KR20100127727A - Plant stem cell derived from cambium of family salicaceae and method of isolating and culturing the same - Google Patents
Plant stem cell derived from cambium of family salicaceae and method of isolating and culturing the same Download PDFInfo
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- KR20100127727A KR20100127727A KR1020100049103A KR20100049103A KR20100127727A KR 20100127727 A KR20100127727 A KR 20100127727A KR 1020100049103 A KR1020100049103 A KR 1020100049103A KR 20100049103 A KR20100049103 A KR 20100049103A KR 20100127727 A KR20100127727 A KR 20100127727A
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- Prior art keywords
- cambium
- stem cells
- derived
- willow
- culture
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/76—Salicaceae (Willow family), e.g. poplar
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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
- C12N5/04—Plant cells or tissues
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- A—HUMAN NECESSITIES
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
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Abstract
Description
본 발명은 버드나무과 (family Salicaceae)의 형성층 유래 줄기세포 및 그 분리배양방법에 관한 것이다.
The present invention relates to stem cells derived from the cambium of the family Salicaceae and to a method of isolation culture.
식물은 과거 식량 자원의 의미였으나, 현재에는 약제, 향료, 색소, 농약, 염료 등을 포함한 광범위한 화학물질의 공급원으로서 그 의미가 확대되고 있다. 특히, 식물 유래 유용 물질의 대부분은 항바이러스, 항박테리아, 항암, 항산화 능력 등의 생리 활성이 있어, 신의약품으로서 발전가능한 이상적인 자원으로 주목받고 있으며, 많은 식물 유래 물질들의 화학 구조와 활성 사이의 관계를 규명하기 위한 연구들이 활발히 진행되고 있다. Plants used to mean food resources in the past, but now they are expanding as a source of a wide range of chemicals, including pharmaceuticals, flavorings, pigments, pesticides, and dyes. In particular, since most of the plant-derived useful substances have physiological activities such as antiviral, antibacterial, anticancer and antioxidant ability, they are attracting attention as an ideal resource for development as a new drug, and the relationship between chemical structure and activity of many plant-derived substances Researches are actively underway to identify the problem.
그러나, 생리 활성 물질은 의약품으로 개발하기 어려운 실정이며, 그 주된 이유는 다음과 같다. 첫째, 식물 내 생리 활성 물질의 함량이 극히 제한적이다. 둘째, 식물의 생장 속도는 매우 느리다. 셋째, 식물 유래 생리 활성 물질은 식물의 특정 기관 내에만 소량 존재한다. 넷째, 자연 훼손이라는 환경 문제가 연루되어 있다. 다섯째, 식물 유래 생리 활성 물질의 경우 화학적인 구조가 매우 복잡하여, 다단계 중합 과정이 요구되어 생산 비용이 매우 높은 경제적인 문제가 있다. 따라서, 식물 유래 생리활성 물질들을 상업적으로 안정적으로 공급하기 매우 어려웠다.However, bioactive substances are difficult to develop as pharmaceuticals, and the main reasons thereof are as follows. First, the content of bioactive substances in plants is extremely limited. Second, the growth rate of plants is very slow. Third, plant-derived bioactive substances are present only in small quantities within certain organs of the plant. Fourth, environmental issues such as natural degradation are involved. Fifth, in the case of a plant-derived physiologically active substance, the chemical structure is very complicated, so a multi-stage polymerization process is required, and thus there is an economic problem in which the production cost is very high. Thus, it has been very difficult to commercially and stably supply plant-derived bioactive substances.
그런데, 생물공학기법의 하나인 식물 세포 배양 방법은 환경문제를 유발시키지 않으면서도 식물 유래 유용물질을 안정적으로 공급할 수 있는, 가장 이상적인 기술로 오랫동안 평가받고 있다. 대한민국 공개특허 1995-0000870 (1995. 01. 03)에 따르면 식물 세포 배양에 의한 유용물질의 생산은 식물에서 직접적인 추출에 의한 방법보다 수많은 장점이 있다. 특히, 기존의 추출법과는 달리, 외부 환경의 영향을 받지 않고도 지속적인 생산이 가능하여, 생태계 파괴와 같은 현안 문제들을 해결할 수 있는 최적의 방법으로 여겨지고 있다. 그러나, 식물 세포 배양에 대한 많은 관심과 노력에도, 산업화에 성공한 예는 아직까지 극히 일부에 불과한 실정이다. 이는 다수의 식물 세포 배양에서 세포 증식과 생산성의 변이가 주요 문제로 여전히 남아있기 때문이다.However, the plant cell culture method, which is one of biotechnology techniques, has long been evaluated as the most ideal technology capable of stably supplying plant-derived useful materials without causing environmental problems. According to Republic of Korea Patent Publication 1995-0000870 (1995. 01. 03) the production of useful substances by plant cell culture has a number of advantages over the method by direct extraction from plants. In particular, unlike conventional extraction methods, it is possible to continuously produce without being influenced by the external environment, and is considered as the best way to solve the problems such as ecosystem destruction. However, despite much interest and efforts in plant cell culture, only a few examples of successful industrialization are still. This is because variation in cell proliferation and productivity remains a major problem in many plant cell cultures.
식물 발현 시스템에 식물 세포를 이용하는 경우, 식물 세포의 분화 조직, 예컨대 잎, 줄기, 종자 등은 분열능이 상실된 영구 조직이므로, 분열능을 가진 세포주로 전환시키기 위해 탈분화 과정이 필수적으로 선행되고 있다. 상기 탈분화 과정은 식물체의 한 조직이나 기관을 이용하여 배양하였을 때 그 조직이나 세포가 이미 특정 기능을 수행하도록 분화된 상태를 해체하는 것을 의미한다. 그러나, 이러한 탈분화 과정 중에 염색체 변이에 의해 세포주의 심각한 변이가 발생될 수 있다.In the case of using plant cells in the plant expression system, since the differentiated tissues of the plant cells, such as leaves, stems, seeds, and the like, are permanent tissues that have lost dividing ability, dedifferentiation process is essentially preceded to convert them into cell lines having dividing ability. The dedifferentiation process means dismantling a state in which a tissue or cell has already been differentiated to perform a specific function when cultured using a tissue or organ of a plant. However, during this dedifferentiation process, severe variations in cell lines can occur due to chromosomal variations.
특히, 식물 세포 배양을 통한 유용물질의 생산은 장기간의 배양기간 동안 빠른 세포 증식과 높은 대사물질 생산능이 안정적으로 유지되어야만 산업화가 가능하지만, 대부분의 세포주는 계대 배양에 의해 본래와는 다른 수많은 변이를 접하게 된다. 따라서, 이와 같은 변이 문제점을 극복하여, 식물 세포 배양을 통한 유용물질 생산에서 유전적으로 안정한 세포주를 획득하기 위한 방안이 절실한 실정이었다. In particular, the production of useful substances through plant cell culture can be industrialized only if the rapid cell proliferation and high metabolite production capacity are maintained stably for a long period of culture, but most cell lines undergo numerous mutations different from the original by passage culture. You will come across. Therefore, in order to overcome such a mutation problem, a method for obtaining a genetically stable cell line in the production of useful substances through plant cell culture was urgently needed.
이에 본 발명자 중 일부는 식물의 줄기에서 채취한 형성층만을 이용하여 캘러스를 유도한 방법을 개발한 바 있으나 (대한민국 등록특허 제0533120호), 이 등록특허는 단순히 목본 식물의 줄기 형성층을 이용하여 캘러스를 유도하였을 뿐이었다. 캘러스(Callus)란 탈분화가 일어나 형성된 조직인 바, 이에 상기 등록특허는 여전히 탈분화에 의한 변이 문제를 보류하고 있는 문제점이 있었다. 또한, 본 발명자 중 일부는 탈분화에 의한 변이의 문제를 해결하고 안정적으로 증식이 가능한 유전적 안정성이 높은 세포주의 제공방법으로서 국제특허출원 PCT/KR 2006/001544호의 발명을 개발한 바 있다. 그러나, 버드나무 과에 속하는 포플러나무는 유용식물로 널리 알려진 것으로서, 이로부터 탈분화에 의한 변이의 문제를 해결하고 안정적으로 증식이 가능한 유전적 안정성이 높은 세포주를 획득하는 것이 요청되었다. Some of the present inventors have developed a method of inducing callus using only the layer formed from the stem of the plant (Korean Patent No. 0533120), but this patent simply uses a stem forming layer of a tree plant to callus. I just induced. Callus (Callus) is a tissue formed by dedifferentiation, so the patent has a problem that withholds the problem of variation by dedifferentiation. In addition, some of the present inventors have developed the invention of International Patent Application No. PCT / KR 2006/001544 as a method of providing a genetically stable cell line that solves the problem of mutation by dedifferentiation and stably proliferates. However, the poplar tree belonging to the willow family is widely known as a useful plant, and from this, it was required to solve the problem of mutation by dedifferentiation and to obtain a cell line with high genetic stability capable of stably proliferating.
이에 본 발명자는 버드나무과 식물의 형성층 유래 줄기세포를 분리함으로써 유용한 식물세포주를 제공하고자 예의노력한 결과, 버드나무과 식물의 형성층 유래 줄기세포를 분리하고, 상기 줄기세포가 안정적으로 증식되고, 배양 시 변이가 없음을 확인하고, 본 발명을 완성하게 되었다.
Accordingly, the present inventors have made efforts to provide useful plant cell lines by isolating stem cells derived from the cambium of the cambium, and as a result, the stem cells are derived from the cambium of the willow, and the stem cells are stably propagated, and the variance in culture. It confirmed that there was no, and completed this invention.
본 발명의 목적은 탈분화 과정 없이 버드나무과의 형성층 유래 줄기세포 및 그 분리배양방법을 제공하는 데 있다.
It is an object of the present invention to provide a cambium-derived stem cell of Willowaceae without dedifferentiation process and a method of isolating the same.
상기와 같은 목적을 달성하기 위하여, 본 발명은 버드나무과 (family Salicaceae)의 형성층에서 유래되고, 탈분화를 거치지 않은 선천적 미분화세포인 것을 특징으로 하는 버드나무과의 형성층 유래 줄기세포를 제공한다.In order to achieve the above object, the present invention provides a stem cell derived from the cambium of Willow family, which is derived from the cambium of the family Salicaceae , and is a natural undifferentiated cell without undergoing dedifferentiation.
본 발명은 또한, 다음의 단계를 포함하는 버드나무과(family Salicaceae)의 형성층 유래 줄기세포의 분리방법을 제공한다:The present invention also provides a method for separating stem-derived stem cells of the family Salicaceae comprising the following steps:
(a) 버드나무과 식물로부터 형성층 함유 조직을 수득하는 단계;(a) obtaining a cambium-containing tissue from willow plants;
(b) 상기 수득된 형성층 함유 조직을 배지에서 배양하는 단계; 및(b) culturing the obtained cambium-containing tissue in a medium; And
(c) 상기 형성층으로부터 세포들을 분리함으로써 형성층 유래 줄기세포를 수득하는 단계. (c) obtaining the cambium-derived stem cells by separating the cells from the cambium.
본 발명은 또한, 상기 버드나무과의 형성층 유래 줄기세포, 그 추출물, 그 파쇄물 및 그 배양물 중 어느 하나 이상을 함유하는 항염증용 조성물을 제공한다.The present invention also provides an anti-inflammatory composition containing any one or more of the stem cells derived from the cambium of the willow, its extract, its lysate and its culture.
본 발명은 또한, 상기 버드나무과의 형성층 유래 줄기세포, 그 추출물, 그 파쇄물 및 그 배양물 중 어느 하나 이상을 함유하는 염증의 예방 또는 개선용 기능성 식품을 제공한다.
The present invention also provides a functional food for preventing or improving inflammation containing any one or more of the stem cells derived from the cambium of the willow, its extract, its lysate and its culture.
본 발명은 탈분화 과정 없이 버드나무과의 형성층 유래 줄기세포 및 그 분리방법을 제공하는 효과가 있다. 본 발명에 따른 버드나무과의 형성층 유래 줄기세포는 탈분화과정없이 미분화상태로 분리되어 장기 배양시에도 세포생장률과 생장패턴에 변이 없이 안정적으로 유지되어 대량 배양이 가능하여 유용하다. 아울러, 본 발명에 따른 버드나무과의 형성층 유래 줄기세포는 LPS(Lipopolysaccharide)를 처리했을 때 발생하는 NO 생성을 억제하는 효과 및 COX-2 염증사이토카인의 유전자 발현을 억제하는 효과가 있는바, 우수한 항염증용 조성물로서 유용하게 사용될 수 있다.
The present invention has the effect of providing a stem cell derived from the cambium of Willow family without dedifferentiation process and its isolation method. Stem cells derived from the cambium of the Willow family according to the present invention are isolated in an undifferentiated state without dedifferentiation process and are useful because they can be stably maintained without change in cell growth rate and growth pattern even during long-term culture. In addition, the cambium-derived stem cells of the present invention according to the present invention has the effect of inhibiting the generation of NO generated when LPS (Lipopolysaccharide) treatment and the effect of inhibiting the gene expression of COX-2 inflammatory cytokines, excellent anti- It can be usefully used as an inflammatory composition.
도 1은 재료식물(포플러나무)의 횡단면에서 형성층을 관찰한 사진이다.
도 2는 본 발명에 따른 줄기세포의 유도 및 분리 사진으로, 2A는 형성층 포함 절편체를 줄기세포 유도배지에 치상한 사진이고, 2B는 형성층 유래 줄기세포와 이질적인 다른 캘러스의 분리가 이루어진 모습을 보이는 사진이고, 2C는 형성층 유래 줄기세포만을 분리하여 3주간 배양한 모습이다.
도 3은 본 발명에 따른 줄기세포 및 포플러나무의 수피 조직에서 유도한 캘러스의 고체 배양 시 사진이다.
도 4는 본 배양 과정의 줄기세포(A) 및 포플러나무 수피 조직에서 유도한 캘러스(B)의 세포응집정도를 관찰한 현미경 사진이다(x100).
도 5는 본 발명에 따른 줄기세포(A) 및 포플러나무 수피 조직에서 유도한 캘러스(B)의 현미경 사진으로, 좌측 하단의 스케일 바는 25㎛이다.
도 6은 Neutral red를 이용하여 액포를 염색한 후 관찰한 본 발명에 따른 줄기세포(A) 및 포플러나무 수피 조직에서 유도한 캘러스(B)의 현미경 사진으로, 우측하단의 스케일 바는 25㎛이다.
도 7은 본 발명에 따른 줄기세포(A) 및 포플러나무 수피 조직에서 유도한 캘러스(B)의 미토콘드리아를 관찰한 사진이다.
도 8은 본 발명에 따른 줄기세포(Stem cell) 및 포플러나무 수피 조직에서 유도한 캘러스(callus)의 생장속도 비를 나타낸 그래프이다.
도 9는 3L 공기부양식(air-lift) 생물반응기(A) 및 20L 공기부양식 생물반응기(B)에서 14일간 배양한 결과를 나타내는 사진이다.
도 10은 본 발명에 따른 줄기세포(Stem cell) 및 포플러나무 수피 조직에서 유도한 캘러스(callus)의 동결보존 후 세포생존율을 나타낸 그래프이다.
도 11은 NO 분석 수행 시 본 발명에 따른 줄기세포 추출물의 농도별 NO 발생억제정도를 보여주는 그래프이다.
도 12는 본 발명에 따른 줄기세포 추출물의 농도별 COX-2 유전자 발현 억제정도를 보여주는 겔 사진이다 (A: 0.5g/ml, B: 1mg/ml, C: 2mg/ml).
1 is a photograph of the formation layer in the cross section of the material plant (poplar tree).
Figure 2 is a photograph of the induction and separation of stem cells according to the present invention, 2A is a photo of the stem cell induction medium containing the cambium layer, 2B shows the separation of the cambium-derived stem cells and heterogeneous other callus Photograph, 2C is a state in which only stem cell-derived stem cells were isolated and cultured for 3 weeks.
Figure 3 is a photograph of a solid culture of callus induced in the bark tissue of stem cells and poplar trees according to the present invention.
Figure 4 is a micrograph observing the degree of cell aggregation of the callus (B) induced in stem cells (A) and poplar bark tissue of the present culture process (x100).
5 is a micrograph of a stem cell (A) and a callus (B) derived from a poplar bark tissue according to the present invention, and the scale bar at the bottom left is 25 μm.
Figure 6 is a micrograph of the stem cells (A) and the callus (B) derived from the poplar bark tissue according to the present invention observed after staining the vacuole using Neutral red, scale bar at the bottom right is 25㎛ .
Figure 7 is a photograph of the mitochondria of the stem cells (A) and callus (B) induced in the poplar bark tissue according to the present invention.
8 is a graph showing the growth rate ratio of the callus (callus) induced in stem cells and poplar bark tissues according to the present invention.
9 is a photograph showing the results of 14 days of culture in a 3L air-lift bioreactor (A) and a 20L air-lift bioreactor (B).
10 is a graph showing the cell survival rate after cryopreservation of callus induced in stem cells and poplar bark tissues according to the present invention.
11 is a graph showing the degree of NO generation inhibition according to the concentration of the stem cell extract according to the present invention when performing the NO analysis.
12 is a gel photograph showing the degree of inhibition of COX-2 gene expression according to the concentration of the stem cell extract according to the present invention (A: 0.5g / ml, B: 1mg / ml, C: 2mg / ml).
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명의 상세한 설명 등에서 사용되는 주요 용어의 정의는 다음과 같다. Definitions of main terms used in the detailed description of the present invention are as follows.
본원에서, 유관속 "형성층"은 식물의 유관속 조직(vascular tissue) 내에 위치하는 측재분열조직으로 줄기와 뿌리에 위치한다. 형성층의 활동에 의해 식물의 비대생장이 일어나며, 그 결과 11,000년 이상의 연륜을 가진 거대 식물체가 존재할 수 있게 된다. 발생학적으로 유관속 형성층은 전형성층으로부터 기원되므로 분열조직적 연속성을 유지하면서 점진적으로 분화된 동일 분열조직으로 편의상 구분될 뿐으로 (식물형태학, 이재두 외 7인 공저, 아카데미 서적, 제10장, 1993), 본 발명에 있어서 형성층은 전형성층을 포함하는 것으로 해석된다. 이러한 형성층과 전형성층은 동일한 1기 분열조직으로서, 본 발명에 있어서 형성층 및 전형성층 조직을 사용하여 동일한 효과를 얻을 것임은 자명한 사항이다.Herein, the vascular "forming layer" is located in the stems and roots as a lateral meristem located in the vascular tissue of a plant. The activity of the cambium causes hypertrophy of the plant, resulting in the presence of large vegetation with more than 11,000 years of age. Genetically speaking, the vascular forming layer originates from the procambium, so that it can be conveniently divided into gradually divided mitotic tissues while maintaining mitotic continuity. In the present invention, the formation layer is interpreted to include a formation layer. It is obvious that such a formation layer and a typical formation layer are the same primary fission structure, and the same effect will be acquired by using a formation layer and a typical formation structure in this invention.
본원에서 식물 "줄기세포" (stem cell)란, 탈분화 과정을 거치지 않아 유전적으로 보다 안정한 선천적인 미분화세포를 말한다. Plant “stem cells” as used herein refer to innate undifferentiated cells that are genetically more stable without undergoing dedifferentiation.
본원에서 "파쇄물"이란 세포를 detergent 등을 이용한 화학적 방법 또는 물리적 방법 등으로 파쇄하여 얻은 세포 용해물을 의미하며, 세포주의 "추출물"이란 세포를 용매에 녹여 분리한 물질로, 증류 또는 증발을 이용하여 농축될 수 있다. 또한, 세포주 "배양액"이란 세포를 배양시킨 다음, 세포를 제외하고 남은 세포 배양용액을 의미한다. 추가적으로 본원에서 "배양물"이란 배양액 및/또는 배양된 세포주를 포함하는 물질로서, 이때 배양된 세포주는 배양조건에 의하여 분화되거나 유용물질의 생산능 및/또는 분비능이 향상된 세포주를 모두 포함하는 개념이다.As used herein, "cracked material" refers to a cell lysate obtained by crushing a cell by a chemical method or a physical method using a detergent or the like, and "extract" of a cell line is a material obtained by dissolving the cell in a solvent and using distillation or evaporation. Can be concentrated. In addition, the cell line "culture medium" means a cell culture solution remaining after culturing the cells, excluding the cells. Additionally, as used herein, the term "culture" is a substance containing a culture medium and / or a cultured cell line, wherein the cultured cell line is a concept including all cell lines that are differentiated by culture conditions or have improved production and / or secretion capacity of useful materials. .
본원에서 "선천적인 미분화 상태(innately undifferentiated)"란 탈분화 과정을 거쳐 미분화 상태로 존재하는 것이 아닌, 본래부터 분화 전 상태를 유지하는 것을 말한다.As used herein, "innately undifferentiated" refers to maintaining a pre-differentiation state, rather than being present in an undifferentiated state through a dedifferentiation process.
본원에서 "캘러스"란, 탈분화 과정을 통하여 분화하지 않은 상태로 된 세포 또는 세포덩어리 (PNAS, 99(25):15843, 2002)를 말한다. As used herein, "callus" refers to cells or cell masses (PNAS, 99 (25): 15843, 2002) that have not been differentiated through dedifferentiation.
본 발명은 일 관점에서, 버드나무과의 형성층 유래 줄기세포에 관한 것으로, 이는 버드나무과의 형성층에서 유래되고, 탈분화를 거치지 않은 선천적 미분화세포이다. The present invention relates to a stem cell derived from the cambium of Willow in one aspect, which is a congenital undifferentiated cell derived from the cambium of Willow and not undergoing dedifferentiation.
이미 분화된 조직인 잎이나 줄기, 뿌리 부분을 사용할 경우, 캘러스를 형성하기 위해서는 분화된 조직에서 미분화조직으로 되돌아가는(rejuvenilation) 탈분화(dedifferentiation) 과정을 거쳐야 하는데 이 탈분화 과정 중에 체세포에 돌연변이가 나타나 세포 불안정성의 원인이 된다. 이에 본 발명자들은 체세포 변이가 거의 없는 식물 세포 시스템을 연구하던 중, 분열조직인 형성층에서만 특이적으로 세포주를 유도할 경우 탈분화를 거치지 않고 분열조직 자체가 가지고 있는 왕성한 분열능을 사용할 수 있으므로 체세포 변이가 없어 유전적으로 안정성이 높고, 생리적으로 균일한 세포주를 유도할 수 있다는 점에 착안하고 형성층 유래 줄기세포를 분리하였다.In the case of using the already differentiated tissues such as leaves, stems, and roots, callus formation requires rejuvenilation and dedifferentiation from the differentiated tissues. Cause. Therefore, the present inventors, while studying a plant cell system with little somatic mutation, inducing cell lines specifically only in the formation layer of the meristem, there is no somatic mutation because the active division of the meristem itself can be used without dedifferentiation. Concentrating on genetically stable, physiologically uniform cell lines, the cambium-derived stem cells were isolated.
본 발명에 따른 버드나무과의 형성층 유래 줄기세포는 다음 중 적어도 하나의 특성을 가지는 것을 특징으로 할 수 있다:Stem cells derived from the cambium of Willow family according to the present invention may be characterized by having at least one of the following characteristics:
(a) 현탁배양 시 버드나무과의 탈분화된 캘러스에 비하여 많은 수의 단세포를 포함하거나 작은 사이즈의 세포 집합체를 포함함;(a) contain large numbers of single cells or small cell aggregates as compared to dedifferentiated callus of Willow family in suspension culture;
(b) 다수의 액포(vacuole)를 가지는 형태학적 특징을 나타냄;(b) exhibit morphological features with multiple vacuoles;
(c) 버드나무과의 탈분화된 캘러스에 비하여 활성이 증가된 미토콘드리아를 가짐;(c) has mitochondria with increased activity compared to dedifferentiated callus of the willow family;
(d) 버드나무과의 탈분화된 캘러스에 비하여 생장속도가 빠르고 오랫동안 성장할 수 있음; 및(d) can grow faster and grow longer compared to dedifferentiated callus of Willow family; And
(e) 버드나무과의 탈분화된 캘러스에 비하여 생물반응기에서 전단 스트레스(shear stress)에 대해 낮은 민감성을 가짐.(e) Has low sensitivity to shear stress in bioreactors compared to dedifferentiated callus from Willow family.
이때, "다수의 액포"를 가진다고 함은, 버드나무과의 탈분화된 캘러스 등과 비교하여 2배 이상의 다수의 액포를 가짐을 말한다. 아울러, 본 발명에 따른 줄기세포는 버드나무과의 캘러스 등과 비교하여 크기면에서 작은 액포를 가진다. 아울러, "다수의 발달된 형태의 미토콘드리아"를 가진다고 함은, 버드나무과의 탈분화된 캘러스와 비교하여 현미경 하에서 활발하게 움직이는 미토콘드리아를 2배 이상의 다수 가짐을 말한다.In this case, "having a large number of vacuoles" means having a plurality of vacuoles two times or more as compared to dedifferentiated callus and the like. In addition, the stem cells according to the present invention has a small vacuole in size compared with the callus of the willow family. In addition, having "a large number of advanced forms of mitochondria" refers to having more than twice the number of mitochondria that actively move under a microscope compared to the dedifferentiated callus of the willow family.
본 발명에 있어서, 상기 줄기세포는 상기 (a) 내지 (e)의 특성 중 적어도 두 개 이상의 특성을 가지는 것을 특징으로 할 수 있으며, 바람직하게는 상기 (a) 내지 (e)의 특성 중 적어도 세 개 이상의 특성을 가지는 것을 특징으로 할 수 있으며, 더욱 바람직하게는 상기 (a) 내지 (e)의 특성 중 적어도 네 개 이상의 특성을 가지는 것을 특징으로 할 수 있다. 아울러, 본 발명에 있어서, 상기 줄기세포는 (a) 내지 (e)의 특성을 모두 가지는 것을 특징으로 할 수 있다. In the present invention, the stem cells may be characterized by having at least two or more of the properties of (a) to (e), preferably at least three of the properties of (a) to (e) It may be characterized by having at least four characteristics, more preferably at least four of the characteristics of the above (a) to (e). In addition, in the present invention, the stem cells may be characterized by having all the properties of (a) to (e).
아울러, 버드나무과 형성층 유래 줄기세포의 분화를 유도하여 도관요소(tracheary elements)로 분화할 수 있는 분화능을 가지고 있음을 확인하였다. 식물줄기세포를 특징짓는 특성에는 자가재생능력(self renewal)이외에 분화능(pluripotency)이 있는바, 이에 본 발명에 따른 버드나무과 형성층 유래 세포가 줄기세포임을 확인하였다. In addition, it was confirmed that the induction of differentiation of stem cells derived from the cambium cambium has a differentiation ability to differentiate into tracheary elements. Characterizing the plant stem cells has a differentiation ability (pluripotency) in addition to the self renewal ability (self renewal), whereby it was confirmed that the willow-derived cell derived from the stem cells according to the present invention.
한편, 본 발명에 따른 줄기세포는 (a) 버드나무과 식물로부터 형성층 함유 조직을 수득하는 단계; (b) 상기 수득된 형성층 함유 조직을 배지에서 배양하는 단계; 및 (c) 상기 형성층으로부터 세포들을 분리함으로써 형성층 유래 줄기세포를 수득하는 단계를 포함하는 분리방법에 의하여 얻어질 수 있는데, 이때, 상기 (b) 단계는 형성층 함유 조직을 배양하여 형성층으로부터 증식되는 배양된 형성층을 유도하는 것을 특징으로 할 수 있고, 상기 (c) 단계는 상기 배양된 형성층을 분리함으로써 형성층 유래 줄기세포를 수득하는 것을 특징으로 할 수 있다. 아울러, 상기 (b)단계는 옥신(auxin)을 포함한 배지에서 배양하는 것을 특징으로 할 수 있는데, 이때, 옥신으로는 NAA (α-Naphtalene acetic acid) 또는 IAA (Indole-3-acetic acid)를 사용할 수 있으며, 이러한 옥신은 1~5㎎/ℓ의 농도로 포함되는 것을 특징으로 할 수 있다. 또한, 상기 (c) 단계는 형성층 이외의 부분으로부터 무정형으로 증식되는 캘러스 층으로부터 상기 배양된 형성층을 분리함으로써 형성층 유래 줄기세포를 수득하는 것을 특징으로 할 수 있다. On the other hand, stem cells according to the present invention comprises the steps of (a) obtaining a cambium-containing tissue from the willow plant; (b) culturing the obtained cambium-containing tissue in a medium; And (c) obtaining the cambium-derived stem cells by separating the cells from the cambium, wherein step (b) is a step of culturing the cambium-containing tissue to grow from the cambium. It may be characterized by inducing the formed layer, the step (c) may be characterized in that to obtain a cambium-derived stem cells by separating the cultured cambium. In addition, the step (b) may be characterized by culturing in a medium containing auxin (auxin), wherein, as the auxin is used NAA (α-Naphtalene acetic acid) or IAA (Indole-3-acetic acid) The auxin may be characterized in that it is included at a concentration of 1 ~ 5mg / ℓ. In addition, the step (c) may be characterized in that to obtain a cambium-derived stem cells by separating the cultured cambium layer from the callus layer that is amorphously proliferated from portions other than the cambium.
본 발명의 일 실시예에서는 버드나무과 사시나무속인 포플러로부터 포플러의 형성층 유래 줄기세포를 분리하였으며, 이에 본 발명은 바람직하게는 사시나무 속 식물의 형성층 유래 줄기세포임를 특징으로 하며, 더욱 바람직하게는 포플러의 형성층 유래 줄기세포임을 특징으로 할 수 있다. In one embodiment of the present invention was isolated from the cambium-derived stem cells of the poplar from the poplar willow and Aspen, and the present invention is preferably characterized in that the stem cells derived from the cambium of the genus Aspen, more preferably poplar It may be characterized that the cambium-derived stem cells.
또한, 본 발명에서는, 상기에서 수득된 버드나무과 사시나무속인 포플러로부터 포플러의 형성층 유래 줄기세포의 항염증 효과를 확인하였으며, 이에 본 발명은 다른 관점에서, 상기 버드나무과의 형성층 유래 줄기세포, 그 추출물, 그 파쇄물 및 그 배양물 중 어느 하나 이상을 함유하는 항염증용 조성물에 관한 것이다. In addition, in the present invention, the anti-inflammatory effect of the cambium-derived stem cells from the poplar, which is obtained from the above-mentioned Willowaceae aspen, was confirmed, and in another aspect, the present invention, stem cells derived from the cambium of the willow family, extracts thereof. The present invention relates to an anti-inflammatory composition containing any one or more of its lysate and its culture.
본 발명에서 상기 배양물은, 상기 줄기세포를 엘리시터로서 3~5중량%의 원당 또는 설탕; 또는 메틸 자스모네이트(methyl jasmonate), 키토산, 페닐알라닌(phenylalanin), 벤조익산, ABA, 살리실산(salicylic acid) 및 아세트산 나트륨(sodium acetate) 중 어느 하나 이상을 포함하는 배지에서 추가로 배양하는 단계를 추가로 수행하여 수득된 것임을 특징으로 할 수 있다. 이때, 바람직하게는 상기 배지는 3~5중량%의 원당 또는 설탕; 및 메틸 자스모네이트(methyl jasmonate), 진균류 추출물, 세균류 추출물, 효모(yeast) 추출물, 키토산, 글루코마난(glucomanan), 글루칸(glucan), 페닐알라닌(phenylalanine), 벤조산(benzoic acid), 살리실산(salicylic acid), 아라키돈산(arachonic acid), STS, mevalonalonate N-benzolyglycine, ABA, SNP, IPP, BHT, CCC, 에테폰(ethephon), 히푸익산(hippuic acid), 암모니움 세릭 니트레이트(amminoium ceric nitrate), AgNO3, 바나딜 설페이트(vanadyl sulfate), p-아미노벤조익산(p-aminobenzoic acid), 브라시노스테로이드(brassinosteroids), 소디움 알지네이트(sodium alginate), 아세트산 나트륨 (sodium acteate)로 구성되는 군에서 선택된 물질;을 포함하는 배지임을 특징으로 할 수 있다.The culture in the present invention, the stem cells as an eliminator 3 to 5% by weight of raw sugar or sugar; Or further culturing in a medium containing any one or more of methyl jasmonate, chitosan, phenylalanine, benzoic acid, ABA, salicylic acid, and sodium acetate. It can be characterized in that obtained by performing. At this time, preferably the medium is 3 to 5% by weight of raw sugar or sugar; And methyl jasmonate, fungal extract, bacterial extract, yeast extract, chitosan, glucomanan, glucan, phenylalanine, benzoic acid, salicylic acid ), Arachonic acid, STS, mevalonalonate N-benzolyglycine, ABA, SNP, IPP, BHT, CCC, ethephon, hipuic acid, ammonium ceric nitrate, Substances selected from the group consisting of AgNO 3 , vanadyl sulfate, p-aminobenzoic acid, brassinosteroids, sodium alginate, sodium acetate It may be characterized in that the medium containing.
본 발명에서 또한, 상기 줄기세포에 엘리시터로서 자외선, 열, 에틸렌, 항진균제, 항생제, 중금속 염 및 높은 염농도 등을 처리하여 물리적 화학적 스트레스를 가하여 수득된 배양물을 이용할 수 있다.In the present invention, it is also possible to use a culture obtained by applying physical and chemical stress to the stem cells by treating ultraviolet rays, heat, ethylene, antifungal agents, antibiotics, heavy metal salts and high salt concentrations as an eliminator.
본 발명에 있어서, 상기 추출물은 증류수, 에탄올 등 알코올, 아세톤, DMSO (Dimethyl Sulfoxide) 및 이들의 혼합용매로 구성된 군에서 선택된 용매를 이용하여 추출된 것을 특징으로 할 수 있다.In the present invention, the extract may be extracted using a solvent selected from the group consisting of distilled water, alcohol such as ethanol, acetone, DMSO (dimethyl sulfoxide) and a mixed solvent thereof.
본 발명의 일 실시예에서는 포플러 유래 형성층 유래 줄기세포 추출물이 LPS(Lipopolysaccharide)를 처리했을 때 발생하는 NO 생성을 억제하는 효과 및 COX-2 염증사이토카인의 유전자 발현을 억제하는 효과를 관찰하여 본 발명에 따른 형성층 유래 줄기세포가 항염증 효과가 있음을 확인하였다. 따라서, 본 발명에 따른 줄기세포 추출물은 매우 우수한 항염증용 조성물로서 유용하게 사용될 수 있다.In one embodiment of the present invention by observing the effect of inhibiting the generation of NO and COX-2 inflammatory cytokine gene expression generated when the poplar-derived cambium-derived stem cell extract is treated with LPS (Lipopolysaccharide) It was confirmed that the cambium-derived stem cells according to the anti-inflammatory effect. Therefore, the stem cell extract according to the present invention can be usefully used as a very excellent anti-inflammatory composition.
이에, 본 발명에서는 상기 줄기세포 또는 그 파쇄물을 함유하는 조성물이 항염증 효과를 나타냄을 제시하는 구체적인 실시예가 없다 하더라도, 상기에서 살펴본 바와 같이 그 줄기세포 추출물이 항염증 활성을 가지는 것을 확인한바, 본 발명에 따른 줄기세포 자체나 그 파쇄물, 그 배양물을 함유한 조성물의 경우에도 항염증 활성을 나타내어 염증을 억제할 수 있다는 것은 당업계에서 통상의 지식을 가진 자에게 자명하다고 할 것이다.Thus, in the present invention, even if there is no specific example suggesting that the composition containing the stem cells or the lysate exhibits an anti-inflammatory effect, as described above, it was confirmed that the stem cell extract has anti-inflammatory activity. It will be apparent to those skilled in the art that the stem cell itself, the lysate thereof, and the composition containing the culture according to the present invention can also exhibit anti-inflammatory activity and suppress inflammation.
본 발명에 따른 줄기세포, 그 추출물, 그 파쇄물 및 그 배양물 중 어느 하나 이상을 유효성분으로 함유하는 항염증용 조성물은 이들을 각각 단독으로 포함하거나 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 포함하여 약학 조성물로 제공될 수 있으며, 상기 줄기세포 등은 질환 및 이의 중증정도, 환자의 연령, 체중, 건강상태, 성별, 투여 경로 및 치료 기간 등에 따라 적절한 약학적으로 유효한 양으로 약학 조성물에 포함될 수 있다. An anti-inflammatory composition containing any one or more of stem cells, extracts thereof, lysates, and cultures thereof according to the present invention as an active ingredient, each of them alone or at least one pharmaceutically acceptable carrier, excipient or diluent It may be provided as a pharmaceutical composition, the stem cells and the like to be included in the pharmaceutical composition in an appropriate pharmaceutically effective amount according to the disease and its severity, the age, weight, health status, sex, route of administration and duration of treatment, etc. Can be.
상기에서 "약학적으로 허용되는"이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 상기 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리톤, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and that, when administered to a human, typically does not cause an allergic reaction such as gastrointestinal disorders, dizziness, or the like. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
상기 약학 조성물은 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다. 또한 본 발명의 약학 조성물은 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사 용액, 멸균 분말의 형태일 수 있다. The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives. Pharmaceutical compositions of the invention can also be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
본 발명은 또 다른 관점에서, 본 발명에 따른 줄기세포, 그 추출물, 그 파쇄물 및 그 배양물 중 어느 하나 이상을 유효성분으로 함유하는 염증의 예방 또는 개선용 기능성 식품에 관한 것이다. In still another aspect, the present invention relates to a functional food for preventing or improving inflammation containing any one or more of stem cells, extracts thereof, lysates and cultures thereof according to the present invention as an active ingredient.
본원에서 '기능성 식품'이란, 일반 식품에 본 발명에 따른 줄기세포 또는 그 추출물, 그 파쇄물 및 그 배양물 중 어느 하나 이상을 첨가함으로써 식품의 기능성을 향상시킨 것을 의미한다.
As used herein, the term "functional food" means that the functionality of the food is improved by adding any one or more of the stem cells or the extract thereof, the lysate, and the culture according to the present invention to a general food.
실시예Example
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.
특히, 하기 실시예에서는 포플러의 형성층 유래 줄기세포 추출물의 항염증 효과를 확인하였으나, 그 줄기세포 자체나 그 파쇄물을 사용해서도 유사한 결과를 얻을 수 있다는 것은 당업계에서 통상의 지식을 가진 자에게 자명한 사항이라 할 것이다.
In particular, in the following examples confirmed the anti-inflammatory effect of the cambium-derived stem cell extract, it is apparent to those skilled in the art that similar results can be obtained using the stem cells themselves or their lysates. One matter.
버드나무과의 형성층 유래 줄기세포의 제조Preparation of Stem Cells Derived from the Formation Layer of Willowaceae
1-1: 식물재료의 준비1-1: Preparation of Plant Materials
버드나무과 사시나무속의 포플러나무의 일 품종인 은백양과 수원사시나무의 교잡종인 현사시나무(P. alba X P. glandulosa , 수원 산림과학원)를 준비하였는데, 이는 도 1과 같이, Phloroglycinol 염색을 통하여 목부(xylem)와 사부(phloem) 섬유를 관찰할 수 있으며, 이 사이에 형성층(cambium)이 존재함을 확인할 수 있다. 이러한 포플러나무로부터 상기 형성층 유래 줄기세포를 수득하기 위하여, 먼저 포플러나무의 줄기를 채취한 후, 즉시 항산화제 100㎎/L 아스코르브산(L-ascorbic acid, DUCHEFA, The Netherlands) 용액에 침적하여 운송·보관하였다. 이때, 전형성층을 이용하여 동일한 줄기세포를 수득하기 위하여는 줄기 대신 잔가지를 채취한다. P. alba ( P. alba) , a hybrid of silver white and Suwon aspen varieties of poplar trees of Willow and Aspen X P. glandulosa , Suwon Forest Science Institute), which can be observed through the phloroglycinol staining (xylem) and phloem fibers (cambium) between them, as shown in Figure 1 You can check it. In order to obtain the cambium-derived stem cells from the poplar tree, the stems of the poplar tree were first collected, and then immediately deposited in 100 mg / L ascorbic acid (L-ascorbic acid, DUCHEFA, The Netherlands) solution for transportation. Stored. At this time, in order to obtain the same stem cells using the typical layer, the twigs are collected instead of the stems.
그 후, 1% 베노밀(benomyl, Dongbu Hannong Chemical, Korea), 1% 다코닐(daconil, Dongbu Hannong Chemical, Korea), 1% 스트렙토마이신(sterptomycin sulphate, DUCHEFA, The Netherlands), 0.1% 세포탁심(cefotaxime sodium DUCHEFA, The Netherlands)의 혼합용액에 24시간 전처리 후 페놀 화합물(phenolic compound)과 잔존 화학물질을 제거하기 위하여 수돗물(tap water)로 30분간 세척하였다. 그리고, 70% 에탄올(ethanol, DC Chemical, Korea)에 1분, 30% 과산화수소(hydrogen peroxide, LG Chemical, Korea) 15분, 1% CLOROX 용액에 15분, 3% CLOROX용액에 5분 표면 살균 후 3~4회 세척하였다.
Then, 1% benomyl (Dongbu Hannong Chemical, Korea), 1% daconil (Dongbu Hannong Chemical, Korea), 1% streptomycin (sterptomycin sulphate, DUCHEFA, The Netherlands), 0.1% cefotaxime sodium After 24 hours of pretreatment in a mixed solution of DUCHEFA, The Netherlands), the mixture was washed with tap water for 30 minutes to remove phenolic compounds and residual chemicals. After 1 minute in 70% ethanol, 15 minutes in 30% hydrogen peroxide (LG Chemical, Korea), 15 minutes in 1% CLOROX solution, 5 minutes in 3% CLOROX solution Washed 3-4 times.
1-2: 줄기로부터 형성층 포함 1-2: Formation layer from stem 절편체의Intersecting 제조 및 조직 분리 Manufacturing and Tissue Separation
상기 실시예 1-1의 살균과정을 거친 줄기 (전형성층 이용시 잔가지)의 바깥조직들을 세로방향으로 잡아당기면 쉽게 벗겨졌다. 벗겨진 조직들은 형성층, 사부, 피층, 표피로 구성되는데, 이러한 형성층 포함 절편체는 도 2A와 같다.
Pulling the outer tissue of the stem (twig when using the typical layer) through the sterilization process of Example 1-1 in the longitudinal direction was easily peeled off. Peeled tissues are composed of the cambium, sand, cortex, and epidermis, the fragment comprising the cambium as shown in Figure 2A.
1-3: 포플러나무의 형성층 유래 줄기세포 유도단계1-3: Induction of stem cells derived from cambium of poplar tree
상기 실시예 1-2에서 준비한 형성층 포함 절편체는 표 1의 줄기세포 유도배지(배지 1)에 치상하여 배양하였다. The fragment comprising the formation layer prepared in Example 1-2 was cultured by denture on the stem cell induction medium (media 1) of Table 1.
Inorganic salts
Vitamin
Amino acid
배지에 생장 조절제로서 NAA, IAA와 같은 옥신(Auxin)은 1~5mg/L의 농도로 첨가할 수 있는데, 바람직하게는 2mg/L의 농도로 첨가한다. 배양은 25±1℃로 조절된 암실에서 실시되었다.Auxins such as NAA and IAA as growth regulators may be added to the medium at a concentration of 1 to 5 mg / L, preferably at a concentration of 2 mg / L. Cultivation was carried out in a dark room controlled at 25 ± 1 ℃.
초기 배양 4~6일째 형성층으로부터 세포 분열이 육안상으로 관찰되고, 배양 14일 이후에 사부·피층 및 표피로 이루어진 층으로부터 탈분화에 의한 무정형의 캘러스가 유도되기 시작하였다. 배양 25일 경과 후 배양된 형성층과 사부를 포함한 윗층, 즉 무정형의 캘러스층으로 분리되기 시작했고(도 2B), 두 층이 자연스럽게 분리될 때까지 기다렸다가 완벽한 분리가 이루어지면 각기 다른 페트리디쉬(petridish)에 분리 배양하였다. 분리 후, 생장률이 좋은 희고 무른 부분을 유도배지와 동일한 새로운 배지로 매 21일째 계대하였다. 도 2C는 형성층 유래 줄기세포만을 분리하여 3주간 배양한 모습이다.Cell division was observed visually from the formation layer on the 4-6th day of initial culture, and after 14 days of culture, amorphous callus by dedifferentiation began to be induced from the layer consisting of sand, cortex and epidermis. After 25 days of incubation, it began to separate into the cultured formation layer and the upper layer, including the quadrant, that is, the amorphous callus layer (Figure 2B), waiting for the two layers to separate naturally, and after complete separation, different petridishes Incubated separately. After separation, the white and soft parts with good growth rate were passaged every 21 days with the same fresh medium as the induced medium. Figure 2C is a state in which only stem cell-derived stem cells were isolated and cultured for 3 weeks.
한편, 비교를 위하여 포플러나무의 수피(bark) 절편체를 소독한 후 상기 <표 1>의 배지에서 배양하였으며, 그 결과, 도 3B에 나타난 바와 같이, 수피 절편체는 탈분화에 의해 캘러스를 형성함을 관찰할 수 있었다. 수피 절편체에서 유도된 캘러스는 사부포함조직과 같이 여러 세포들 간의 분열 속도에 차이로 무정형을 이루었고, 생장률이 불안전하며 쉽게 갈변되는 경향을 보였다. 갈변 및 응집되어진 수피에서 유도된 캘러스는 자신이 분비하는 페놀 화합물에 의해 생장이 둔해지다가 종국에는 괴사하였다. 즉, 6개월 후부터 수피(bark)로부터 유도된 캘러스들은 유지 및 배양이 어려웠다. 반면, 도 3A에 나타난 바와 같이, 포플러나무의 형성층 유래 줄기세포는 장기 배양시 세포의 생장률, 생장 패턴, 응집 정도에 변이 없이 안정적으로 유지되어 대량 배양이 가능했다.
On the other hand, the bark sections of the poplar tree were sterilized for comparison and then cultured in the medium of Table 1, as a result, as shown in FIG. 3B, the bark sections form callus by dedifferentiation. Could be observed. Callus derived from bark fragments was amorphous due to differences in the rate of division between cells, such as tetrahedral tissue, and showed unstable growth rate and easy browning. Callus derived from browned and agglomerated bark was slowed down by the phenolic compounds it secreted and eventually became necrotic. That is, callus derived from bark from 6 months later was difficult to maintain and incubate. On the other hand, as shown in FIG. 3A, the cambium-derived stem cells of the poplar tree were stably maintained without change in cell growth rate, growth pattern and degree of aggregation during long-term culture, thereby enabling mass culture.
버드나무과의 형성층 유래 줄기세포의 증식 및 특성관찰Proliferation and Characterization of Stem Cells Derived from the Formation Layer of Willowaceae
상기 실시예 1에서 분리한 형성층 유래 줄기세포를 하기 <표 2>의 액상배지가 함유된 플라스크에 넣어 암조건에서 25±1℃에서 100rpm의 회전 교반기(shaker)에서 배양하였다. 계대배양 주기는 2주일로 고정함으로써 배양세포가 항상 대수생장기 상태에서 높은 활력을 유지할 수 있도록 하였다.The stem cell-derived stem cells isolated in Example 1 were placed in a flask containing the liquid medium of the following <Table 2> and cultured in a rotary shaker of 100 rpm at 25 ± 1 ℃ under dark conditions. The subculture period was fixed at 2 weeks so that the cultured cells could always maintain high vitality in the algebraic state.
Inorganic salts
Vitamin
Amino acid
세포 응집정도(biological microscope CX31, Olympus, Japan)를 살펴 볼 때, 본 발명에 따른 줄기세포는, 고체배양에서 응집도를 관찰하였을 때, 도 4A에 나타난 바와 같이 관찰되어 고체배양과정 중에도 많은 수의 단세포를 포함하며, 일부는 작은 사이즈의 세포 집합체로 존재하는 것을 확인할 수 있었다. 그러나, 이에 반하여 대조군인 포플러나무의 수피 조직에서 유도한 캘러스의 경우, 도 4B에 나타난 바와 같이 응집됨을 관찰할 수 있었다. 아울러, 본 발명에 따른 줄기세포는 표 2의 배지 2로 현탁배양하였을 때, 초기 현탁 배양 시 통상 300㎛의 크기를 가지는 작은 세포집합체로 존재하여, 초기 현탁 배양 시 통상 450㎛ 이상의 크기를 가지는 세포집합체를 보이는 캘러스보다 작은 세포집합체를 보임을 확인할 수 있었다. 특히, 배양이 계속됨에 따라 캘러스의 경우 훨씬 더 뭉쳐진 형태로 집합체를 이루어나간 반면, 본 발명에 따른 줄기세포는 다수의 단세포를 포함하거나 작은 사이즈의 세포집합체를 이루는 것을 확인할 수 있었다. When examining the degree of cell aggregation (biological microscope CX31, Olympus, Japan), the stem cells according to the present invention, when observed the degree of aggregation in solid culture, is observed as shown in Figure 4A, a large number of single cells during the solid culture process Included, some were confirmed to exist as a small size cell aggregate. On the contrary, in the case of callus induced in the bark tissue of the control poplar tree, it was observed to aggregate as shown in FIG. 4B. In addition, the stem cells according to the present invention, when suspended in culture in
한편, 도 5A에 나타난 바와 같이, 본 발명에 따른 줄기세포는 다수개의 액포(vacuole)를 가지는 형태학적 특징을 관찰할 수 있었다. 이러한 특징은 식물체 내에 존재하는 미분화된 세포에서 압력 등과 같은 원인에 의하여 나타나는 특징으로서, 이에 본 발명에 따른 줄기세포는 미분화상태임을 확인할 수 있었다. 일반 포플러나무의 체세포(수피 조직에서 유도한 캘러스)의 경우, 도 5B에 나타난 바와 같이, 이러한 특징을 확인할 수 없었다. 이를 더 상세히 살펴보기 위하여, 액포를 neutral red로 염색한 결과, 본 발명에 따른 줄기세포는 도 6A에 나타난 바와 같이 적색의 다수개의 작은 액포(vacuole)를 확인할 수 있었으며, 일반 포플러나무의 체세포(수피 조직에서 유도한 캘러스)의 경우 도 6B에 나타난 바와 같이, 하나의 큰 중심 액포가 존재함을 확인할 수 있었다.On the other hand, as shown in Figure 5A, stem cells according to the present invention was able to observe the morphological characteristics having a plurality of vacuole (vacuole). This feature is a feature that appears by causes such as pressure in the undifferentiated cells present in the plant, it was confirmed that the stem cells according to the present invention is in an undifferentiated state. In the case of somatic cells (callus derived from bark tissue) of the general poplar tree, as shown in FIG. 5B, such a feature could not be confirmed. To examine this in more detail, as a result of staining the vacuole with neutral red, stem cells according to the present invention were able to identify a number of small vacuoles of red as shown in FIG. 6A, and the somatic cells of the poplar tree (bark) Tissue-induced callus), as shown in FIG. 6B, one large It could be confirmed that a central vacuole exists.
한편, 본 발명에 따른 줄기세포는 광학현미경 BX41TF를 통해 관찰한 결과, 움직임 면에서 활성이 매우 높은 미토콘드리아를 다수 개 관찰할 수 있었다. 도 7A는 본 발명에 따른 줄기세포가 다수개의 미토콘드리아를 가짐을 나타내는 것으로 화살표는 미토콘드리아를 나타낸다. 이에 반하여, 일반 포플러나무의 체세포(수피 조직에서 유도한 캘러스)의 경우, 도 7B에 나타난 바와 같이, 이러한 특징을 확인할 수 없었다. On the other hand, stem cells according to the present invention was observed through the optical microscope BX41TF, as a result, it was able to observe a large number of mitochondria very active in terms of movement. 7A shows that stem cells according to the present invention have a plurality of mitochondria, and arrows indicate mitochondria. In contrast, in the case of the somatic cells (callus derived from the bark tissue) of the general poplar tree, as shown in FIG. 7B, these characteristics could not be confirmed.
한편, 대량 배양 가능성을 살펴보기 위하여, 3L의 내용적을 갖는 공기 부양식 생물반응기(airlift bioreactor, 삼성과학, Korea)에서 포플러 수피조직에서 유도한 캘러스와 본 발명에 따른 포플러의 형성층 유래 줄기세포를 배양하였다. 배지는 <표 2>의 액상배지를 사용하였고, 암조건에서 25±1℃로 일정하게 유지하였다.On the other hand, in order to examine the possibility of mass cultivation, incubated stem cells derived from the poplar cambium-derived stem cells of the poplar according to the present invention in a 3L airlift bioreactor (Samsung Science, Korea) It was. The medium used was the liquid medium of <Table 2>, it was kept constant at 25 ± 1 ℃ under dark conditions.
그 결과, <표 3> 및 도 8에 나타난 바와 같이, 포플러 수피조직에서 유도한 캘러스의 생장속도는 4.2배이고, GI (growth index = (maximum DCW - initial DCW) / initial DCW )는 3에 불과하였으나, 본 발명에 따른 형성층 유래 줄기세포는 생장속도가 4.79배이고, GI가 3.79로 포플러나무 수피조직에서 유도한 캘러스에 비하여 높음을 확인할 수 있었다. 통상 반응기의 경우 <표 4>의 포플러의 수피조직에서 유도한 캘러스의 생존율 결과에서 알 수 있듯이 반응기 내에서의 생장고리 (growth ring) 생성과 배양중의 식물 배양체 응집성과 세포벽이 단단하여 전단에 대한 민감성으로 세포 생존율(cell viability)이 급격히 감소하나, 형성층 유래 줄기세포 배양물은 생물반응기 내의 생장고리 면적을 아주 작게 형성하고, 배양기에 간단한 자극을 주어 배지를 움직여주면 내벽의 링(ring)이 간단하게 해소되었다. 또한 응집이 작고, 많은 액포(vacuole)를 가지고 있어 전단에 대한 민감성이 약하여 표 4에서 볼 수 있듯이 세포 생존율(cell viability)의 약간의 감소만을 보였다.As a result, as shown in Table 3 and FIG. 8, the growth rate of callus induced from poplar bark tissue was 4.2 times, and GI (growth index = (maximum DCW-initial DCW) / initial DCW) was only 3. , The cambium-derived stem cells according to the present invention had a growth rate of 4.79 times and a GI of 3.79, which was higher than that of callus induced from poplar bark tissue. In the case of a conventional reactor, as shown in the survival rate of callus derived from the poplar bark tissue of <Table 4>, the growth ring in the reactor and the cohesiveness of the plant culture during the culture and the cell wall are hard, Sensitivity dramatically decreases cell viability, but stem cell cultures from cambium form very small growth areas in bioreactors, and give simple stimulation to the incubator to move the media, resulting in a simple ring on the inner wall. Has been eliminated. In addition, the aggregation was small and had many vacuoles, so that the sensitivity to shear was weak. As shown in Table 4, only a slight decrease in cell viability was shown.
아울러, <표 5> 및 도 9에서 볼 수 있듯이, 3L 공기부양식(air-lift) 생물반응기 및 20L 공기부양식 생물반응기에서 14일간 배양한 결과, 20L의 대량배양시에도 14일 배양 시 오히려 리터당 건조세포 양이 증가한 것으로 나타났다. In addition, as shown in Table 5 and FIG. 9, 14 days of culture in a 3L air-lift bioreactor and a 20L air-lift bioreactor resulted in 14 days of culture even in a large volume culture of 20L. The amount of dry cells per liter was found to increase.
즉, 본 발명에 따른 형성층 유래 줄기세포는 대량배양을 위한 생물 반응기에서 전단 스트레스에 대하여 낮은 민감성을 가지므로, 생물반응기 내에서 급속 대량 생장이 가능함을 확인하였다. 따라서, 본 발명에 따른 형성층 유래 줄기세포가 포플러의 탈분화된 캘러스에 비하여 전단스트레스에 대하여 낮은 민감성을 가짐을 알 수 있었다.That is, the cambium-derived stem cells according to the present invention have low sensitivity to shear stress in the bioreactor for mass culture, and thus, it was confirmed that rapid mass growth is possible in the bioreactor. Therefore, it was found that the cambium-derived stem cells according to the present invention have low sensitivity to shear stress as compared to the dedifferentiated callus of poplar.
한편, 포플러 수피조직에서 유도한 캘러스와 본 발명에 따른 포플러 형성층 유래 줄기세포에 대하여 동결보존을 실시하였다. 현탁배양물은 배양 6일에서 8일 된 것을 사용하며, 동결보존제는 0.5M glycerol(DUCHEFA, The Netherlands)과 0.5M DMSO(DUCHEFA, The Netherlands)와 1M sucrose(DUCHEFA, The Netherlands) 포함된 배지이고, 5ml cryovial(Duran, USA)에 옮겼다. 동결보존제에 처리되는 세포 접종량은 200mg/ml 이다. 동결보존제 처리된 현탁세포는 30분간 냉동고에 유지한 다음 deep freezer에 3시간 보관 후 액체질소에 침지시켜 냉동시켰다. On the other hand, cryopreservation was performed on the callus derived from the poplar bark tissue and the stem cells derived from the poplar forming layer according to the present invention. Suspension culture is used for 6 to 8 days of culture, cryopreservative is a medium containing 0.5M glycerol (DUCHEFA, The Netherlands) and 0.5M DMSO (DUCHEFA, The Netherlands) and 1M sucrose (DUCHEFA, The Netherlands) , 5 ml cryovial (Duran, USA). The amount of cell inoculation treated with cryopreservative is 200 mg / ml. The cryopreservative treated suspension cells were kept in a freezer for 30 minutes and then stored in a deep freezer for 3 hours and then frozen by immersion in liquid nitrogen.
그 후 해빙을 위하여 액체질소에 20분 이상 유지된 배양세포를 꺼내어 40℃ 항온수조에 넣고 1~2분간 해동시켰다. 세포 재생장을 위해, 세포 현탁액을 무균상태의 깔때기 및 여과지를 사용하였다. 여과된 세포는 filter paper가 포함된 고형생장배지상에 적용시키고 30분간 실온에서 안정화 시킨 다음, 다시 신선한 고형생장배지로 다시 옮겨졌다.After that, the cultured cells kept in liquid nitrogen for 20 minutes or more were taken out for thawing and thawed in a 40 ° C. constant temperature water bath for 1 to 2 minutes. For cell regeneration, the cell suspension was used aseptic funnel and filter paper. The filtered cells were applied on solid growth media containing filter paper, stabilized at room temperature for 30 minutes, and then transferred back to fresh solid growth media.
Stem cell
Callus
9.47%
그 결과, 도 10 및 표 6에서 볼 수 있듯이, 포플러 수피조직에서 유도한 캘러스는 본 발명의 포플러 형성층 유래 줄기세포에 비하여 매우 낮은 생존율을 보임을 확인할 수 있었다. As a result, as shown in Figure 10 and Table 6, it was confirmed that the callus induced in the poplar bark tissue shows a very low survival rate compared to the stem cells derived from the poplar forming layer of the present invention.
아울러, 식물줄기세포를 특징짓는 특성에는 자가재생능력(self renewal)이외에 분화능(pluripotency)이 있다. 이에 포플러나무 형성층 유래 줄기세포의 도관요소 분화를 유도하기 위하여,생장조절제를 첨가한 MS medium 조건에서 25±1℃, 암조건에서 배양한 결과 형성층 줄기세포로부터 도관요소가 분화됨을 확인할 수 있었다.
In addition, the characteristics that characterize the plant stem cells have a pluripotency in addition to self renewal (self renewal). In order to induce the conduit element differentiation of the stem cells derived from the poplar tree, it was confirmed that the conduit elements were differentiated from the cambium stem cells as a result of culturing in the condition of 25 ± 1 ℃, dark conditions under the growth medium added MS.
엘리시터의Elisitarian 처리 및 포플러의 형성층 유래 줄기세포의 추출물 제조 Treatment and Preparation of Extracts from Stem Cells Derived from the Formation Layer of Poplar
3-1: 3-1: 엘리시터의Elisitarian 처리 process
실시예 2와 같이 14일간 현탁배양한 줄기세포를 수거하여 2가지 처리구로 나누어 실험을 수행하였다. 즉, (1) 상기 14일간 현탁배양한 세포주(growth phase), (2) 상기 14일간 현탁배양한 세포주를 멸균수에 원당 3~5중량%(g/L) 및 메틸 자스모네이트 100μM를 첨가한 배지에서 14일간 암배양한 세포주(elicitation phase)를 이용하였다.
Stem cells suspended in culture for 14 days were collected as in Example 2, and the experiment was performed by dividing into two treatment groups. That is, (1) the cell phase suspended in culture for 14 days (growth phase), (2) 3 to 5% by weight (g / L) of methyl jasmonate and 100 μM of methyl jasmonate were added to sterile water in the cell line suspended in culture for 14 days Cancer cells were used for 14 days in the elicitation phase (elicitation phase).
3-2: 추출물의 제조3-2: Preparation of Extract
상기 두 세포주를 각각 배양액을 제거시킨 다음 동결건조한 동결건조세포주(Dry) 2g에 50ml의 80% 에탄올로 15℃에서 48시간 교반시키면서 용해시켰다. 상기 용해 후, 3,000rpm에서 20분간 원심분리시켜 상층액을 동결건조하여 얻은 추출분말을 PBS에 용해시킴으로써, 상기 두 세포주에 대한 에탄올 추출물을 각각 수득하여 사용하였다.
Each of the two cell lines was removed from the culture solution and then lyophilized in 2 g of lyophilized lyophilized cell line (Dry) with 50 ml of 80% ethanol at 15 ° C. for 48 hours. After the lysis, the extract powder obtained by lyophilization of the supernatant by centrifugation at 3,000 rpm for 20 minutes was dissolved in PBS, and ethanol extracts of the two cell lines were obtained and used, respectively.
NONO ( ( NitricNitric oxideoxide ) 생성 억제효과 확인 - 항염증 효과의 확인 (1)) Confirmation of production inhibitory effect-Confirmation of anti-inflammatory effect (1)
NO 분석은 대식세포 (Macropharge) 활성을 측정하는 방법 중의 하나로 대식세포의 중요한 기능 중 하나인 식세포작용(phagocytosis)와 관련된 간접적인 활성측정 방법이다. 실시예 3의 포플러 형성층 유래 줄기세포의 에탄올 추출물이, RAW264.7 cell에 LPS(Lipopolysaccharide)를 처리했을 때 발생하는 NO 생성을 저해하는 효과를 상청액(supernatant)상에 녹아있는 NO 양으로 측정하였다.The NO assay is a method of measuring macrophage activity and is an indirect activity measurement method associated with phagocytosis, which is one of the important functions of macrophages. The ethanol extract of the stem cells derived from the poplar forming layer of Example 3 was measured by the amount of NO dissolved in the supernatant to inhibit the production of NO generated when LPS (Lipopolysaccharide) was treated in RAW264.7 cells.
먼저, 24-well 배양플레이트(culture plate)에 RAW264.7 cell(2X105/ml)과 시료를 농도별로 30분간 전처리 배양한 후 LPS(1ug/ml)를 처리하여 24시간 배양시킨 세포배양액 100ul과 Griess reagent 100ul를 넣어 15분 동안 실온에서 암반응 시킨 후 Microplate reader(540nm)에서 흡광도를 측정 (Packard, USA)하였다. 배양액만 넣은 well을 대조군으로 사용하여 NO 농도를 백분율로 나타내었다.First, RAW264.7 cells (2X10 5 / ml) and samples were pretreated for 30 minutes by concentration in a 24-well culture plate, followed by 100ul of cell culture solution treated with LPS (1ug / ml) for 24 hours. 100 μl of Griess reagent was added for dark reaction at room temperature for 15 minutes, and then the absorbance was measured in a microplate reader (540 nm) (Packard, USA). The concentration of NO was expressed as a percentage using the well containing only the culture as a control.
그 결과, 도 11에 나타난 바와 같이, 포플러 형성층 유래 줄기세포의 에탄올 추출물 1mg/ml에서 22%, 1.3mg/ml에서 42% LPS에 의해 유도되는 NO 생성을 억제함을 확인할 수 있었으며, 5mg/ml농도에서는 LPS에 의해 유도된 NO 생성이 80%까지 저해됨을 확인할 수 있었다. 이러한 실험결과는 본 발명에 따른 형성층 유래 줄기세포가 우수한 항염증 효과가 있음을 제시한다.
As a result, as shown in Figure 11, the ethanol extract of the poplar forming layer-derived stem cells were found to inhibit NO production induced by 22% at 1mg / ml, 42% LPS at 1.3mg / ml, 5mg / ml In the concentration, it was confirmed that NO production induced by LPS was inhibited by 80%. These experimental results suggest that the cambium-derived stem cells according to the present invention has an excellent anti-inflammatory effect.
COXCOX -2 발현효과 확인 - 항염증 효과의 확인 (2)-2 Confirmation of expression effect-Confirmation of anti-inflammatory effect (2)
COX-2 염증사이토카인의 유전자 발현 억제효과를 조사하기 위해 6 well 배양플레이트(culture plate)에 RAW264.7 cell(2X106/ml)과 시료를 농도별(A: 0.5g/ml, B: 1mg/ml, C: 2mg/ml)로 30분간 전처리 배양한 후, LPS(1ug/ml)를 처리하여 24시간 배양시킨 세포를 수거하여 TRIzol로부터 전체 RNA를 분리하여 cDNA를 합성하였다. 그 다음, COX-2 Primer를 이용하여 PCR을 수행하였다.To investigate the gene expression inhibitory effect of COX-2 inflammatory cytokines, RAW264.7 cells (2X10 6 / ml) and samples were collected in 6 well culture plates (A: 0.5g / ml, B: 1mg). / ml, C: 2mg / ml) for 30 minutes pre-culture, cells treated with LPS (1ug / ml) for 24 hours to collect the total RNA was separated from TRIzol to synthesize cDNA. Then, PCR was performed using COX-2 Primer.
<COX-2 Primer><COX-2 Primer>
mouse COX2 F: 5'-AAGAAGAAAGTTCATTCCTGATCCC-3' (서열번호 1)mouse COX2 F: 5'-AAGAAGAAAGTTCATTCCTGATCCC-3 '(SEQ ID NO: 1)
mouse COX2 R: 5'-TGACTGTGGGAGGATACATCTCTCC-3' (서열번호 2)mouse COX2 R: 5'-TGACTGTGGGAGGATACATCTCTCC-3 '(SEQ ID NO: 2)
그 결과, 도 12에 나타난 바와 같이, 포플러 형성층 유래 줄기세포의 에탄올 추출물 2mg/ml농도에서 LPS에 의해 유도된 COX-2 염증 유전자 발현이 감소됨을 확인할 수 있었다.
As a result, as shown in Figure 12, it was confirmed that the expression of COX-2 inflammatory genes induced by LPS in the ethanol extract 2mg / ml concentration of the poplar forming layer-derived stem cells.
약학제제Pharmaceutical 제조예Production Example
제제예Formulation example 6-1: 정제의 제조 6-1: Preparation of Tablets
실시예 3에서 제조된 줄기세포 추출물 100㎎을 옥수수 전분 100㎎, 유당 100㎎ 및 스테아린산 마그네슘 2㎎을 혼합하여 통상의 정제제조방법에 따라 제조하였다.100 mg of the stem cell extract prepared in Example 3 was mixed according to the conventional tablet manufacturing method by mixing
제제예Formulation example 6-2: 캡슐제의 제조 6-2: Preparation of Capsule
실시예 3에서 제조된 줄기세포 추출물 500㎎을 연질 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.500 mg of the stem cell extract prepared in Example 3 was filled into a soft gelatin capsule to prepare a capsule.
제제예Formulation example 6-3: 시럽제의 제조 6-3: Preparation of Syrup
실시예 1에서 수득한 줄기세포 1g, 이성화당 10g, 만니톨 5g, 적량의 정제수의 함량으로 통상적인 액제제의 제조방법에 따라 100㎖의 시럽제를 제조하였다.100 ml of syrup was prepared according to a conventional method of preparing a liquid formulation with the content of 1 g of stem cells obtained in Example 1, 10 g of isomerized sugar, 5 g of mannitol, and an appropriate amount of purified water.
제제예Formulation example 6-4: 주사제의 제조 6-4: Preparation of Injection
실시예 3에서 제조된 줄기세포 추출물 200㎎을 폴리옥시에틸렌 수소화 카스트로 오일을 함유하는 생리 식염수 200㎎에 가열 용해시켜 혼합 추출물을 0.1%의 농도로 함유하는 주사제를 제조하였다.
200 mg of the stem cell extract prepared in Example 3 was heat-dissolved in 200 mg of physiological saline containing polyoxyethylene hydrogenated castro oil to prepare an injection containing the mixed extract at a concentration of 0.1%.
기능성 식품의 제조 Preparation of Functional Foods
제조예Production Example 7-1: 기능성 음료의 제조 7-1: Preparation of Functional Beverages
실시예 1에서 수득한 줄기세포 200㎎을 96㎖의 물에 용해시킨 후 보조제로서 비타민 C 500㎎, 교미제로서 구연산, 올리고당을 각각 1g 가하고, 보존재로서 나트륨벤조에이트 0.05g을 가한 후 정제수를 가하여 전량을 100㎖로 만들어 기능성 음료를 제조하였다.After dissolving 200 mg of the stem cells obtained in Example 1 in 96 ml of water, 500 mg of vitamin C as an adjuvant, 1 g of citric acid and oligosaccharides as a copulating agent were added, and 0.05 g of sodium benzoate was added as a preservative. The amount was added to 100ml to prepare a functional beverage.
제조예Production Example 7-2: 기능성 음료의 제조 7-2: Preparation of Functional Beverages
실시예 3에서 제조한 줄기세포 추출물 200㎎을 96㎖의 물에 용해시킨 후 보조제로서 비타민 C 500㎎, 교미제로서 구연산, 올리고당을 각각 1g 가하고, 보존재로서 나트륨벤조에이트 0.05g을 가한 후 정제수를 가하여 전량을 100㎖로 만들어 기능성 음료를 제조하였다.
After dissolving 200 mg of the stem cell extract prepared in Example 3 in 96 ml of water, 500 mg of vitamin C as an adjuvant, 1 g of citric acid and oligosaccharides were added as a coagent, and 0.05 g of sodium benzoate was added as a preservative. The amount was added to 100ml to prepare a functional beverage.
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
<110> UNHWA CORPORATION <120> Plant Stem Cell Derived from Cambium of Family Salicaceae and Method of Isolating the Same <130> P10-B144 <150> KR10-2009-0045955 <151> 2009-05-26 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> mouse COX2 F primer <400> 1 aagaagaaag ttcattcctg atccc 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> mouse COX2 R primer <400> 2 tgactgtggg aggatacatc tctcc 25 <110> UNHWA CORPORATION <120> Plant Stem Cell Derived from Cambium of Family Salicaceae and Method of isolating the same <130> P10-B144 <150> KR10-2009-0045955 <151> 2009-05-26 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> mouse COX2 F primer <400> 1 aagaagaaag ttcattcctg atccc 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> mouse COX2 R primer <400> 2 tgactgtggg aggatacatc tctcc 25
Claims (23)
Stem cells derived from the cambium of the willow family derived from the cambium of the family Salicaceae , characterized in that they are congenital undifferentiated cells without dedifferentiation.
(a) 현탁배양 시 버드나무과의 탈분화된 캘러스에 비하여 많은 수의 단세포를 포함하거나 작은 사이즈의 세포 집합체를 포함함;
(b) 다수의 액포(vacuole)를 가지는 형태학적 특징을 나타냄;
(c) 버드나무과의 탈분화된 캘러스에 비하여 활성이 증가된 미토콘드리아를 가짐;
(d) 버드나무과의 탈분화된 캘러스에 비하여 생장속도가 빠르고 오랫동안 성장할 수 있음; 및
(e) 버드나무과의 탈분화된 캘러스에 비하여 생물반응기에서 전단 스트레스(shear stress)에 대해 낮은 민감성을 가짐.
The stem-derived stem cell of claim 1, further comprising at least one of the following characteristics:
(a) contain large numbers of single cells or small cell aggregates as compared to dedifferentiated callus of Willow family in suspension culture;
(b) exhibit morphological features with multiple vacuoles;
(c) has mitochondria with increased activity compared to dedifferentiated callus of the willow family;
(d) can grow faster and grow longer compared to dedifferentiated callus of Willow family; And
(e) Has low sensitivity to shear stress in bioreactors compared to dedifferentiated callus from Willow family.
According to claim 2, the cambium-derived stem cells, characterized in that it has at least two of the properties of (a) to (e).
According to claim 2, the cambium-derived stem cells, characterized in that it has at least three of the properties of (a) to (e).
According to claim 2, the cambium-derived stem cells, characterized in that it has at least four of the properties of (a) to (e).
According to claim 2, the cambium-derived stem cells, characterized in that it has all the properties of (a) to (e).
(a) 버드나무과 식물로부터 형성층 함유 조직을 수득하는 단계;
(b) 상기 수득된 형성층 함유 조직을 배지에서 배양하는 단계; 및
(c) 상기 형성층으로부터 세포들을 분리함으로써 형성층 유래 줄기세포를 수득하는 단계.
According to claim 1, The cambium-derived stem cells of the willow family cambium-derived stem cells, characterized in that separated by the separation method comprising the following steps:
(a) obtaining a cambium-containing tissue from willow plants;
(b) culturing the obtained cambium-containing tissue in a medium; And
(c) obtaining the cambium-derived stem cells by separating the cells from the cambium.
8. The stem cell according to any one of claims 1 to 7, wherein the willow is a genus Populus .
8. The stem cell according to any one of claims 1 to 7, wherein the willow family is Populus .
(a) 버드나무과 식물로부터 형성층 함유 조직을 수득하는 단계;
(b) 상기 수득된 형성층 함유 조직을 배지에서 배양하는 단계; 및
(c) 상기 형성층으로부터 세포들을 분리함으로써 형성층 유래 줄기세포를 수득하는 단계.
Isolation method of stem cells derived from cambium of family Salicaceae comprising the following steps:
(a) obtaining a cambium-containing tissue from willow plants;
(b) culturing the obtained cambium-containing tissue in a medium; And
(c) obtaining the cambium-derived stem cells by separating the cells from the cambium.
The method of claim 10, wherein step (b) is characterized in that the culture in a medium containing auxin (auxin).
The method of claim 11, wherein the auxin is contained at a concentration of 1-5 mg / l.
The method according to claim 10, wherein the step (c) divides the cultured formation layer from an amorphous callus layer which is derived from a portion other than the formation layer, and then separates the cells from the formation layer to obtain stem cell-derived stem cells. How to.
The method of claim 10, wherein the willow is genus Populus .
11. The method of claim 10, wherein the willow is Populus .
An anti-inflammatory composition containing any one or more of the stem cells derived from the cambium of the willow family of any one of claims 1 to 7, its extract, its lysate and its culture.
The method of claim 16, wherein the culture is the stem cells of 3 to 5% by weight of raw sugar or sugar; Or methyl jasmonate, fungus extract, bacterial extract, yeast extract, chitosan, glucomanan, glucan, phenylalanine, benzoic acid, salicylic acid ), Arachonic acid, STS, mevalonalonate N-benzolyglycine, ABA, SNP, IPP, BHT, CCC, ethephon, hipuic acid, ammonium ceric nitrate, At least one of AgNO 3 , vanadyl sulfate, p-aminobenzoic acid, brassinosteroids, sodium alginate, and sodium acteate Anti-inflammatory composition, characterized in that obtained by further culturing in the medium.
The anti-inflammatory composition according to claim 16, wherein the willow is genus Populus .
The anti-inflammatory composition of claim 16, wherein the willow is Populus .
Functional food for the prevention or improvement of inflammation containing any one or more of the stem cells derived from the cambium of the willow family of any one of claims 1 to 7, its extract, its lysate and its culture.
The method of claim 20, wherein the culture of the stem cells 3 to 5% by weight of raw sugar or sugar; Or methyl jasmonate, fungus extract, bacterial extract, yeast extract, chitosan, glucomanan, glucan, phenylalanine, benzoic acid, salicylic acid ), Arachonic acid, STS, mevalonalonate N-benzolyglycine, ABA, SNP, IPP, BHT, CCC, ethephon, hipuic acid, ammonium ceric nitrate, At least one of AgNO 3 , vanadyl sulfate, p-aminobenzoic acid, brassinosteroids, sodium alginate, and sodium acteate Functional food for the prevention or improvement of inflammation, characterized in that obtained by further culturing in the medium.
21. The functional food for preventing or improving inflammation of claim 20, wherein the willow is genus Populus .
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