresponding inducer genes (e.g. epi some vectors), messenger RNAs produced by in vitro transcrt ion, or proteins produced in various cell lines It can be used in the form of.
  • the method of directly injecting the reprogramming inducer into the differentiated cells may be used by selecting any method known in the art, but is not limited thereto, and may include, but are not limited to, micro crosting method, electroporation method ( El ectroporat ion, particle injection method, direct muscle injection method, insulator and transposon method may be appropriately selected and applied. -... —
  • the differentiated cells into which the reprogramming factor is introduced may be cultured according to appropriate media and culture conditions known in the art, and may be treated by adding to Sagunjatanol medium during such culture.
  • culture conditions for inducing reprogramming hES (human embryonic stem cell) culture conditions may be used, but are not particularly limited thereto.
  • Sagunjatang of the present invention effectively enhance the efficiency of induced pluripotent stem cells, can be effectively used in the field of production of induced pluripotent stem cells.
  • Figure 1 shows the iPSc manufacturing process of the present invention.
  • Figure 2 shows the results of confirming the expression level of oct4, Sox-2 and Nanog in human fibroblasts introduced with 0ct3 / 4, sox-2, lf4 and Myc by immunocytochemical analysis.
  • FIG. 3 is a diagram illustrating the expression of iPSc marker genes Nanog, sox-2, oct4 and Klf4 by treating Sagunja-tang with human fibroblasts into which 0ct3 / 4, sox-2, Klf4 and Myc were introduced.
  • FIG. 4 is a diagram showing the treatment of Sagunja-tang in human fibroblasts into which 0ct3 / 4, sox-2, lf4 and Myc were introduced, and the degree of induction into iPSc was confirmed by AP lkaline phosphatase staining.
  • HFF Human foreskin-derived fibroblasts
  • Episomal vector purchased from addgene for iPSc induction in subculture of human HFF cells (pCXLE-0ct3 / 4, pCXLE-Sox-2, pCXLE-K.lf4, pCXLE-Myc, hereinafter referred to as 'OSK') After transforming each of the bacteria to grow, episomal DNA was isolated from each of the measured concentrations and stored at -70 ° C.
  • Immunostaining was performed on gelatin-coated four well chamber slides and was specifically performed as follows.
  • cells induced by 0SKM were fixed with 4% paraformaldehyde, washed with PBS, and then permeabilized (in PBS / 0.2% BSA, 0.1% Triton X-100), and blocking with 4% BSA at room temperature. 1 hour at. After blocking, antibodies bound to oc, Sox-2 or Nanog, respectively, were stained by diluting at a ratio of 1: 100 in PBS / 0.2% BSA. After washing, the secondary antibody was stained with FITC- or Alexa 488, 594-conjugated secondary antibody at room temperature for 1 hour, and then contrast-stained (act in) and observed under a microscope.
  • the group which introduced 0SKM (iPSc) and Sagunjatang here As a result, as shown in FIG. 3, the group which introduced 0SKM (iPSc) and Sagunjatang here.
  • the treated group of the present invention iPSC + SGT-4) showed the result of effectively expressing all markers of iPSc.
  • AP staining was purchased from System Bisciences and performed in the prescribed order. Specifically, the cells treated with SGT-4 by concentration at the same conditions as those transfected with 0SKM were incubated for 20 days, and then washed with PBS buffer, followed by citrate-acetone-formaldehyde (cit rate-acetone-formaldehyde). After fixing for 2 minutes), the AP staining solution was treated in the dark for 15 minutes, and the stained cells were observed under a microscope. As a result, as shown in Fig. 4, when treated with Sagunjatang of the present invention to fibroblasts transfected with 0SKM, the concentration-dependent AP stained colony number (purple: undifferentiated cells; colorless: differentiated cells) increased.

Abstract

The present invention relates to a composition containing Sagunjatang as an active ingredient for promoting the reprogramming of differentiated cells into induced pluripotent stem cells (iPSc), and to a method for preparing the induced pluripotent stem cells using the same. According to the present invention, the Sagunjatang of the present invention effectively promotes the efficiency of induced pluripotent stem cells, and thus can be effectively used in the field of preparing induced pluripotent stem cells.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
사군자탕을 유효성분으로 포함하는， 세포의 유도만능줄기세포로의 리프로그래밍 촉진용 조성물 및 이를 이용한 유도만능줄기세포의 제조방법  Composition for promoting reprogramming of cells into induced pluripotent stem cells, including Sagunja-tang as an active ingredient, and a method for producing induced pluripotent stem cells using the same
【기술분야】 ' [Technology] '
본 발명은 대한민국 미래창조과학부 지원하에 과제번호 C13040에 의해 이루어진 것으로서， 상기 과제의 연구관리전문기관은 한국한의학연구원， 연구사업명은 "창의연구사업" ， 연구과제명은 "한약처방 기반 만능유도 줄기세포 (ips) 효율 증진 기술개발" ， 주관기관은 한국한의학연구원， 연구기간은 2013.02.01 ~ 2013.11.30이다.  The present invention was made by the task number C13040 in support of the Ministry of Science, ICT and Future Planning, the research management specialized organization of the task is the Korea Institute of Oriental Medicine, the research project name is "creative research project", the research title is "medicinal herbs based pluripotent induction stem cells ( ips) Development of technology to enhance efficiency ", The lead institution is Korea Institute of Oriental Medicine. The period of research is 2013.02.01 ~ 2013.11.30.
본 발명은 사군자탕을 유효성분으로 포함하는， 분화된 세포의 유도만능줄기세포 (iPSc)로의 리프로그래밍 촉진용 조성물 및 이를 이용한 유도만능줄기세포의 제조방법에 관한 것이다.  The present invention relates to a composition for promoting reprogramming into induced pluripotent stem cells (iPSc) of differentiated cells, including Sagunja-tang as an active ingredient, and a method for producing induced pluripotent stem cells using the same.
[배경기술] [Background]
유도만능출기세포는， 만능분화능 (pluripotency)을 가지고 있지 않던 분화된 세포들이 인위적인 역분화 과정을 통해 만능분화능을 가지도록 유도된 세포들을 일컫는 말로서， ① 세포의 모양 (등근 모양, 큰 핵과 인, 적은 세포질)과 자라는 속도 (배아줄기세포의 분열시간: 17 hr)가 배아즐기세포와 유사하며, ② 유전자 발현 (gene expression) 과 염색체 변형 (chromatin modification) 패턴이 배아줄기세포와 유사하며， ③ 만능분화능을 가지며， ④ 면역 결핍 생쥐에서 테라토마를 형성할 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 progenitor cells refer to cells in which differentiated cells that did not have pluripotency were induced to have pluripotent capacity through artificial dedifferentiation process. ① Cell shape (isometric, large nucleus and phosphorus, small number) Cytoplasm) and growth rate (embryonic stem cell division time: 17 hr) are similar to embryonic stem cells, ② gene expression and chromatin modification patterns are similar to embryonic stem cells, ③ pluripotent differentiation ability ④ Can form teratoma in immunodeficient mice, ⑤ form chimera mouse when inserted into blastocyst of mouse, ⑥ has germ line transmission of gene .
2006년 세계 최초로 일본 교토대 야마나카 교수팀에 의해 다분화능 (Pluripotency)에 핵심적인 역할을 하는 리프로그래밍 유도 전사인자들 (0ct4, 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, a team of researchers from Yamanaka, Kyoto University, Japan, induced pluripotency from somatic cells through the complex overexpression of reprogramming induction transcription factors (0ct4, Sox-2, Klf4, cMyc), which play a key role in pluripotency. With the development of reverse differentiation technology capable of producing stem cells (Cell, 126: 663-676, 2006), Based on the technology, competition for the development of technology from all over the world is intensifying to lead the development of cell therapies and new drugs.
이러한 역분화 기술은 환자에게 신체적 손상 및 불편함이 거의 없는 비교적 손쉬운 방법으로 자가—체세포를 확보하고， 이로부터 인간배아즐기 세포와 같은 특성의 유도만능줄기세포를 제조하는 것을 가능케 함으로써， 환자- 체세포로부터 맞춤형 자가 전분화능 줄기세포주를 확립하는 전략을 획기적으로 진화시켰으며, 인간배아줄기세포 이용 시 야기될 수 있는 생명윤리 논란 및 면역 적합성 문제를 극복할 수 있는 최적의 해법으로 인식되면서 미래 재생의료 기술 개발 분야에 무한한 가능성을 제시하고 있다. 또한, 이러한 역분화 만능줄기세포는 환자 면역 저합형 세포 치료제 개발을 가능케 하며， 세포를 얻기 어려운 기관인 뇌나 심장 등의 세포로 분화 등을 가져을 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 남아있는 실정이어서， 유도만능줄기세포의 제조 효율을 증진시킬 수 있는 기술의 개발이 요구되어 왔다. 사군자탕은， 한국의 「동의보감」 에 수톡된 인삼 (Panax ginseng) , 복령 (Per i a cocos) , 백출 (At ractylodi s j aponi ca) 및 감초 (Glycyrrhi za uralens i s)를 구성 약재로 하는 탕제로서， 원기 보층을 가져올 수 있어， 기력 약화, 소화불량， 위염， 위산과다， 위궤양， 냉증 및 부종 등에 사용되어 왔다. 그러나， 이러한 사군자탕을 줄기세포에 적용한 예는 아직까지 보고된 바 없다. 이러한 배경 하에 본 발명자들은 유도만능줄기세포의 효율 증진을 가지고 을 수 있는 물질을 개발하고자 하였고， 그 결과 수십 종의 한약 처방 중에서 사군자탕을 리프로그래밍 인자를 발현하는 분화된 세포에 처리하는 경우， 유도만능줄기세포의 제조 효율이 대조군에 비하여 현저하게 증가하는 것을 확인하여, 본 발명을 완성하였다. 【발명의 상세한 설명】 This dedifferentiation technique enables the patient-somatic cells to obtain autologous somatic cells in a relatively easy way, with little physical damage and discomfort to the patient, and thereby to produce induced pluripotent stem cells having characteristics such as human embryonic cells. The company has evolved a strategy to establish a customized self-differentiating stem cell line, and it is recognized as an optimal solution to overcome the bioethics controversy and immunocompatibility problems that may occur when using human embryonic stem cells. It presents unlimited possibilities in the field of development. In addition, these dedifferentiated pluripotent stem cells enable the development of immune immunosynthetic cell therapeutics for patients, and may cause differentiation into cells such as brain or heart, which are difficult to obtain cells, to identify the causes and treatment methods for brain diseases and heart diseases. Has the advantage to do that. However, despite the excellence of dedifferentiation technology, there are still problems to be overcome such as low dedifferentiation efficiency and long time frame for dedifferentiation process in order to utilize it in the stage of cell therapy and drug development. Subsequently, there has been a demand for the development of a technology capable of improving the production efficiency of induced pluripotent stem cells. Sagunja-tang is a medicinal herb that consists of ginseng (Panax ginseng), bokyeong (Per ia cocos), baekrye (At ractylodi sj aponi ca) and licorice (Glycyrrhi za uralens is). Has been used to weaken, dyspepsia, gastritis, gastric acid, stomach ulcers, cold and edema. However, an example of applying Sagunja-tang to stem cells has not been reported. Against this background, the present inventors have developed a substance capable of improving the efficiency of induced pluripotent stem cells, and as a result, when treating Sagunja-tang with differentiated cells expressing reprogramming factors among dozens of Chinese medicine prescriptions, induced pluripotency It was confirmed that the production efficiency of stem cells is significantly increased compared to the control, to complete the present invention. [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
본 발명의 하나의 목적은 사군자탕을 유효성분으로 포함하는， 분화된 세포의 유도만능줄기세포 (iPSc)로의 리프로그래밍 촉진용 조성물을 제공하는 것이다.  One object of the present invention is to provide a composition for promoting reprogramming into induced pluripotent stem cells (iPSc) of differentiated cells, including Sagunja-tang as an active ingredient.
본 발명의 다른 목적은 리프로그래밍 인자가 도입된 분화된 세포에 사군자탕을 처리하는 단계를 포함하는， 분화된 세포의 유도만능줄기세포로의 리프로그래밍 촉진 방법을 제공하는 것이다.  Another object of the present invention is to provide a method for promoting reprogramming of differentiated cells into induced pluripotent stem cells, comprising the step of treating Sagunja-tang to differentiated cells into which reprogramming factors have been introduced.
【기술적 해결방법】 Technical Solution
상기의 목적을 달성하기 위한 하나의 양태로서， 본 발명은 사군자탕을 유효성분으로 포함하는， 분화된 세포의 유도만능줄기세포 (iPSc)로의 리프로그래밍 촉진용 조성물을 제공한다. 본 발명에서 용어， "사군자탕"은 인삼 (Panax ginseng), 복령 (Peria cocos), 백출 (Atractylodis japonica) 및 감초 (Glycyrrhiza uralensis)를 구성 약재로' 하는 한방유래의 탕약을 의미한다. 본 발명의 사군자탕은 인삼, 복령， 백출 및 감초의 열수 추출물인 것이 바람직하다. 구체적으로 열수 추출은 1-2:1-2:1—2:1- 2 중량비의 인삼， 복령， 백출 및 감초를 5~15배의 물에 넣고 90~100°C 온도로 1~4시간 가열하여 이루어질 수 있다. 본 발명에서 상기 사군자탕은 'SGT-4'와 흔용될 수 있다. 본 발명에서는， 이러한 사군자탕을 리프로그래밍 유도 인자를 도입한 분화된 세포에 처리하는 경우， 유도만능즐기세포로의 리프로그래밍을 촉진시킬 수 있음을 규명하여， 이를 분화된 세포의 유도만능줄기세포로의 리프로그래밍 또는 역분화 촉진 용도로 사용할 수 있음을 최초로 규명하였다ᅳ 본 발명에서 용어， "유도만능줄기세포 (induced pluri otent stem cell, iPSc)"는 분화가 끝난 체세포 또는 제한된 분화능을 가지는 세포에 세포 분화 관련 유전자를 주입하여 리프로그래밍 과정을 통해 분화 이전의 세포 단계로 되돌린， 배아 줄기세포처럼 만능성을 유도해낸 세포를 말한다. 이러한 유도만능줄기세포는 배아줄기세포와 거의 같은 특성올 가지고 있는데， 구체적으로 비슷한 세포모양을 보여주고, 유전자 및 단백질 발현 패턴이 유사하며ᅳ 생체 내외에서 다분화능을 가지는 특성을 가지고 있다. 이러한 유도만능줄기세포는 인간 배아줄기세포와 같은 만능성을 가지고 있으나, 여성의 난자 등으로부터 획득하는 것이 아니라 이미 분화가 끝난 체세포 등에서 유도된 것이기 때문에 인간 배아줄기세포와 같은 윤리적 문제점을 수반하지 않으며, 환자의 체세포에서 줄기세포로 전환할 수 있기 때문에 면역거부 반응의 문제도 적다. 다만， 이러한 유도만능줄기세포의 유용성에도 불구하고 역분화 효율이 낮아 층분한 수의 유도만능즐기세포를 얻을 수 없다는 단점이 있었으나， 본 발명에서는 사군자탕을 리프로그래밍 인자를 포함하는 분화된 세포에 처리하였올 때， 유도만능줄기세포로의 리프로그래밍 효율을 효과적으로 증진시킬 수 있음을 확인하였다. 본 발명에서 용어， "리프로그래밍 (reprogramming) "이란 분화된 세포를 만능성 (plur ipotent )을 가지는 상태로 복원 또는 전환될 수 있는 프로세스를 의미한다. 본 발명에서 상기 리프로그래밍이란 0% 내지 100% 미만의 분화능을 가지는 분화된 세포들을 미분화 상태로 되돌리는 과정이라면 모두 이에 포함하며， 바람직하게는 0%의 분화능을 가지는 분화된 세포 또는 0% 초과 내지 100% 미만의 분화능을 가지는 일정부분 분화된 세포를 100% 분화능을 가지는 세포로 복원 또는 전환시키는 것을 모두 포함한다. As one aspect for achieving the above object, the present invention provides a composition for promoting reprogramming of differentiated cells into induced pluripotent stem cells (iPSc) comprising Sagunja-tang as an active ingredient. In the present invention, the term "Sagunja-tang " means herbal medicines derived from ' Ginseng (Panax ginseng), Bokryeong (Peria cocos), Baekchul (Atractylodis japonica) and licorice (Glycyrrhiza uralensis) as a medicinal herb. Sagunjatang of the present invention is preferably a hot water extract of ginseng, Bokyeong, Baekchul and Licorice. Specifically, hot water extraction is carried out with 1-2: 1-2: 1—2: 1- 2 weight ratio of ginseng, bokyeong, baekrye and licorice in 5 ~ 15 times of water and heated at 90 ~ 100 ° C for 1 ~ 4 hours. It can be done by. In the present invention, the Sagunjatang may be commonly used with 'SGT-4'. In the present invention, when treating the Sagunja-tang to differentiated cells introduced with a reprogramming induction factor, it was found that the reprogramming to induced pluripotent cells can be promoted, and the recombination of the differentiated cells into induced pluripotent stem cells It was first identified that it can be used for promoting programming or reverse differentiation. In the present invention, the term “induced pluri otent stem cell (iPSc)” refers to cell differentiation into differentiated somatic cells or cells with limited differentiation capacity. It is a cell that induces pluripotency, such as embryonic stem cells, which is returned to the cell stage before differentiation through injection of related genes and reprogramming. These induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically, show similar cell shapes, gene and protein expression patterns are similar, and have the characteristics of differentiation in and out of the body. These induced pluripotent stem cells have the same pluripotency as human embryonic stem cells, but do not involve ethical problems such as human embryonic stem cells because they are derived from already differentiated somatic cells rather than obtained from female eggs. The problem of immune rejection is less because it can switch from somatic cells to stem cells. However, despite the usefulness of such induced pluripotent stem cells, there was a disadvantage in that a low number of induced pluripotent enjoyable cells could not be obtained due to a low reverse differentiation efficiency, but in the present invention, Sagunja-tang was treated to differentiated cells containing a reprogramming factor. When it came, it was confirmed that the reprogramming efficiency to induced pluripotent stem cells can be effectively enhanced. As used herein, the term "reprogramming" refers to a process by which differentiated cells can be restored or converted into a state having pluripotent. In the present invention, the reprogramming includes any process of returning differentiated cells having a differentiation capacity of 0% to less than 100% to an undifferentiated state, preferably, differentiated cells having a differentiation capacity of 0% or more than 0%. It includes both restoring or converting partially differentiated cells having a differentiation capacity of less than 100% into cells having 100% differentiation ability.
이러한 리프로그래밍은 리프로그래밍 유도인자 (reprogramming factor )에 의하여 수행될 수 있다.  Such reprogramming may be performed by a reprogramming factor.
본 발명에서 용어， "리프로그래밍 유도인자1'는 분화된 세포에 처리 또는 발현되는 경우 iPSc 특이적 유전자의 발현을 가져와 만능성을 가지는 유도만능줄기세포로 유도하는 물질을 의미한다. 상기 리프로그래밍 유도인자는 분화된 세포의 리프로그래밍을 유도하는 물질이면 본 발명에 제한 없이 포함될 수 있으며， 그 예로 0ct3/4 , sox-2 , c-Myc 및 Kl f-4 등을 들 수 있으나， 적용할 세포의 종류에 따라 선택할 수 있으며 상기 예에 제한되는 것은 아니다. As used herein, the term "reprogramming inducer 1 " refers to a substance that, when processed or expressed in differentiated cells, induces iPSc-specific gene expression and induces induced pluripotent stem cells having pluripotency. Induction of reprogramming Factors may be included in the present invention as long as the substance induces reprogramming of differentiated cells, and examples thereof may include 0ct3 / 4, sox-2, c-Myc, and Kl f-4. It can select according to the kind of cell and is not limited to the above example.
이러한 리프로그래밍 유도인자가 도입되어 리프로그래밍이 유도되는 분화된 세포에， 본 발명의 사군자탕을 처리하는 경우， 리프로그래밍 유도인자에 의한 iPSc 세포로의 유도 효율을 증진되게 된다.  When such a reprogramming inducer is introduced to the differentiated cells in which reprogramming is induced, when the Sagunjatang of the present invention is treated, induction efficiency into iPSc cells by the reprogramming inducer is enhanced.
상기 리프로그래밍 유도인자가 도입될 수 있는 분화된 세포는， 인간, 원숭이， 돼지， 말, 소, 양, 개， 고양이， 생쥐 및 토끼 등의 다양한 동물로부터 유래한 세포일 수 있으며， 바람직하게는 인간으로부터 유래한 세포일 수 있으나， 이에 의하여 유도만능줄기세포로 역분화시킬 수 있는 분화된 세포가 제한되는 것은 아니다.  Differentiated cells into which the reprogramming inducer may be introduced may be cells derived from various animals such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits, preferably humans It may be a cell derived from, but is not limited to the differentiated cells that can be differentiated into induced pluripotent stem cells.
또한， 이러한 분화된 세포는 체세포일 수 있다. 이러한 체세포는 성체를 구성하는 세포로서 분화능 및 자가 생산능이 제한된 세포를 의미하며， 특별히 그 종류가 제한되는 것은 아니나， 그 예로 섬유 아세포 일 수 있다. 본 발명의 일 실시예에서는, 0ct3/4 , c-Myc , Kl f4 및 sox-2를 인간 섬유아세포에 도입하였고， 도입된 세포에서 oct4 , sox-2 및 Nanog 발현을 확인한 결과， 상기 3가지 유도만능줄기세포 마커 단백질을 발현하고 았는 결과를 나타내었으며 (도 2)， 또한， 상기 4가지 유전자가 도입된 섬유아세포에 사군자탕올 처리한 경우, 유도만능줄기세포 마커 유전자인 Nanog , sox-2 , oct4 및 Kl f4를 발현하는 결과를 나타내었다 (도 3) . 또한, 상기 4가지 단백질을 발현하는 인간 섬유아세포를 hEhES (human embryoni c stem cel l ) 배지 조건 하에서 배양하면서， 사군자탕을 농도별로 처리하고 AP(Alkal ine phosphase) 염색을 수행하여 유도만능줄기세포로의 전환 효율을 확인한 결과， 상기 4가지 유전자만 도입된 대조군에 비하여 본 발명의 사군자탕을 처리한 군의 유도만능줄기세포로의 리프로그래밍 효율이 현저히 높았으며， 사군자탕의 농도에 의존적인 결과를 나타내어， 본 발명의 사군자탕이 리프로그래밍 인자에 의한 유도만능줄기세포 유도에 있어 그 효율을 증진시켜줄 수 있는 물질임을 시사하였다 (도 4) . 상기와 같은 결과들은 본 발명의 사군자탕은 분화된 세포의 유도만능줄기세포로의 리프로그래밍을 촉진하는 물질로서 사용될 수 있음을 시사한다ᅳ 또 하나의 양태로서, 본 발명은 리프로그래밍 인자가 도입된 분화된 세포에 사군자탕을 처리하는 단계를 포함하는 , 분화된 세포의 유도만능줄기세포로의 리프로그래밍 촉진 방법을 제공한다. In addition, such differentiated cells may be somatic cells. Such a somatic cell is a cell constituting an adult means a cell having limited differentiation and self-producing ability, and the kind thereof is not particularly limited, but may be, for example, fibroblast. In one embodiment of the present invention, 0ct3 / 4, c-Myc, Kl f4 and sox-2 were introduced into human fibroblasts, and oct4, sox-2 and Nanog expression was confirmed in the introduced cells. The results showed that the pluripotent stem cell marker protein was expressed (FIG. 2). Also, when four groups of fibroblasts into which four genes were introduced were treated with four-fold ethanol, induced pluripotent stem cell marker genes Nanog, sox-2, and oct4. And Kl f4 were expressed (FIG. 3). In addition, while culturing human fibroblasts expressing the four proteins under hEhES (human embryonic stem cell) medium conditions, Sagunja-tang was treated by concentration and AP (Alkal ine phosphase) staining to induced pluripotent stem cells. As a result of confirming the conversion efficiency, the reprogramming efficiency to induced pluripotent stem cells of the group treated with Sagunja-tang of the present invention was significantly higher than that of the control group into which only the four genes were introduced, and the results were dependent on the concentration of Sagunja-tang. It was suggested that Sagunja-tang of the present invention is a substance that can enhance the efficiency in induction of induced pluripotent stem cells by reprogramming factors (FIG. 4). These results suggest that Sagunja-tang of the present invention can be used as a substance for promoting reprogramming of differentiated cells into induced pluripotent stem cells. In another aspect, the present invention provides a method for promoting reprogramming of differentiated cells into induced pluripotent stem cells, comprising the step of treating Sagunja-tang to differentiated cells into which reprogramming factors have been introduced.
상기 리프로그래밍 인자， 분화된 세포， 사군자탕， 유도만능즐기세포 및 리프로그래밍에 대해서는 앞서 설명한 바와 같다.  The reprogramming factor, differentiated cells, Sagunja-tang, induced pluripotent cells and reprogramming are as described above.
상기 방법은 구체적으로， (a) 분화된 세포에 리프로그래밍 인자를 도입하는 단계; 및 (b) 상기 리프로그래밍 인자가 도입된 분화된 세포에 사군자탕을 처리하는 단계를 포함할 수 있다.  The method specifically includes the steps of: (a) introducing a reprogramming factor into differentiated cells; And (b) treating Sagunja-tang with the differentiated cells into which the reprogramming factor is introduced.
여기서, 분화된 세포에 리프로그래밍 인자를 도입하는 방법은 당업계에서 통상적으로 사용되는 세포에 물질을 제공하는 방법을 제한없이 사용할 수 있으며， 리프로그래밍 유도인자의 종류에 따라， 리프로그래밍 유도인자를 분화된 세포의 배양액에 투여하는 방법 또는 리프로그래밍 유도인자를 분화된 세포 내에 직접 주입하는 방법 둥을 사용할 수 있으며, 이때 사용되는 리프로그래밍 유도인자는 해당 인자의 유전자를 삽입한 바이러스 백터로 형질감염시킨 패키징 세포로부터 수득한 바이러스， 해당 유도인자 유전자를 포함하는 플라스미드 백터 (예， epi some vector ) , 시험관 내 전사 ( in vi t ro transcr ipt ion)에 의해 생산한 메신저 RNA , 또는 다양한 세포주 내에서 생산된 단백질 등의 형태로 사용할 수 있다.  Here, the method of introducing a reprogramming factor into the differentiated cells can be used without limitation the method of providing a substance to the cells commonly used in the art, according to the type of reprogramming inducer, differentiation of the reprogramming inducer The method of administering to the culture of the cells, or the method of directly injecting the reprogramming inducer into the differentiated cells can be used, wherein the reprogramming inducer used is packaged transfected with a viral vector inserted the gene of the factor Viruses obtained from cells, plasmid vectors containing the corresponding inducer genes (e.g. epi some vectors), messenger RNAs produced by in vitro transcrt ion, or proteins produced in various cell lines It can be used in the form of.
상기 리프로그래밍 유도인자를 분화된 세포에 직접 주입하는 방법은 당업계에 공지된 임의의 방법을 선택하여 사용할 수 있으며， 이에 제한되지는 않으나， 미세주입법 (mi croi j ect ion) , 전기천공법 (el ectroporat ion) , 입자 분사법 (part i cle bombardment ) , 직접근육주입법 , 인슐레이터 ( insulator ) 및 트랜스포존을 이용한 방법 중에서 적절하게 선택하여 적용할 수 있다. -…— 상기와 같은 과정 등으로 리프로그래밍 인자가 도입된 분화된 세포는 당업계에 알려진 적당한 배지와 배양조건에 따라 배양될 수 있으며， 이러한 배양 과정 중에 사군자탕올 배지에 첨가하여 처리할 수 있다. 리프로그래밍을 유도하기 위한 배양 조건으로, hES (human embryoni c stem cel l ) 배양 조건을 사용할 수 있으나, 특별히 이에 제한되는 것은 아니다. 【유리한 효과】 The method of directly injecting the reprogramming inducer into the differentiated cells may be used by selecting any method known in the art, but is not limited thereto, and may include, but are not limited to, micro crosting method, electroporation method ( El ectroporat ion, particle injection method, direct muscle injection method, insulator and transposon method may be appropriately selected and applied. -… — The differentiated cells into which the reprogramming factor is introduced may be cultured according to appropriate media and culture conditions known in the art, and may be treated by adding to Sagunjatanol medium during such culture. As culture conditions for inducing reprogramming, hES (human embryonic stem cell) culture conditions may be used, but are not particularly limited thereto. Advantageous Effects
본 발명의 사군자탕은, 유도만능줄기세포의 효율을 효과적으로 증진시키므로， 유도만능줄기세포의 제조 분야에 효과적으로 이용될 수 있다 . 【도면의 간단한 설명】  Sagunjatang of the present invention, effectively enhance the efficiency of induced pluripotent stem cells, can be effectively used in the field of production of induced pluripotent stem cells. [Brief Description of Drawings]
도 1은， 본 발명의 iPSc 제조 과정을 나타낸 것이다.  Figure 1 shows the iPSc manufacturing process of the present invention.
도 2는, 0ct3/4, sox-2 , lf4 및 Myc을 도입한 인간 섬유아세포에 대하여 oct4, Sox-2 및 Nanog의 발현 정도를 면역세포화학 분석법으로 확인한 결과를 나타낸 것이다.  Figure 2 shows the results of confirming the expression level of oct4, Sox-2 and Nanog in human fibroblasts introduced with 0ct3 / 4, sox-2, lf4 and Myc by immunocytochemical analysis.
도 3은, 0ct3/4, sox-2, Klf4 및 Myc을 도입한 인간 섬유아세포에 사군자탕을 처리하여， iPSc 마커 유전자인 Nanog, sox-2, oct4 및 Klf4의 발현을 확인한 도이다.  3 is a diagram illustrating the expression of iPSc marker genes Nanog, sox-2, oct4 and Klf4 by treating Sagunja-tang with human fibroblasts into which 0ct3 / 4, sox-2, Klf4 and Myc were introduced.
도 4는， 0ct3/4, sox-2, lf4 및 Myc을 도입한 인간 섬유아세포에 사군자탕을 처리하고， iPSc로의 유도 정도를 AP lkaline phosphatase) 염색으로 확인한 도이다.  4 is a diagram showing the treatment of Sagunja-tang in human fibroblasts into which 0ct3 / 4, sox-2, lf4 and Myc were introduced, and the degree of induction into iPSc was confirmed by AP lkaline phosphatase staining.
【발명의 실시를 위한 형태】 [Form for implementation of invention]
이하 본 발명을 하기 예에 의해 상세히 설명한다. 다만 하기 예는 본 발명을 예시하기 위한 것일 뿐， 하기 예에 의해 본 발명의 범위가 제한되는 것은 아니다. 실시예: 사군자탕 (SGT-4)의 제조  Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples. Example: Preparation of Sagunjatang (SGT-4)
사누자탕의 구성 한약재를 하기 표 1과 같은 무게 비율로 배합하여， 약탕기 (Cosmos 660， Inchon, Korea)에 넣고 , 물을 시료의 10배로 첨가하여 100°C에서 2시간 전탕한 후， 농축 및 동결건조하여 22.3%의 수율로 추출물을 얻었다. 얻어진 추출물 중 스록 (stock)의 농도는 2mg/mL이었으며， 시료는 - 70°C에 넣어 보관하였다. 【표 1】 Constituents of Sanuja-tang mixed in the weight ratio as shown in Table 1 below, put into a medicine bath (Cosmos 660, Inchon, Korea), add water 10 times of the sample and warmed at 100 ° C for 2 hours, concentrated and frozen Dried to obtain an extract in 22.3% yield. The concentration of stock in the obtained extract was 2 mg / mL, and the sample was stored at -70 ° C. Table 1
Figure imgf000010_0001
실험예: 사군자탕의 iPS (induced pluripotent stem cell) 효율 증진 효과
Figure imgf000010_0001
Experimental Example: Effect of Sagunja-tang on Enhancing iPS (induced pluripotent stem cell) Efficiency
(1) 세포 배양 (1) cell culture
인간 foreskin 유래 섬유아세포 (HFF)는 System Biosciences에서 구입하였으며， 항생제를 추가하지 않은 조건에 FBS(fetal bovine serum) 10%가 포함된 DMEM(Dulbecco's Modified Eagle Medium) 배지를 사용하여 계대 배양을 수행하였다.  Human foreskin-derived fibroblasts (HFF) were purchased from System Biosciences, and passaged using Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% FBS (fetal bovine serum) under conditions without antibiotics.
(2) 에피솜 백터 (episomal vector)를사용한 iPSc의 제조 (2) Preparation of iPSc Using Episomal Vector
인간 HFF 세포를 계대 배양하면서， iPSc 유도를 위해 addgene 사에서 구입한 에피솜 백터 (pCXLE-0ct3/4, pCXLE-Sox-2 , pCXLE-K.lf4, pCXLE-Myc, 이하 'OSK '으로 명명)를 각각 박테리아에 형질전환하여 키운 후， 에피솜 DNA를 각각 분리하여 농도를 측정한 후 -70°C에 보관하였다. 그 다음， 세포 (6X105개 /ml)에 각각 lyg/ml 농도로 마이크로포레이터로 전기천공올 수행하였으며， 배양 후 5일째 된 세포를 트립신 처리하고， 세포수를 계수한 후， 재분주 (reseeding)하였다. 그 다음, hES(human embryonic stem cell) 배지 조건 하에서 SGT-4를 농도별 처리하였으며, 매일 배지를 갈아주면서 약물을 농도별로 처리하였고， 이렇게 20일 배양한 다음， AP(Mkaline Phosphatase)-염색을 수행하였다. 또한， 도 3에 표시된 해당 기간에 RNA 분석 역시 수행하였다. 이러한 iPSc의 제조 과정을 도 1에 모식도로 나타내었다. Episomal vector purchased from addgene for iPSc induction in subculture of human HFF cells (pCXLE-0ct3 / 4, pCXLE-Sox-2, pCXLE-K.lf4, pCXLE-Myc, hereinafter referred to as 'OSK') After transforming each of the bacteria to grow, episomal DNA was isolated from each of the measured concentrations and stored at -70 ° C. Then, cells (6X10 5 / ml) were electroporated with microporator at a concentration of lyg / ml, respectively, and trypsinized the cells 5 days after culture, counting the number of cells, and then reseeding ) Subsequently, the concentration of SGT-4 was treated under human embryonic stem cell (hES) medium conditions. After 20 days of incubation, AP (Mkaline Phosphatase) -staining was performed. In addition, RNA analysis was also performed during the period indicated in FIG. 3. The manufacturing process of this iPSc is shown in the schematic diagram in FIG.
(3) 면역 염색 (3) immunostaining
면역염색 수행은, 젤라틴-코팅된 4 웰 챔버 슬라이드 상에서 수행하였으며， 구체적으로 하기와 같은 과정으로 수행하였다.  Immunostaining was performed on gelatin-coated four well chamber slides and was specifically performed as follows.
구체적으로， 0SKM에 의해 유도된 세포를 4% 파라포름알데히드로 고정한 다음 PBS로 세척하였고 그 다음 permeabilized(in PBS/0.2% BSA, 0.1% Triton X-100)하였으며, 4% BSA로 블톡킹을 상온에서 1시간 수행하였다. 블록킹 후에 각각 oc , Sox-2 또는 Nanog에 결합하는 항체를 PBS/0.2% BSA에서 1:100의 비율로 희석해서 염색하였다. 세척 후 2차 항체에 FITC- 혹은 Alexa 488, 594- 접합된 2차 항체로 상온에서 1시간 염색하였고， 그 다음 대비 염색 (act in)을 수행한 후 현미경으로 관찰하였다.  Specifically, cells induced by 0SKM were fixed with 4% paraformaldehyde, washed with PBS, and then permeabilized (in PBS / 0.2% BSA, 0.1% Triton X-100), and blocking with 4% BSA at room temperature. 1 hour at. After blocking, antibodies bound to oc, Sox-2 or Nanog, respectively, were stained by diluting at a ratio of 1: 100 in PBS / 0.2% BSA. After washing, the secondary antibody was stained with FITC- or Alexa 488, 594-conjugated secondary antibody at room temperature for 1 hour, and then contrast-stained (act in) and observed under a microscope.
그 결과， 도 2에 나타낸 바와 같이， 0SKM을 형질감염시킨 세포는 모두 oct4, Sox-2 및 Nanog를 충분히 발현하고 있는 것을 나타내었다.  As a result, as shown in FIG. 2, all cells transfected with 0SKM fully expressed oct4, Sox-2 and Nanog.
(4) iPSc 클론의 iPSc마커 유전자 발현 분석 (4) iPSc marker gene expression analysis of iPSc clone
각각의 조건에서 배양된 iPSc 콜로니에서 전체 RNA를 분리하여 정량한 다음， iPSc의 마커로 알려진 유전자의 발현을 RT-PCR로 조사하였다. 구체적으로， 각각의 조건에서 배양된 iPSc 콜로니에서 Total RNA를 분리하여 정량한 다음 iPSc의 마커로 알려진 Nanog, sox-2, oct4, KIF4와 interal 대조군으로 GAPDH를 사용하여 "RTPCR을 수행하였으며 먼저 정량된 RNA 1 / 을 RT primix kit( ntron Inc, korea)를 사용하여 cDNA를 합성하고， 이를 주형으로 하여 , 하기 표 2의 Nanog, sox-2, oct4, KIF4, GAPDH 프라이머를 이용하여 SolgTM 2X Taq PCR(Solgent)을 사용하여 PCR 반웅을 수행하였다. PCR 수행 후 1 % 아가로스 겔로 전기영동 하여 bi으 rad densitometry로 정량하였고， 그 결과를 도 3에 나타내었다. 【표 2】 Total RNA was isolated and quantified from iPSc colonies cultured under the respective conditions, and then expression of genes known as markers of iPSc was examined by RT-PCR. Specifically, as quantified by separating the Total RNA from the iPSc colonies cultured in each condition, and then known as the iPSc markers Nanog, sox-2, oct4, KIF4 with interal control using the GAPDH "was performing RTPCR the first amount RNA 1 / was synthesized using the RT primix kit (ntron Inc, korea) cDNA, using as a template, SolgTM 2X Taq PCR using Nanog, sox-2, oct4, KIF4, GAPDH primers in Table 2 Solgent) was used to perform PCR reaction, followed by electrophoresis on 1% agarose gel and quantified by bi rad densitometry, and the results are shown in FIG. 3. Table 2
Figure imgf000012_0001
그 결과， 도 3에 나타낸 바와 같이， 0SKM을 도입한 군 ( iPSc) 및 여기에 사군자탕을. 처리한 본 발명의 군 ( iPSC+SGT-4)은 iPSc의 마커를 모두 효과적으로 발현하고 있는 결과를 나타내었다.
Figure imgf000012_0001
As a result, as shown in FIG. 3, the group which introduced 0SKM (iPSc) and Sagunjatang here. The treated group of the present invention (iPSC + SGT-4) showed the result of effectively expressing all markers of iPSc.
(5) AP (Alkal ine Phosphatase) 염색 (5) Alkal ine Phosphatase (AP) staining
AP 염색은 System Bi osci ences에서 구입하여 정해진 순서로 수행하였다. 구체적으로， 먼저 0SKM으로 형질감염한 조건과 같은 조건에서 SGT-4를 농도별로 처리한 세포를 20일 배양한 다음, PBS 완충액으로 세척한 후 시트레이트ᅳ아세톤ᅳ 포름알데히드 (c i t rate-acetone-formaldehyde)로 2분 고정한 다음， AP 염색 용액을 암실에서 15분 처리하였고， 이렇게 염색된 세포를 현미경에서 관찰하였다. 그 결과， 도 4에 나타낸 바와 같이, 0SKM을 형질감염시킨 섬유아세포에 본 발명의 사군자탕을 처리한 경우， 농도 의존적으로 AP 염색된 콜로니 수 (보라색: 미분화 세포; 무색: 분화된 세포)가 증가하는 결과를 보여 사군자탕이 농도 의존적으로 iPSc의 생산 효율을 중진시킬 수 있음을 확인하였다. 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. AP staining was purchased from System Bisciences and performed in the prescribed order. Specifically, the cells treated with SGT-4 by concentration at the same conditions as those transfected with 0SKM were incubated for 20 days, and then washed with PBS buffer, followed by citrate-acetone-formaldehyde (cit rate-acetone-formaldehyde). After fixing for 2 minutes), the AP staining solution was treated in the dark for 15 minutes, and the stained cells were observed under a microscope. As a result, as shown in Fig. 4, when treated with Sagunjatang of the present invention to fibroblasts transfected with 0SKM, the concentration-dependent AP stained colony number (purple: undifferentiated cells; colorless: differentiated cells) increased. The results showed that Sagunja-tang could increase the production efficiency of iPSc in a concentration-dependent manner. From the above description, those skilled in the art to which the present invention pertains should make the present invention in another specific form without changing the technical spirit or essential features. It will be appreciated that it may be practiced. In this regard, the above-described embodiments are to be understood as illustrative in all respects and not as restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims

【청구의 범위】 【Scope of Claim】
【청구항 1】 【Claim 1】
사군자탕을 유효성분으로 포함하는, 분화된 세포의 유도만능줄기세포 ( iPSc) 로의 리프로그래밍 촉진용 조성물. A composition for promoting reprogramming of differentiated cells into induced pluripotent stem cells (iPSc), comprising Sagunjatang as an active ingredient.
【청구항 21 【Claim 21
제 1항에 있어서， 상기 사군자탕은 인삼， 복령， 백출 및 감초의 열수 추출물인 것인 조성물. The composition of claim 1, wherein the Sagunjatang is a hot water extract of ginseng, Bokryeong, Baekchul, and licorice.
【청구항 3】 【Claim 3】
제 1항에 있어서， 상기 조성물은 0ct3/4 , sox-2 , c-Myc 및 Kl f4를 도입한 세포에 처리하는 것인 조성물. The composition of claim 1, wherein the composition is applied to cells into which 0ct3/4, sox-2, c-Myc, and Kl f4 have been introduced.
【청구항 4] [Claim 4]
제 1항에 있어서， 상기 세포는 인간으로부터 유래된 것인 조성물. The composition according to claim 1, wherein the cells are derived from humans.
【청구항 5】 【Claim 5】
제 1항에 있어서， 상기 분화된 세포는 섬유 아세포인 것인 조성물ᅳ The composition of claim 1, wherein the differentiated cells are fibroblasts.
【청구항 6】 【Claim 6】
리프로그래밍 인자가 도입된 분화된 세포에 사군자탕을 처리하는 단계를 포함하는， 분화된 세포의 유도만능줄기세포 ( iPSc)로의 리프로그래밍 촉진 방법. A method of promoting reprogramming of differentiated cells into induced pluripotent stem cells (iPSc), comprising the step of treating Sagunjatang to differentiated cells into which reprogramming factors have been introduced.
【청구항 7】 【Claim 7】
제 6항에 있어서， 상기 리프로그래밍은 0ct3/4 , sox-2 , c-Myc 및 Kl f4 인 방법. 【청구항 8] The method of claim 6, wherein the reprogramming is 0ct3/4, sox-2, c-Myc, and Kl f4. [Claim 8]
제 6항에 있어서， 상기 분화된 세포는 인간으로부터 유래된 것인 방법. 【청구항 9】 The method of claim 6, wherein the differentiated cells are derived from humans. 【Claim 9】
제 6항에 있어서, 상기 분화된 세포는 섬유 아세포인 것인 방법 The method of claim 6, wherein the differentiated cells are fibroblasts.
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Citations (3)

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