KR20190098028A - A composition comprising an exosome derived from adipose-derived stem cell as an active ingredient and its application for improving Ulcerative Colitis - Google Patents
A composition comprising an exosome derived from adipose-derived stem cell as an active ingredient and its application for improving Ulcerative Colitis Download PDFInfo
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- KR20190098028A KR20190098028A KR1020180126890A KR20180126890A KR20190098028A KR 20190098028 A KR20190098028 A KR 20190098028A KR 1020180126890 A KR1020180126890 A KR 1020180126890A KR 20180126890 A KR20180126890 A KR 20180126890A KR 20190098028 A KR20190098028 A KR 20190098028A
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- exosomes
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Abstract
Description
본 발명은 지방줄기세포 유래의 엑소좀을 유효성분으로 포함하는 조성물의 궤양성 대장염의 개선 용도에 관한 것이다.The present invention relates to the improvement of ulcerative colitis of a composition comprising an exosome derived from adipose stem cells as an active ingredient.
또한, 본 발명은 상기 조성물을 포함하는 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료용 약학 조성물에 관한 것이다.The present invention also relates to a pharmaceutical composition for preventing, inhibiting, alleviating, improving or treating ulcerative colitis comprising the composition.
추가로, 본 발명은 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료에 있어서 임상적 적용이 가능한 고순도이면서 입자크기 분포가 균일한 지방줄기세포 유래의 엑소좀을 대량으로 수득할 수 있고, 이와 같이 수득된 기능적 활성이 우수한 지방줄기세포 유래의 엑소좀을 유효성분으로 포함하는 조성물을 저가로 대량으로 제공할 수 있는 임상 및 상업적으로 뛰어난 기술에 관한 것이다.In addition, the present invention can obtain a large amount of exosomes derived from adipose stem cells of high purity and uniform particle size distribution that can be clinically applied in the prevention, inhibition, alleviation, improvement or treatment of ulcerative colitis. The present invention relates to a clinical and commercially superior technology capable of providing a large amount of a composition containing exosomes derived from adipose stem cells having excellent functional activity as an active ingredient at low cost.
염증성 장질환은 대장 및/또는 소장의 점막에 만성 염증 및/또는 궤양을 일으키는 원인 불명의 질환이다. 대표적인 염증성 장질환으로는 궤양성 대장염(UC; Ulcerative Colitis)과 크론병이 있다. 두 질환 모두 복통, 설사 등 유사한 위장관 증상을 나타내고 스테로이드나 면역억제제 같은 치료에 반응한다는 공통점이 있다. 그러나, 유전적 배경 및 병태생리에 차이가 있고 침범부위, 합병증 등의 임상적 양상이 다르다.Inflammatory bowel disease is an unknown cause of chronic inflammation and / or ulceration in the mucosa of the large intestine and / or small intestine. Representative inflammatory bowel diseases include ulcerative colitis (UC) and Crohn's disease. Both diseases have similar gastrointestinal symptoms, such as abdominal pain and diarrhea, and they have in common that they respond to treatments such as steroids and immunosuppressants. However, there are differences in genetic background and pathophysiology, and clinical features such as involvement sites and complications.
궤양성 대장염은 대장의 점막 또는 점막하층에 국한된 염증을 특징으로 하고, 호전과 악화가 반복되는 혈성 설사, 대변 급박감 및 복통 등이 주증상이다. 궤양성 대장염은 항문직장 경계부위부터 병변이 시작되어 연속적, 대칭적으로 근위부로 진행하며, 염증 및 궤양 부위와 정상 부위의 경계가 명확한 경우가 대부분이다. 궤양성 대장염의 경우 대부분 직장을 침범하며, 점막의 부종, 발적, 궤양 등의 병변이 직장으로부터 연속되어 존재한다. 반면에 크론병은 입안에서 항문까지 위장관의 어느 곳에서도 국소적으로 침범할 수 있고, 점막에 염증이 국한되는 궤양성 대장염과는 달리 장의 전층에 염증이 침윤할 수 있다. 또한, 크론병은 궤양성 대장염과는 달리 점막 발적은 경미하고, 아프타성 궤양, 종주형 궤양 등이 비연속적, 비대칭적으로 분포한다.Ulcerative colitis is characterized by inflammation localized in the mucosa or submucosa of the large intestine, and the main symptoms include bloody diarrhea, stool urgency, and abdominal pain, with repeated improvement and deterioration. Ulcerative colitis begins with the lesion at the anal rectal margin and progresses continuously and symmetrically. In most cases, ulcerative and ulcerative and normal areas have clear boundaries. Most cases of ulcerative colitis invade the rectum, and swelling of the mucous membrane, redness, ulcers, and the like continue from the rectum. On the other hand, Crohn's disease can locally invade anywhere in the gastrointestinal tract, from the mouth to the anus. In contrast to ulcerative colitis, where inflammation is limited to the mucous membranes, inflammation can invade the entire intestine. In addition, unlike ulcerative colitis, Crohn's disease is mild in mucosal redness, and discontinuous and asymmetrical distribution of aphthous ulcers and columnar ulcers.
궤양성 대장염과 크론병은 다양한 원인에 기인하는 만성적인 전신질환으로 수술 및 치료방법과 예후가 다르지만 면역반응 조절 이상과 관련이 있는 것으로 보고되고 있다.Ulcerative colitis and Crohn's disease are chronic systemic diseases caused by various causes. However, ulcerative colitis and Crohn's disease have been reported to be related to abnormal immune response control.
궤양성 대장염의 치료를 위해 수술이나 식사요법 외에 항염증제, 5-아미노살리실산 계통 약물(예를 들어, 설파살라진), 스테로이드, 면역억제제 등의 약물요법이 이용되고 있다. 그러나 항염증제, 설파살라진이나 스테로이드는 충분한 치료 효과를 얻을 수 없을 뿐만 아니라, 위장관 장애, 간질환, 신장병, 당뇨병, 두통, 발진, 백혈구 감소증과 같은 부작용이 있다. 최근에는 생물학적 제제로서, TNF-α 저해제 등의 면역억제제가 개발되어 사용되고 있으나, 이러한 면역억제제는 인체의 면역을 억제하기 때문에 약물을 장시간 사용할 경우에 인체의 면역기능의 저하로 인하여 암, 결핵 등을 포함하여 여러 가지 감염증 등을 일으킬 수 있다는 단점이 있다.For the treatment of ulcerative colitis, anti-inflammatory drugs, 5-aminosalicylic acid-based drugs (eg, sulfasalazine), steroids, immunosuppressants and the like are used in addition to surgery and diet therapy. However, anti-inflammatory drugs, sulfasalazine or steroids do not have sufficient therapeutic effects, but also have side effects such as gastrointestinal disorders, liver disease, kidney disease, diabetes, headaches, rashes and leukopenia. Recently, immunosuppressive agents, such as TNF-α inhibitors, have been developed and used as biological agents. However, these immunosuppressive agents suppress the body's immunity. It has the disadvantage that it can cause various infections.
이러한 문제점을 감안하여 천연물을 이용한 궤양성 대장염 치료제에 대한 연구가 진행되고 있다. 이러한 천연물을 원료로 하는 궤양성 대장염 치료제의 경우, 천연 추출물 내의 유효성분 함량이 적은 관계로 궤양성 대장염 치료 효과를 얻기 위해서는 많은 양의 사용이 필요하고, 이들 중 대부분은 천연물 소재라는 점을 마케팅에 활용하고 있을 뿐 궤양성 대장염 치료의 실질적 효능에 대해서는 과학적 연구가 좀더 필요한 상황이다.In view of these problems, research on the treatment of ulcerative colitis using natural products is underway. In the case of the treatment of ulcerative colitis which is made from such natural materials, the amount of active ingredient in the natural extract is low, so that a large amount of use is necessary to obtain the therapeutic effect of ulcerative colitis, and most of them are natural materials. More scientific research is needed on the practical efficacy of treating ulcerative colitis.
한편, 줄기세포를 이용한 조직 재생 및 치료 방법이 제안되고 있다. 배아줄기세포 또는 태아조직 유래 줄기세포는 분화능력 및 재생치료능력이 우수하고 거부반응이 적지만, 윤리적 문제로 임상에 적용될 수 없고 종양을 형성할 수 있는 위험성이 존재한다. 이에 대한 대안으로서 성체줄기세포를 이용한 조직 재생 및 치료 방법이 제안되었다. 그러나, 환자 자신의 성체줄기세포가 이닌 타인의 성체줄기세포를 사용한 경우 이식편대숙주병(graft-versus-host disease)을 일으킬 위험이 있고, 자가 성체줄기세포를 이용하여 치료를 하기 위해서는 환자로부터 성체줄기세포를 채취한 후 이를 배양하는 과정이 필요하여 복잡하고 비용이 많이 드는 문제가 있다. On the other hand, tissue regeneration and treatment using stem cells has been proposed. Although embryonic stem cells or fetal tissue-derived stem cells have excellent differentiation and regenerative treatment ability and low rejection, they are not applicable to clinical practice due to ethical issues and there is a risk of forming tumors. As an alternative, tissue regeneration and treatment methods using adult stem cells have been proposed. However, if adult stem cells of the patient's own adult stem cells are used, there is a risk of developing graft-versus-host disease. There is a complicated and expensive problem because the process of culturing stem cells after harvesting them.
최근에는 전술한 바와 같은 줄기세포의 문제점을 감안하여 성체줄기세포를 배양하여 얻은 배양액을 이용하여 조직 재생 및 치료를 하고자 하는 시도가 있다. 그러나, 성체줄기세포 배양액에는 성체줄기세포가 분비하는 다양한 단백질, 사이토카인, 성장인자 등이 함유되어 있는 반면, 세포가 성장하면서 분비한 노폐물, 오염방지를 위해 첨가된 항생제, 동물유래 혈청 등의 성분도 포함되어 있기 때문에 손상된 조직에 사용할 경우 각종 위험에 노출될 가능성이 높다. Recently, in view of the problems of stem cells as described above, there is an attempt to regenerate and treat tissue using a culture medium obtained by culturing adult stem cells. However, the adult stem cell culture medium contains various proteins, cytokines, and growth factors secreted by the adult stem cells, while other components such as waste products secreted as the cells grow, antibiotics added to prevent contamination, and animal-derived serum. As it is included, it is likely to be exposed to various risks when used in damaged tissues.
최근 세포 분비물(secretome)에 세포의 행동(behavior)을 조절하는 다양한 생체활성인자가 포함되어 있다는 연구가 보고되고 있으며, 특히 세포 분비물 내에는 세포 간 신호전달 기능을 갖는 '엑소좀(exosome)'이 포함되어 있어 그 성분과 기능에 대한 연구가 활발히 진행 중에 있다. Recently, research has been reported that the cell secretome contains various bioactive factors that regulate the behavior of the cells. Especially, in the cell secretion, 'exosomes', which have intercellular signaling functions, As it is included, research on its components and functions is actively underway.
세포는 세포외 환경에 다양한 막(membrane) 유형의 소포체를 방출하는데, 통상 이러한 방출 소포체들을 세포외 소포체(Extracellular vesicles, EV)라고 부르고 있다. 세포외 소포체는 세포막 유래 소포체, 엑토좀(ectosomes), 쉐딩 소포체(shedding vesicles), 마이크로파티클(microparticles), 엑소좀 등으로 불려지기도 하며, 경우에 따라서는 엑소좀과는 구별되어 사용되기도 한다.Cells release various membrane types of endoplasmic reticulum into the extracellular environment, which are commonly referred to as extracellular vesicles (EVs). Extracellular vesicles are called cell membrane-derived vesicles, ectosomes, shedding vesicles, microparticles, exosomes, and the like, and in some cases, may be used separately from exosomes.
엑소좀은 세포막의 구조와 동일한 이중인지질막으로 이루어진 수십 내지 수백 나노미터 크기의 소포체로서 내부에는 엑소좀 카고(cargo)라고 불리는 단백질, 핵산(mRNA, miRNA 등) 등이 포함되어 있다. 엑소좀 카고에는 광범위한 신호전달 요소들(signaling factors)이 포함되며, 이들 신호전달 요소들은 세포 타입에 특이적이고 분비세포의 환경에 따라 상이하게 조절되는 것으로 알려져 있다. 엑소좀은 세포가 분비하는 세포 간 신호전달 매개체로서 이를 통해 전달된 다양한 세포 신호는 표적 세포의 활성화, 성장, 이동, 분화, 탈분화, 사멸(apoptosis), 괴사(necrosis)를 포함한 세포 행동을 조절한다고 알려져 있다. 엑소좀은 유래된 세포의 성질 및 상태에 따라 특이적인 유전물질과 생체활성 인자들이 포함되어 있다. 증식하는 줄기세포 유래 엑소좀의 경우 세포의 이동, 증식 및 분화와 같은 세포 행동을 조절하고, 조직 재생과 관련된 줄기세포의 특성이 반영되어 있다(Nature Review Immunology 2002 (2) 569-579). Exosomes are tens to hundreds of nanometers of endoplasmic reticulum consisting of a double phospholipid membrane identical to the structure of a cell membrane, and include proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo. Exosome cargo includes a wide range of signaling factors, which are known to be specific for cell types and differently regulated by the environment of the secretory cell. Exosomes are intercellular signaling media secreted by cells, and the various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Known. Exosomes contain specific genetic material and bioactive factors depending on the nature and state of the cells from which they are derived. Stem cell-derived exosomes, which proliferate, regulate cell behavior such as cell migration, proliferation and differentiation, and reflect stem cell characteristics related to tissue regeneration (Nature Review Immunology 2002 (2) 569-579).
그러나, 엑소좀을 이용한 일부 질환의 치료에 대한 가능성 제시 등 다양한 연구가 이루어지고 있음에도 불구하고, 보다 면밀한 임상 및 비임상 연구가 필요하며, 특히 엑소좀이 작용하는 다양한 표적을 과학적으로 규명하여 엑소좀을 다양한 질환 치료에 응용할 수 있는 기술의 개발이 필요한 실정이다.However, despite various studies, such as suggesting the possibility of treating some diseases using exosomes, more detailed clinical and nonclinical studies are needed, and in particular, exosomes are scientifically identified for various targets on which exosomes work. It is necessary to develop a technology that can be applied to the treatment of various diseases.
본 발명자들은 궤양성 대장염과 관련하여 종래 알려진 치료제에 비하여 효과가 뛰어나고 안전한 치료제를 개발하고자 노력하였다. 이에 본 발명자들은 지방줄기세포로부터 유래된 엑소좀의 새로운 용도에 대해 예의 연구를 거듭하던 중, 지방줄기세포 배양액으로부터 분리된 엑소좀이 전술한 바와 같은 줄기세포 자체나 줄기세포 배양액의 안전성 문제를 해결할 수 있고, 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료에 효과적임을 확인하여 본 발명을 완성하였다.The present inventors have tried to develop an effective and safe therapeutic agent compared to the conventionally known therapeutic agent with respect to ulcerative colitis. Accordingly, the present inventors have intensively studied new uses of exosomes derived from adipose stem cells, and the exosomes isolated from adipose stem cell cultures solve the safety problems of the stem cells themselves or stem cell cultures as described above. The present invention was completed by confirming that it is effective in preventing, suppressing, alleviating, improving or treating ulcerative colitis.
한편, 상기한 배경기술로서 설명된 사항들은 본 발명의 배경에 대한 이해 증진을 위한 것일 뿐, 본 발명의 "선행 기술"로서 이용될 수 있다는 승인으로서 인용한 것은 아님을 이해하여야 한다.On the other hand, it is to be understood that the matters described as the background art are only for the purpose of improving the understanding of the background of the present invention and are not cited as an approval that they can be used as the "prior art" of the present invention.
본 발명의 목적은 지방줄기세포 유래의 엑소좀을 유효성분으로 포함하는 조성물의 궤양성 대장염의 개선 용도를 제공하는데 있다.It is an object of the present invention to provide an improved use of ulcerative colitis of a composition comprising an exosome derived from adipose stem cells as an active ingredient.
본 발명의 다른 목적은 상기 조성물을 포함하는 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료용 약학 조성물을 제공하는데 있다.Another object of the present invention to provide a pharmaceutical composition for the prevention, inhibition, alleviation, improvement or treatment of ulcerative colitis comprising the composition.
본 발명의 또 다른 목적은 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료에 있어서 임상적 적용이 가능한 고순도이면서 입자크기 분포가 균일한 지방줄기세포 유래의 엑소좀을 대량으로 수득할 수 있고, 이와 같이 수득된 기능적 활성이 우수한 지방줄기세포 유래의 엑소좀을 유효성분으로 함유하는 조성물을 제공하는데 있다. Another object of the present invention is to obtain a large amount of exosomes derived from adipose stem cells of high purity and uniform particle size distribution that can be clinically applied in the prevention, inhibition, alleviation, improvement or treatment of ulcerative colitis, It is to provide a composition containing an exosome derived from adipose stem cells excellent in functional activity thus obtained as an active ingredient.
본 발명의 또 다른 목적은 상기 조성물을 이용하여 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료하는 방법을 제공하는데 있다.Still another object of the present invention is to provide a method for preventing, suppressing, alleviating, improving or treating ulcerative colitis using the composition.
그러나, 전술한 바와 같은 본 발명의 과제는 예시적인 것으로, 이에 의해 본 발명의 범위가 제한되는 것은 아니다. 또한, 본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.However, the problems of the present invention as described above are exemplary, and the scope of the present invention is not limited thereby. Further objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
상기와 같은 목적을 달성하기 위하여, 본 발명은 지방줄기세포 유래의 엑소좀을 유효성분으로 포함하는, 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료용 조성물을 제공한다. In order to achieve the above object, the present invention provides a composition for preventing, inhibiting, alleviating, improving or treating ulcerative colitis, comprising an exosome derived from adipose stem cells as an active ingredient.
또한, 본 발명은 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료에 있어서 임상적 적용이 가능한 고순도이면서 입자크기 분포가 균일한 지방줄기세포 유래의 엑소좀을 대량으로 수득할 수 있고, 이와 같이 수득된 기능적 활성이 우수한 지방줄기세포 유래의 엑소좀을 유효성분으로 포함하는 조성물을 저가로 대량으로 제공할 수 있는 임상 및 상업적으로 뛰어난 신규한 기술을 제공한다. In addition, the present invention can obtain a large amount of exosomes derived from adipose stem cells with high purity and uniform particle size distribution that can be clinically applied in the prevention, inhibition, alleviation, improvement or treatment of ulcerative colitis. It provides a novel clinical and commercially superior technology that can provide a large amount of a composition comprising an exosome derived from adipose stem cells with excellent functional activity as an active ingredient in a large amount at low cost.
본 명세서에서 용어, "엑소좀(exosomes)"은 세포막의 구조와 동일한 이중인지질막으로 이루어진 수십 내지 수백 나노미터(바람직하게는 대략 30~200 nm) 크기의 소포체를 의미한다(단, 분리 대상이 되는 세포 종류, 분리방법 및 측정방법에 따라 엑소좀의 입자 크기는 가변될 수 있음)(Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, (2015) DOI 10.1007/s00216-015-8535-3). 엑소좀에는 엑소좀 카고(cargo)라고 불리는 단백질, 핵산(mRNA, miRNA 등) 등이 포함되어 있다. 엑소좀 카고에는 광범위한 신호전달 요소들(signaling factors)이 포함되며, 이들 신호전달 요소들은 세포 타입에 특이적이고 분비세포의 환경에 따라 상이하게 조절되는 것으로 알려져 있다. 엑소좀은 세포가 분비하는 세포 간 신호전달 매개체로서 이를 통해 전달된 다양한 세포 신호는 표적 세포의 활성화, 성장, 이동, 분화, 탈분화, 사멸(apoptosis), 괴사(necrosis)를 포함한 세포 행동을 조절한다고 알려져 있다.As used herein, the term "exosomes" refers to vesicles of a size ranging from tens to hundreds of nanometers (preferably approximately 30 to 200 nm) consisting of a double phospholipid membrane identical to the structure of a cell membrane, provided that Particle size of exosomes may vary depending on the cell type, isolation method and measurement method) (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, (2015) DOI 10.1007 / s00216-015-8535-3). Exosomes include proteins called exosome cargo (cargo), nucleic acids (mRNA, miRNA, etc.). Exosome cargo includes a wide range of signaling factors, which are known to be specific for cell types and differently regulated by the environment of the secretory cell. Exosomes are intercellular signaling media secreted by cells, and the various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Known.
한편, 본 명세서에서 사용된 "엑소좀"이란 용어는 지방줄기세포에서 분비되어 세포외 공간으로 방출된 나노 크기의 베지클 구조를 갖고 있고 엑소좀과 유사한 조성을 갖는 베지클(예를 들어, 엑소좀-유사 베지클)을 모두 포함하는 것을 의미한다. On the other hand, the term "exosome" as used herein has a nano-size vesicle structure secreted by the adipose stem cells and released into the extracellular space and a vesicle (eg, exosomes) having a composition similar to exosomes -Like vesicles).
그 동안 지방줄기세포가 아닌 신경줄기세포 유래의 엑소좀이 뇌손상 치료, 줄기세포 이식거부에 따른 염증 질환 치료에 효과가 있다는 보고는 있으나, 현재까지 지방줄기세포 배양액으로부터 분리 정제된 엑소좀을 사용할 때 "궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료"에 효과가 있다는 것에 대해서는 공지된 바가 없었다.Exosomes derived from neural stem cells, not fat stem cells, have been reported to be effective in treating brain damage and inflammatory diseases caused by stem cell transplant rejection. However, when using exosomes purified from adipose stem cell culture, There is no known effect on "preventing, inhibiting, alleviating, improving or treating ulcerative colitis."
또한, 줄기세포 이식을 위해 자궁내막 재생세포 유래의 엑소좀을 줄기세포와 함께 환자에게 투여하여 면역조절(줄기세포로 인한 거부반응 조절)을 하고자 한 사례가 있다. 그러나 이러한 종래기술에서는 줄기세포를 환자에게 투여할 때 발생하는 면역거부를 조절하기 위해 자궁내막 재생세포 유래의 엑소좀을 줄기세포와 병용하여 투여하는 것을 제안하고 있을 뿐이다.In addition, there is a case where an exosome derived from endometrial regenerative cells along with stem cells is administered to a patient for stem cell transplantation and immunomodulation (regulation of stem cell rejection). However, in the prior art, it is only proposed to administer exosomes derived from endometrial regenerative cells in combination with stem cells in order to control immunorejection generated when the stem cells are administered to the patient.
현재까지 대량 배양이 가능한 지방줄기세포를 배양한 후, 지방줄기세포 배양액으로부터 대량으로 경제적으로 분리 정제된 엑소좀을 궤양성 대장염 치료에 임상적으로 적용가능하게 구현한 치료제는 개발된 바가 없다. 지방에서 유래한 지방줄기세포(adipose-derived stem cells)는 지방 흡입술과 같은 간단한 시술에 의해 대량으로 입수가 가능하고 골수, 제대 또는 제대혈 등에 비해 약 40배 정도의 줄기세포가 있어 상업적으로는 가장 원가가 저렴하고 생산량이 많지만, 지방 내에 세포 잔해물, 노폐물, 단백질 및 거대입자와 같은 불순물이 많아 지방 유래 줄기세포 배양액으로부터 고순도이면서 입자크기 분포가 균일한 엑소좀을 경제적으로 대량으로 분리하는 것이 어렵다. 따라서, 경제성 측면에서 지방줄기세포 배양액으로부터 고순도의 입자크기 분포가 균일한 엑소좀을 대량으로 분리하는 것에는 기술적 장벽이 있었다. To date, no therapeutic agent has been developed that has been clinically applicable to the treatment of ulcerative colitis after culturing fat stem cells capable of mass cultivation, and then purifying exosomes that have been economically separated and purified from fat stem cell culture in large quantities. Adipose-derived stem cells derived from adipose are available in large quantities by simple procedures such as liposuction, and have about 40 times more stem cells than bone marrow, umbilical cord or cord blood. Although it is inexpensive and has a high yield, it is difficult to economically separate a large amount of exosomes with high purity and uniform particle size from fat-derived stem cell culture because of abundant impurities such as cell debris, waste products, proteins and macroparticles in fat. Therefore, in terms of economics, there was a technical barrier to separating large quantities of exosomes with a uniform distribution of high-purity particles from adipose stem cell culture.
본 발명의 조성물은 약학 조성물로 적용될 때, 유효성분으로 함유된 지방줄기세포 유래의 엑소좀이 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료에 유의적인 효과를 나타내며, 줄기세포 자체나 줄기세포 배양액의 안전성 문제를 해결할 수 있다. 또한, 본 발명은 궤양성 대장염 치료제로서의 적용이 가능한 고순도이면서 입자크기 분포가 균일한 지방줄기세포 유래의 엑소좀을 유효성분으로 함유하는 조성물을 저가로 대량으로 제공할 수 있고, 엑소좀 치료제의 임상 적용 및 상업화를 가능하게 하는 점에서 종래기술과 명백히 차별화되는 기술이다.When the composition of the present invention is applied as a pharmaceutical composition, exosomes derived from adipose stem cells contained as an active ingredient have a significant effect on the prevention, inhibition, alleviation, improvement or treatment of ulcerative colitis, stem cells themselves or stem cells The safety problem of the culture solution can be solved. In addition, the present invention can provide a high-cost, high-purity and uniform particle size distribution of exosomes derived from adipose stem cells, which can be applied as a therapeutic agent for ulcerative colitis at low cost in large quantities, clinical It is a distinct technology from the prior art in that it enables application and commercialization.
본 발명의 일 구체예의 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료용 조성물은 지방줄기세포 유래의 엑소좀을 유효성분으로 포함한다.The composition for preventing, inhibiting, alleviating, improving or treating ulcerative colitis according to one embodiment of the present invention includes an exosome derived from adipose stem cells as an active ingredient.
본 발명의 일 구체예의 조성물에 있어서, 상기 엑소좀은 궤양성 대장염의 원인이 되는 사이토카인인 TNF-α, IL-6, IL-12 및 IL-23의 생성량 또는 발현량을 감소시키는 것을 특징으로 한다.In the composition of one embodiment of the present invention, the exosomes are characterized by reducing the amount of production or expression of cytokines TNF-α, IL-6, IL-12 and IL-23, which cause ulcerative colitis do.
본 발명의 일 구체예의 조성물에 있어서, 상기 엑소좀은 하기의 단계들을 수행하여 수득될 수 있다: (a) 지방줄기세포 배양액에 트레할로오스를 첨가하는 단계, (b) 상기 트레할로오스가 첨가된 지방줄기세포 배양액을 여과하는 단계, (c) 상기 여과된 지방줄기세포 배양액으로부터, TFF(Tangential Flow Filtration)를 이용하여 엑소좀을 분리하는 단계, 및 (d) 탈염과 버퍼교환(diafiltration)에 사용되는 완충용액에 트레할로오스를 첨가하고, 상기 트레할로오스가 첨가된 완충용액을 이용한 TFF(Tangential Flow Filtration)를 이용하여, 상기 분리된 엑소좀에 대한 탈염과 버퍼교환(diafiltration)을 수행하는 단계. In the composition of one embodiment of the present invention, the exosomes can be obtained by performing the following steps: (a) adding trehalose to the adipose stem cell culture, (b) the trehalose Filtering the added fat stem cell culture medium, (c) separating exosomes from the filtered fat stem cell culture medium using tangential flow filtration (TFF), and (d) desalting and diafiltration. Trehalose was added to the buffer solution used in the above), and desalting and diaphragm exchange (diafiltration) of the separated exosomes were performed by using TFF (Tangential Flow Filtration) using the buffer solution containing trehalose. Step).
본 발명의 일 구체예의 조성물에 있어서, 상기 (d) 단계에서 탈염과 버퍼교환(diafiltration)에 사용되는 완충용액에 트레할로오스를 첨가하면, 입자크기 분포가 균일하고 순도가 높은 엑소좀을 효과적으로 수득할 수 있다(도 6 참조). 본 발명에서는 TFF에 의한 엑소좀 분리 전의 사전 여과 과정((b) 단계)과 엑소좀 분리 후 TFF에 의한 탈염 및 버퍼교환 과정((d) 단계)에서 트레할로우스를 사용하는 것에 의해 고순도의 입자크기 분포가 균일한 엑소좀을 높은 수율로 수득할 수 있다. In the composition of one embodiment of the present invention, when trehalose is added to the buffer solution used for desalting and diafiltration in step (d), the exosomes having a uniform particle size distribution and high purity are effectively Can be obtained (see FIG. 6). In the present invention, particles of high purity are obtained by using trehalose in the pre-filtration process (step (b)) before separation of exosomes by TFF and the desalting and buffer exchange process (t) step by TFF after separation of exosomes. Exosomes with uniform size distribution can be obtained in high yield.
한편, 본 발명에 있어서 트레할로오스는 세포 잔해물, 노폐물, 단백질 및 거대 입자와 같은 불순물에 대해 엑소좀을 효율적으로 분별할 수 있는 기능을 부여한다. On the other hand, trehalose in the present invention provides a function capable of efficiently separating exosomes against impurities such as cell debris, waste products, proteins and large particles.
본 발명의 일 구체예의 조성물에 있어서, 상기 탈염과 버퍼교환은 연속적으로 수행하거나 단속적으로 수행할 수 있다. 시작 부피(starting volume)에 대하여 적어도 4배, 바람직하게는 6배 내지는 10배 이상, 보다 바람직하게는 12배 이상의 부피를 갖는 완충용액을 이용하여 탈염과 버퍼교환을 수행할 수 있다. In the composition of one embodiment of the present invention, the desalting and buffer exchange may be performed continuously or intermittently. Desalting and buffer exchange can be carried out using a buffer solution having a volume of at least 4 times, preferably 6 times to 10 times or more preferably 12 times or more relative to the starting volume.
본 발명의 일 구체예의 조성물에 있어서, TFF를 위해 MWCO(molecular weight cutoff) 100,000 Da(Dalton), 300,000 Da, 500,000 Da 또는 750,000 Da의 TFF 필터, 또는 0.05 μm TFF 필터를 사용할 수 있다. In the composition of one embodiment of the invention, a molecular weight cutoff (MWCO) 100,000 Da (Dalton), 300,000 Da, 500,000 Da or 750,000 Da of a TFF filter, or 0.05 μm TFF filter can be used for the TFF.
본 발명의 일 구체예의 조성물에 있어서, 상기 (c) 단계는 TFF(Tangential Flow Filtration)를 이용하여 1/100 내지 1/25의 부피까지 농축하는 과정을 더 포함할 수 있다. In the composition of one embodiment of the present invention, step (c) may further comprise the step of concentrating to a volume of 1/100 to 1/25 using TFF (Tangential Flow Filtration).
본 발명의 일 구체예의 조성물에서 상기 지방줄기세포의 종류는 병원체에 의한 감염의 위험이 없고 면역 거부 반응을 일으키지 않는 것이라면 제한되지 않으나, 바람직하게는 인간지방 유래 줄기세포일 수 있다.The type of the adipose stem cells in the composition of one embodiment of the present invention is not limited as long as there is no risk of infection by the pathogen and does not cause an immune rejection reaction, but preferably, human fat-derived stem cells.
본 발명의 일 구체예의 조성물은 다양한 원인에 기인하여 발병되는 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료에 효과적으로 이용될 수 있다. The composition of one embodiment of the present invention can be effectively used for the prevention, inhibition, alleviation, amelioration or treatment of ulcerative colitis caused by various causes.
본 발명의 일 구체예의 조성물은 약학 조성물로 제조될 수 있다. 본 발명의 일 구체예의 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료용 조성물이 약학 조성물로 제조되는 경우, 본 발명의 일 구체예에 따른 약학 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. The composition of one embodiment of the present invention may be prepared into a pharmaceutical composition. When the composition for preventing, inhibiting, alleviating, improving or treating ulcerative colitis of one embodiment of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition according to one embodiment of the present invention may be various oral or parenteral formulations. .
본 발명의 일 구체예의 약학 조성물은 약학적으로 허용 가능한 담체, 부형제 또는 희석제 등을 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 트레할로오스, 수크로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 카보네이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스(microcrystalline cellulose), 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 미네랄 오일 등을 들 수 있으며, 이에 제한되지 않는다. 본 발명의 일 구체예의 약학 조성물은 통상의 방법에 따라 산제, 환제, 정제, 캡슐제, 현탁제, 에멀젼, 시럽, 과립제, 엘릭시르제(elixirs), 에어로졸 등의 경구투여용 제제, 외용제, 좌제, 또는 멸균 주사용액의 형태로 제형화하여 사용할 수 있다.The pharmaceutical composition of one embodiment of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent. The carrier, excipient and diluent may include lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like, but are not limited thereto. . Pharmaceutical compositions of one embodiment of the present invention, powders, pills, tablets, capsules, suspensions, emulsions, syrups, granules, oral preparations such as elixirs, aerosols, external preparations, suppositories, according to a conventional method Or in the form of sterile injectable solutions.
본 발명의 일 구체예의 약학 조성물의 투여는 어떠한 적절한 방법으로 환자에게 소정의 물질을 도입하는 것을 의미하며, 상기 약학 조성물의 투여경로는 약물이 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 예를 들어, 본 발명의 일 구체예의 약학 조성물은 경구 또는 비경구 투여할 수 있으며, 비경구 투여로는 경피 투여, 복강 내 투여, 정맥 내 투여, 동맥 내 투여, 림프 내 투여, 근육 내 투여, 피하 투여, 피내 투여, 국소 투여, 직장 내 투여 등을 거론할 수 있다. 그러나, 이에 제한되는 것이 아니며 당업계에 알려진 다양한 투여 방법을 배제하지 않는다. 또한, 본 발명의 일 구체예의 약학 조성물은 활성 물질이 표적 조직 또는 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다. 또한, 본 발명의 일 구체예의 약학 조성물의 유효량은 치료 효과를 기대하기 위하여 투여에 요구되는 양을 의미한다.Administration of the pharmaceutical composition of one embodiment of the present invention means introducing a predetermined substance into the patient in any suitable manner, the route of administration of the pharmaceutical composition via any general route as long as the drug can reach the target tissue. Can be. For example, the pharmaceutical composition of one embodiment of the present invention may be administered orally or parenterally, and parenteral administration may include transdermal administration, intraperitoneal administration, intravenous administration, intraarterial administration, lymphatic administration, intramuscular administration, Subcutaneous administration, intradermal administration, topical administration, rectal administration and the like can be discussed. However, it is not limited thereto and does not exclude various methods of administration known in the art. In addition, the pharmaceutical compositions of one embodiment of the present invention may be administered by any device in which the active substance may migrate to the target tissue or cell. In addition, an effective amount of the pharmaceutical composition of one embodiment of the present invention means an amount required for administration in order to expect a therapeutic effect.
본 발명의 일 구체예의 약학 조성물의 비경구 투여용 제제는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제, 또는 좌제일 수 있다. 본 발명의 일 구체예의 약학 조성물의 비경구 투여용 제제는 주사제로도 제조될 수 있다. 본 발명의 일 구체예의 주사제는 수성 주사제, 비수성 주사제, 수성 현탁 주사제, 비수성 현탁 주사제, 또는 용해 또는 현탁하여 사용하는 고형 주사제 등일 수 있으나, 이에 제한되는 것은 아니다. 본 발명의 일 구체예의 주사제는 그 종류에 따라 주사용 증류수, 식물유(예를 들어, 낙화생유, 참기름, 동백기름 등), 모노글리세리드, 디글리세리드, 프로필렌글리콜, 캄퍼, 벤조산에스트라디올, 차살리실산비스무트, 아르세노벤졸나트륨, 또는 황산스트렙토마이신 중 적어도 1종을 포함할 수 있고, 선택적으로 안정제나 방부제를 포함할 수 있다.Formulation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may be a sterile aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized formulation, or suppository. Formulations for parenteral administration of the pharmaceutical compositions of one embodiment of the invention may also be prepared by injection. Injectables of one embodiment of the present invention may be, but are not limited to, aqueous injections, non-aqueous injections, aqueous suspension injections, non-aqueous suspension injections, or solid injections used by dissolution or suspension. Injectables of one embodiment of the present invention, depending on the type of distilled water for injection, vegetable oil (for example, peanut oil, sesame oil, camellia oil, etc.), monoglycerides, diglycerides, propylene glycol, camphor, ester benzoic acid benzoic acid, bismuth succilate It may include at least one of sodium arsenobenzol, or streptomycin, and optionally may include a stabilizer or preservative.
본 발명의 일 구체예의 약학 조성물의 배합비율은 전술한 바와 같은 추가 성분들의 종류나 양, 형태 등에 따라서 적당하게 선택할 수 있다. 예를 들어, 주사제 전량에 대해, 본 발명의 약학 조성물은 약 0.1 내지 99 중량%, 바람직하게는 약 10 내지 90 중량% 정도 포함될 수 있다. 또한, 본 발명의 일 구체예의 약학 조성물의 적합한 투여량은 환자의 질환 종류, 질환의 경중, 제형의 종류, 제제화 방법, 환자의 연령, 성별, 체중, 건강 상태, 식이, 배설률, 투여 시간 및 투여 방법에 따라 조절될 수 있다. 예를 들어, 성인에게 본 발명의 일 구체예의 약학 조성물을 투여하는 경우, 하루에 0.001 mg/kg ~ 100 mg/kg의 용량으로 1 내지 수회에 나누어 투여할 수 있다.The blending ratio of the pharmaceutical composition of one embodiment of the present invention may be appropriately selected depending on the kind, amount, form, etc. of the additional ingredients as described above. For example, with respect to the total amount of injection, the pharmaceutical composition of the present invention may be included in an amount of about 0.1 to 99% by weight, preferably about 10 to 90% by weight. In addition, suitable dosages of the pharmaceutical compositions of one embodiment of the present invention include the type of disease of the patient, the severity of the disease, the type of formulation, the method of formulation, the age, sex, weight, health condition, diet, excretion rate, time of administration and administration of the patient. It can be adjusted according to the method. For example, when administering the pharmaceutical composition of an embodiment of the present invention to adults, it may be administered in one to several divided doses of 0.001 mg / kg to 100 mg / kg per day.
본 발명의 다른 구체예는 상기 약학 조성물의 치료학적으로 유효한 양을 포유동물에게 투여하는 단계를 포함하는, 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료하는 방법을 제공한다. 상기 포유동물은 인간, 개, 고양이, 설치류, 말, 소, 원숭이, 또는 돼지일 수 있다.Another embodiment of the invention provides a method of preventing, inhibiting, alleviating, ameliorating or treating ulcerative colitis, comprising administering to a mammal a therapeutically effective amount of said pharmaceutical composition. The mammal may be a human, dog, cat, rodent, horse, cow, monkey, or pig.
본 발명의 조성물은 궤양성 대장염의 원인이 되는 다양한 염증성 사이토카인 및 염증관련 인자의 생성을 감소시키고, 궤양성 대장염 증상 관련 지표들을 개선하여 궤양성 대장염을 예방, 억제, 완화, 개선 또는 치료할 수 있다. 즉, 본 발명의 조성물은 궤양성 대장염의 원인이 되는 염증성 사이토카인인 IL-6, TNF-α, IL-12 및 IL-23에 대해 작용하여 다양한 원인에 기인하는 궤양성 대장염에 대한 광범위한 적용이 가능하고 효과적으로 궤양성 대장염을 예방, 억제, 완화, 개선 또는 치료하는데 사용될 수 있다.The composition of the present invention can reduce the production of various inflammatory cytokines and inflammation-related factors that cause ulcerative colitis, and improve indicators related to ulcerative colitis symptoms to prevent, inhibit, alleviate, ameliorate or treat ulcerative colitis. . That is, the composition of the present invention acts against the inflammatory cytokines IL-6, TNF-α, IL-12, and IL-23, which cause ulcerative colitis, and thus has wide application to ulcerative colitis due to various causes. It can be used to prevent, inhibit, alleviate, ameliorate or treat ulcerative colitis as possible and effectively.
또한, 본 발명은 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료에 있어서 임상적 적용이 가능한 고순도이면서 입자크기 분포가 균일한 지방줄기세포 유래의 엑소좀을 저가로 대량으로 수득할 수 있다. 따라서, 본 발명은 기능적 활성이 우수한 지방줄기세포 유래의 엑소좀을 유효성분으로 포함하는 조성물을 저가로 대량으로 제공할 수 있다. 또한, 본 발명의 조성물은 스케일-업(scale-up)이 가능하고 GMP(Good Manufacturing Practice)에도 적합하다. In addition, the present invention can obtain a large amount of exosomes derived from adipose stem cells of high purity and uniform particle size distribution that can be clinically applied in the prevention, inhibition, alleviation, improvement or treatment of ulcerative colitis. Therefore, the present invention can provide a large amount of a composition containing exosomes derived from adipose stem cells excellent in functional activity as an active ingredient at low cost. In addition, the compositions of the present invention are capable of scale-up and are also suitable for Good Manufacturing Practice (GMP).
한편, 전술한 바와 같은 효과들에 의해 본 발명의 범위가 제한되는 것은 아니다.On the other hand, the scope of the present invention is not limited by the effects as described above.
도 1은 본 발명의 일 구체예에 따라 지방줄기세포 배양액으로부터 엑소좀을 제조하는 방법에 있어서 엑소좀을 분리 및 정제하는 과정을 설명하는 플로우챠트이다.
도 2는 본 발명의 일 구체예에 따라 지방줄기세포 배양액으로부터 엑소좀을 제조하는 단계(step)별로 용액 내에 포함되어 있는 단백질의 총량 비율(Relative amount of protein)을 측정한 결과를 나타낸다. 각 단계별 단백질 총량의 비율은 지방줄기세포 배양액 전체에 대한 단백질 총량의 상대적 비율로 나타내었다. 실험 결과는 2개의 서로 다른 배치에서 얻어진 결과를 각각 도시하였다.
도 3은 본 발명의 일 구체예에 따라 얻어진 엑소좀의 생산성(productivity)과 순도(purity)를 측정한 결과를 도시한 것이다. 엑소좀의 생산성은 "지방줄기세포 배양액(CM) 단위 mL 당 얻어진 엑소좀의 입자수"로 계산하였고, 엑소좀의 순도는 "최종 분획물에 포함되어 있는 단백질 단위 μg 당 엑소좀의 입자수"로 계산하였다. 실험 결과는 5개의 서로 다른 배치(batch)에서 얻어진 결과를 도시하였다.
도 4는 본 발명의 일 구체예에 따라 얻어진 엑소좀의 물리적 특성 분석 결과를 도시한 것이다. "도 4A"는 TRPS(tunable resistive pulse sensing) 분석에 의한 입자 크기 분포와 입자수를 나타낸다. "도 4B"는 NTA(nanoparticle tracking analysis) 분석에 의한 입자 크기 분포와 입자수를 나타낸다. "도 4C"는 TEM(transmitted electron microscopy) 분석에 의한 입자 이미지를 배율에 따라 도시하였다. "도 4D"는 본 발명의 일 구체예에 따라 얻어진 엑소좀의 웨스턴 블랏 결과를 나타낸다. "도 4E"는 본 발명의 일 구체예에 따라 얻어진 엑소좀에 대한 마커 분석에 있어서 CD63 및 CD81에 대한 유세포분석 결과를 나타낸다.
도 5는 트레할로오스 첨가에 따라 입자크기 분포가 균일하고 순도가 높은 엑소좀이 수득되는 것을 보여주는 입자 크기 분포에 관한 NTA 분석 결과를 도시한다. 첨가된 트레할로오스의 양이 증가함에 따라 단일한 피크를 갖는 입자 크기 분포 결과를 얻을 수 있다.
도 6은 본 발명의 일 구체예에 따른 엑소좀의 제조과정에서 트레할로오스 첨가 여부에 따른 입자 크기 분포를 나타내는 NTA 분석 결과를 도시한다. "도 6A"는 제조 과정 전과정에서 트레할로오스를 첨가한 경우, "도 6B"는 세포 배양액을 동결 보관하였다가 해동한 후 트레할로오스를 첨가한 경우, "도 6C"는 트레할로오스를 첨가하지 않고 제조한 결과를 나타낸다. "도 6D"에는 도 6A 내지 도 6C 방법에 의하여 분리한 엑소좀의 상대적인 생산성(Relative productivity)과 상대 농도(Relative concentration)를 비교한 결과를 도시하였다. "도 6E"에는 도 6A 내지 도 6C 방법에 의하여 분리한 엑소좀의 평균 입자크기(Mean size)를 도시하였다.
도 7은 인체 피부섬유아세포인 HS68 세포에 본 발명의 일 구체예에 따른 엑소좀을 처리한 후 세포 독성이 없음을 확인한 결과를 도시한다.
도 8은 RAW 264.7 세포에 대해 LPS와 함께 본 발명의 엑소좀을 처리한 경우 LPS에 의해 유도되는 TNF-α 및 IL-6의 mRNA 발현량이 감소한 것을 확인한 리얼타임 PCR 결과를 도시한 그래프이다.
도 9는 본 발명의 일 구체예에 따른 엑소좀에 의한, 염증 반응의 일종인 NO 형성의 감소 효과를 확인한 실험 결과를 도시한다. 도 9에서 PBS는 인산염 완충용액(phosphate-buffered saline), DEX는 덱사메타손(dexamethasone), EXO는 엑소좀, CM은 지방줄기세포 배양액(conditioned media), CM-EXO는 엑소좀이 제거된 지방줄기세포 배양액(exosome-depeleted conditioned media)을 나타낸다.
도 10은 본 발명의 일 구체예에 따른 엑소좀에 의한, 염증성 사이토카인인 TNF-α 형성의 감소 효과를 확인한 실험 결과를 도시한다. 도 10에서, PBS는 인산염 완충용액(phosphate-buffered saline), DEX는 덱사메타손(dexamethasone), 각 숫자는 엑소좀 처리량(μg/mL)을 나타낸다.
도 11은 본 발명의 일 구체예에 따라 분리된 엑소좀에 의한 NO 형성 감소 효과와, 종래의 침전법(PPT)에 의해 분리된 엑소좀에 의한 NO 형성 감소 효과를 비교한 실험 결과를 도시한다. 도 11A는 종래의 침전법에 의해 분리된 엑소좀의 NTA 분석 결과이고, 도 11B는 본 발명의 일 구체예에 따른 방법에 의해 분리 정제된 엑소좀의 NTA 분석 결과이며, 도 11C는 NO 형성 감소 효과를 비교 도시한 그래프이다. NO 형성의 감소 정도는 양성대조군인 덱사메타손(Dex)에 의한 NO 형성 감소 정도에 대한 상대 비율(%)로 표시하였다.
도 12는 궤양성 대장염이 유발된 생쥐에 본 발명의 일 구체예에 따른 엑소좀을 처리한 결과, 궤양성 대장염에 의한 개체 사망율이 감소하고 개체 사망을 지연하는 효과가 있음을 나타내는 그래프이다.
도 13은 THP-1 단핵구를 대식세포로 분화시킨 후 얻은 THP-1 유래 대식세포에 대해, LPS 및 IFN-γ와 함께 본 발명의 엑소좀을 처리(co-treatment)한 경우, 친염증성 사이토카인인 IL-6의 mRNA의 발현량이 감소한 것을 확인한 리얼타임 PCR 결과를 도시한 그래프이다.
도 14 내지 도 17은 THP-1 단핵구를 대식세포로 분화시킨 후 얻은 THP-1 유래 대식세포에 대해, 본 발명의 엑소좀을 전처리(pre-treatment)하고 일정 시간 배양한 다음 LPS 및 IFN-γ를 처리한 경우, 친염증성 사이토카인인 IL-6, TNF-α, IL-12 및 IL-23의 mRNA의 발현량이 감소한 것을 확인한 리얼타임 PCR 결과를 도시한 그래프이다.1 is a flowchart illustrating a process for separating and purifying exosomes in a method for producing exosomes from adipose stem cell culture according to one embodiment of the present invention.
Figure 2 shows the results of measuring the relative amount of protein (Relative amount of protein) contained in the solution for each step of preparing the exosomes from adipose stem cell culture according to an embodiment of the present invention. The ratio of the total amount of protein in each step is expressed as the relative ratio of the total amount of protein to the whole adipose stem cell culture. The experimental results show the results obtained in two different batches, respectively.
Figure 3 shows the results of measuring the productivity (purity) and (productivity) of the exo-some obtained in accordance with an embodiment of the present invention. The productivity of exosomes was calculated as "the number of particles of exosomes per mL of fat stem cell culture (CM)" and the purity of exosomes was "the number of particles of exosomes per μg of protein contained in the final fraction". Calculated. The experimental results show the results obtained in five different batches.
Figure 4 shows the results of physical characterization of the exosomes obtained according to one embodiment of the present invention. 4A shows particle size distribution and particle number by tunable resistive pulse sensing (TRPS) analysis. 4B shows particle size distribution and particle number by NTA (nanoparticle tracking analysis) analysis. FIG. 4C shows the particle image by magnification by means of the transmitted electron microscopy (TEM) analysis. 4D shows Western blot results of exosomes obtained according to one embodiment of the invention. 4E shows the results of flow cytometry for CD63 and CD81 in marker analysis for exosomes obtained according to one embodiment of the invention.
FIG. 5 shows NTA analysis results for particle size distribution showing that exosomes with uniform particle size distribution and high purity are obtained with trehalose addition. As the amount of trehalose added increases, particle size distribution results with a single peak can be obtained.
Figure 6 shows the NTA analysis results showing the particle size distribution according to whether or not added trehalose in the manufacturing process of the exosomes according to an embodiment of the present invention. FIG. 6A shows the addition of trehalose throughout the manufacturing process, FIG. 6B shows freezing of the cell culture and thawing after thawing, FIG. 6C shows trehalo. The result obtained without adding oss is shown. 6D shows the results of comparing relative productivity and relative concentration of exosomes isolated by the methods of FIGS. 6A to 6C. 6E shows the mean particle size of the exosomes isolated by the methods of FIGS. 6A-6C.
Figure 7 shows the results confirmed that there is no cytotoxicity after treatment with exosomes according to one embodiment of the present invention to HS68 cells, human skin fibroblasts.
FIG. 8 is a graph showing real-time PCR results confirming that the mRNA expression levels of TNF-α and IL-6 induced by LPS were reduced when the exosome of the present invention was treated with LPS for RAW 264.7 cells.
Figure 9 shows the experimental results confirming the effect of reducing the formation of NO, a kind of inflammatory response by the exosomes according to an embodiment of the present invention. In Figure 9, PBS is a phosphate buffer (phosphate-buffered saline), DEX is dexamethasone (dexamethasone), EXO is exosomes, CM is a fat stem cell culture (conditioned media), CM-EXO is a fat stem cells from which exo-some is removed Exosome-depeleted conditioned media is shown.
Figure 10 shows the experimental results confirming the effect of reducing the inflammatory cytokine TNF-α formation by the exosomes according to an embodiment of the present invention. In FIG. 10, PBS represents phosphate-buffered saline, DEX represents dexamethasone, and each number represents exosome throughput (μg / mL).
FIG. 11 shows the results of experiments comparing the effect of reducing NO formation by exosomes isolated according to one embodiment of the present invention and the effect of reducing NO formation by exosomes isolated by conventional precipitation methods (PPT). . Figure 11A is a result of NTA analysis of exosomes separated by a conventional precipitation method, Figure 11B is a result of NTA analysis of exosomes separated and purified by a method according to an embodiment of the present invention, Figure 11C is NO formation reduction It is a graph comparing the effect. The decrease in NO formation was expressed as a relative percentage (%) to the reduction in NO formation by the positive control dexamethasone (Dex).
12 is a graph showing that the treatment of exosomes according to an embodiment of the present invention in mice induced with ulcerative colitis results in a decrease in individual mortality caused by ulcerative colitis and a delay in individual death.
Figure 13 is a proinflammatory cytokine when co-treatment of the exosome of the present invention with LPS and IFN-γ for THP-1 derived macrophages obtained after differentiating THP-1 monocytes into macrophages It is a graph showing the results of real-time PCR confirmed that the expression level of the mRNA of IL-6 is reduced.
Figures 14 to 17 shows that the THP-1 derived macrophages obtained after differentiating THP-1 monocytes into macrophages, pre-treatment of the exosomes of the present invention and cultured for a predetermined time, and then LPS and IFN-γ Is a graph showing real-time PCR results confirming that the expression levels of the proinflammatory cytokines IL-6, TNF-α, IL-12 and IL-23 mRNAs are reduced.
이하 본 발명을 하기 실시예에서 보다 상세하게 기술한다. 다만, 하기 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 권리범위를 제한하거나 한정하는 것이 아니다. 본 발명의 상세한 설명 및 실시예로부터 본 발명이 속하는 기술분야의 통상의 기술자가 용이하게 유추할 수 있는 것은 본 발명의 권리범위에 속하는 것으로 해석된다. 본 발명에 인용된 참고문헌들은 본 발명에 참고로서 통합된다.Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples merely illustrate the contents of the present invention and do not limit or limit the scope of the present invention. From the detailed description and examples of the present invention, those skilled in the art to which the present invention pertains can be easily inferred to be within the scope of the present invention. References cited in the present invention are incorporated herein by reference.
명세서 전체에서, 어떤 부분이 어떤 구성 요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성 요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.Throughout the specification, when a part is said to "include" a certain component, it means that it can further include other components, except to exclude other components unless specifically stated otherwise.
실시예Example
실시예 1: 세포의 배양Example 1 Culture of Cells
마우스 대식세포주인 RAW 264.7은 한국세포주은행에서 구입하여 배양하였다. 세포 배양을 위해 10% 우태아 혈청 (fetal bovine serum: ThermoFisher Scientific에서 구입) 및 1% 항생제-항진균제 (antibiotics-antimycotics: ThermoFisher Scientific에서 구입)가 함유된 DMEM (ThermoFisher Scientific에서 구입) 배지에 5% CO2, 37℃ 조건에서 계대 배양하였다.RAW 264.7, a mouse macrophage, was purchased from the Korea Cell Line Bank and cultured. 5% CO in DMEM (purchased from ThermoFisher Scientific) medium containing 10% fetal bovine serum (purchased from ThermoFisher Scientific) and 1% antibiotic-antimycotics (purchased from ThermoFisher Scientific) for cell culture Subcultured at 2 , 37 ° C.
인체 피부 섬유아세포(human dermal fibroblast)인 HS68 세포는 ATCC에서 구입하여, 10% 우태아 혈청 (fetal bovine serum: ThermoFisher Scientific에서 구입) 및 1% 항생제-항진균제 (antibiotics-antimycotics: ThermoFisher Scientific에서 구입)가 함유된 DMEM (ThermoFisher Scientific에서 구입) 배지에 5% CO2, 37℃ 조건에서 계대 배양하였다.HS68 cells, which are human dermal fibroblasts, were purchased from ATCC and were prepared by 10% fetal bovine serum (purchased from ThermoFisher Scientific) and 1% antibiotic-antimycotics (purchased from ThermoFisher Scientific). Passage was carried out in DMEM (purchased from ThermoFisher Scientific) medium containing 5% CO 2 at 37 ° C.
당해 발명이 속하는 기술분야에 알려진 세포배양 방법에 따라 5% CO2, 37℃ 조건에서 지방 유래 줄기세포를 배양하였다. 그 다음, 인산염 완충용액(phosphate-buffered saline)(ThermoFisher Scientific에서 구입)으로 세척 후, 무혈청, 무페놀레드 배지로 교체하여 1일 내지 10일간 배양하고 그 상층액(이하, 배양액)을 회수하였다.Fat-derived stem cells were cultured at 5% CO 2 , 37 ° C. according to cell culture methods known in the art. Then, washed with phosphate-buffered saline (purchased from ThermoFisher Scientific), replaced with serum-free, phenol-free medium, cultured for 1 to 10 days, and the supernatant (hereinafter, culture) was recovered. .
엑소좀의 분리 과정에서 입자크기 분포가 균일하고 순도가 높은 엑소좀을 수득하기 위하여 배양액에 트레할로오스를 2 중량% 첨가하였다. 트레할로오스를 첨가한 후 배양액을 0.22 μm 필터로 여과하여 세포 잔해물, 노폐물 및 거대 입자 등의 불순물을 제거해 주었다. 여과된 배양액은 즉시 분리 과정을 통해 엑소좀을 분리하였다. 또한, 여과된 배양액은 냉장고(영상 10℃ 이하)에서 보관한 후 엑소좀 분리에 사용하였다. 또한, 여과된 배양액은 -60℃ 이하의 초저온 냉동고에서 동결 보관하였다가 해동시킨 후 엑소좀 분리를 수행하였다. 이후, 배양액으로부터 접선흐름여과장치(Tangential Flow Filtration; TFF)를 이용하여 엑소좀을 분리하였다.In the separation of exosomes, 2% by weight of trehalose was added to the culture to obtain exosomes with uniform particle size distribution and high purity. After the addition of trehalose, the culture solution was filtered through a 0.22 μm filter to remove impurities such as cell debris, waste, and large particles. The filtered culture immediately separated the exosomes through a separation process. In addition, the filtered culture was stored in the refrigerator (image 10 ℃ or less) and then used for exosome separation. In addition, the filtered culture solution was stored frozen in an cryogenic freezer of -60 ℃ or less and thawed and then exosomes were separated. Thereafter, exosomes were separated from the culture using a tangential flow filtration device (TFF).
실시예 2: TFF 방법에 의한 엑소좀의 분리 및 정제Example 2: Isolation and Purification of Exosomes by TFF Method
실시예 1에서 0.22 μm 필터로 여과된 배양액으로부터 엑소좀을 분리, 농축, 탈염과 버퍼교환(diafiltration)을 위해 TFF(Tangential Flow Filtration) 방법을 사용하였다. TFF 방법을 위한 필터로는 카트리지 필터(cartridge filter, 일명 hollow fiber filter; GE Healthcare에서 구입) 또는 카세트 필터(cassette filter; Pall 또는 Sartorius 또는 Merck Millipore에서 구입)를 사용하였다. TFF 필터는 다양한 분자량 차단(molecular weight cutoff; MWCO)에 의해 선택될 수 있다. 선택된 MWCO에 의해 선별적으로 엑소좀을 분리, 농축하였고, MWCO보다 작은 입자나 단백질, 지질, 핵산, 저분자 화합물 등은 제거하였다.In Example 1, the exosomes were separated from the culture medium filtered with a 0.22 μm filter, and the TFF (Tangential Flow Filtration) method was used for concentration, desalting and diafiltration. The filter for the TFF method was a cartridge filter (aka hollow fiber filter; purchased from GE Healthcare) or a cassette filter (purchased from Pall or Sartorius or Merck Millipore). TFF filters can be selected by various molecular weight cutoffs (MWCO). Exosomes were selectively isolated and concentrated by the selected MWCO, and particles, proteins, lipids, nucleic acids, and small molecule compounds smaller than MWCO were removed.
엑소좀을 분리, 농축하기 위하여 MWCO 100,000 Da(Dalton), 300,000 Da, 또는 500,000 Da의 TFF 필터를 사용하였다. 배양액을 TFF 방법을 이용하여 1/100 내지 1/25 정도의 부피가 될 때까지 농축하면서, MWCO보다 작은 물질들은 제거하여 엑소좀을 분리하였다.MWCO 100,000 Da (Dalton), 300,000 Da, or 500,000 Da TFF filter was used to isolate and concentrate the exosomes. The culture solution was concentrated to a volume of 1/100 to 1/25 by using the TFF method, while exosomes were separated by removing substances smaller than MWCO.
분리, 농축된 엑소좀 용액은 TFF 방법을 이용하여 추가로 탈염과 버퍼교환(diafiltration)을 수행하였다. 이때, 탈염과 버퍼교환은 연속적으로 수행(continuous diafiltration)하거나 단속적으로 수행(discontinuous diafiltration)하였으며, 시작 부피(starting volume)에 대하여 적어도 4배, 바람직하게는 6배 내지는 10배 이상, 보다 바람직하게는 12배 이상의 부피를 갖는 완충용액을 이용하여 수행하였다. 완충용액에는 입자크기 분포가 균일하고 순도가 높은 엑소좀을 수득하기 위하여 PBS에 녹인 2 중량%의 트레할로오스를 첨가하였다. 트레할로오스 처리에 따라 고순도이면서 입자크기 분포가 균일한 엑소좀을 높은 수율로 수득할 수 있는 효과를 확인한 결과는 도 6에 도시하였다.The separated and concentrated exosome solution was further subjected to desalting and diafiltration using the TFF method. At this time, desalting and buffer exchange were carried out continuously (discontinuous diafiltration) or at least 4 times, preferably 6 times to 10 times, more preferably, relative to the starting volume. It was performed using a buffer solution having a volume of 12 times or more. To the buffer solution was added 2% by weight of trehalose dissolved in PBS to obtain exosomes with uniform particle size distribution and high purity. The results of confirming the effect of obtaining a high purity and uniform particle size distribution of exosomes according to the trehalose treatment in a high yield is shown in FIG.
실시예 3: 분리된 엑소좀의 특성 분석Example 3: Characterization of Isolated Exosomes
분리된 엑소좀, 배양액, 및 TFF 분리과정의 분획물에서 단백질의 양은 BCA 발색법(ThermoFisher Scientific에서 구입) 또는 플루오로프로파일(FluoroProfile) 형광법(Sigma에서 구입)을 이용하여 측정하였다. 본 발명의 일 구체예의 TFF 방법에 의해 엑소좀이 분리, 농축되고 단백질, 지질, 핵산, 저분자 화합물 등이 제거되는 정도는 단백질 정량법에 의하여 모니터링하여 그 결과를 도 2에 도시하였다. 그 결과 본 발명의 일 구체예의 TFF 방법에 의하여 매우 효과적으로 배양액에 존재하는 단백질이 제거됨을 알 수 있었다.The amount of protein in the isolated exosomes, cultures, and fractions of TFF separation was measured using BCA coloration (purchased from ThermoFisher Scientific) or FluoroProfile fluorescence (purchased from Sigma). Exosome is isolated and concentrated by the TFF method of one embodiment of the present invention, and the degree of protein, lipid, nucleic acid, low molecular weight compounds, etc. is monitored by protein quantitation and the results are shown in FIG. As a result, it was found that the protein present in the culture medium was effectively removed by the TFF method of one embodiment of the present invention.
본 발명의 일 구체예의 TFF 방법에 의해 엑소좀을 분리하는 경우 생산성과 순도를 독립적인 다섯 배치에서 비교한 결과를 도 3에 도시하였다. 독립적인 다섯 배치로부터 얻어진 결과를 분석한 결과, 본 발명의 일 구체예의 TFF 방법에 의하여 매우 안정적으로 엑소좀을 분리할 수 있음을 확인하였다.When the exosomes were separated by the TFF method of one embodiment of the present invention, the results of comparing the productivity and purity in five independent batches are shown in FIG. 3. Analysis of the results obtained from the five independent batches, it was confirmed that the exosomes can be isolated very stably by the TFF method of one embodiment of the present invention.
분리된 엑소좀은 나노입자 트랙킹 분석(nanoparticle tracking analysis: NTA; Malvern에서 구입) 또는 가변 저항펄스 감지(tunable resistive pulse sensing: TRPS; Izon Science에서 구입)에 의해 입자의 크기와 농도를 측정하였다. 분리된 엑소좀의 균일도와 크기는 투과전자현미경(transmitted electron microscopy: TEM)을 이용하여 분석하였다. 본 발명의 일 구체예에 따라 분리된 엑소좀의 TRPS, NTA, TEM 분석 결과는 도 4A 내지 도 4C에 도시하였다.The isolated exosomes were measured for particle size and concentration by nanoparticle tracking analysis (NTA; purchased from Malvern) or tunable resistive pulse sensing (TRPS; purchased from Izon Science). The uniformity and size of the isolated exosomes were analyzed using a transmitted electron microscopy (TEM). TRPS, NTA, TEM analysis results of the exosomes isolated in accordance with one embodiment of the present invention are shown in Figures 4A to 4C.
TFF 방법으로 엑소좀을 분리한 후, 트레할로오스의 첨가 여부에 따른 엑소좀의 크기 분포를 NTA 분석한 결과를 도 5에 도시하였다. 트레할로오스 농도를 0 중량%, 1 중량% 및 2 중량%로 증가시켰고(도 5의 위에서부터 아래), 3회 반복하여 실험하였다. 트레할로오스가 존재하지 않은 경우 300 nm 이상의 크기를 갖는 입자가 확인되는 반면, 트레할로오스의 첨가량을 늘려주면 300 nm 이상의 크기를 갖는 입자가 줄어들고 엑소좀의 크기 분포가 균일해지는 것을 확인하였다.After separating the exosomes by the TFF method, the results of NTA analysis of the size distribution of the exosomes depending on whether trehalose was added are shown in FIG. 5. Trehalose concentrations were increased to 0 wt%, 1 wt% and 2 wt% (from top to bottom in FIG. 5) and were repeated three times. When trehalose is not present, particles having a size of 300 nm or more are identified, while increasing the amount of trehalose added decreases the particles having a size of 300 nm or more and makes the size distribution of exosomes uniform. .
TFF 방법으로 엑소좀을 분리하는 과정에 트레할로오스의 첨가에 따른 효과를 추가로 조사하였다. 도 6에서 보는 바와 같이 엑소좀의 제조과정 전과정에 PBS에 녹인 2 중량%의 트레할로오스를 첨가한 경우, 균일한 크기 분포를 갖는 엑소좀을 얻을 수 있었다(도 6A). 반면 트레할로오스를 첨가하지 않고 동결 보관하였던 배양액을 사용하되, 탈염과 버퍼교환 과정에서만 트레할로오스를 첨가하여 TFF 과정을 진행한 경우나, 트레할로오스를 전혀 첨가하지 않고 TFF 과정을 진행한 경우, 크기가 큰 입자가 많이 포함된 불균일한 엑소좀을 얻었다(도 6B 및 도 6C).The effect of the addition of trehalose in the process of separating exosomes by the TFF method was further investigated. As shown in FIG. 6, when 2% by weight of trehalose dissolved in PBS was added to the entire process of manufacturing an exosome, an exosome having a uniform size distribution was obtained (FIG. 6A). On the other hand, when the TFF process was performed by adding trehalose only in the desalting and buffer exchange process, the TFF process was used without the addition of trehalose. In case of progress, a non-uniform exosome containing a lot of large particles was obtained (FIGS. 6B and 6C).
분리된 엑소좀의 상대적인 생산성과 농도를 비교한 결과, 엑소좀의 제조과정 전과정에 트레할로오스를 첨가한 경우 매우 높은 생산성으로 엑소좀을 얻을 수 있었으며, 얻어진 엑소좀의 농도도 5배 이상 높았다(도 6D). NTA 분석 결과에서 나타난 바와 같이, 분리된 엑소좀의 평균 크기도 엑소좀의 제조과정 전과정에 트레할로오스를 첨가한 경우 200 nm로 균일하게 확인되었다(도 6E).As a result of comparing the relative productivity and concentration of the isolated exosomes, when trehalose was added to the entire process of manufacturing the exosomes, the exosomes were obtained with very high productivity, and the concentration of the obtained exosomes was more than five times higher. (FIG. 6D). As shown in the NTA analysis results, the average size of the separated exosomes was also uniformly confirmed at 200 nm when trehalose was added to the entire process of manufacturing the exosomes (FIG. 6E).
도 4D는 본 발명의 일 구체예의 방법에 따라 분리된 엑소좀에 대해 웨스턴 블랏을 수행한 결과로서, CD9, CD63, CD81 및 TSG101 마커의 존재를 확인하였다. 각 마커에 대한 항체로는 각각 항-CD9 (Abcam에서 구입), 항-CD63 (System Biosciences에서 구입), 항-CD81 (System Biosciences에서 구입), 및 항-TSG101 (Abcam에서 구입)을 사용하였다.Figure 4D shows the results of Western blot for exosomes isolated according to the method of one embodiment of the present invention, confirming the presence of CD9, CD63, CD81 and TSG101 markers. Anti-CD9 (purchased from Abcam), anti-CD63 (purchased from System Biosciences), anti-CD81 (purchased from System Biosciences), and anti-TSG101 (purchased from Abcam) were used as antibodies to each marker.
도 4E는 본 발명의 일 구체예의 방법에 따라 분리된 엑소좀에 대해 유세포분석기를 이용하여 분석한 결과로서 CD63 및 CD81 마커의 존재를 확인하였다. CD63에 대해 양성(positive)인 엑소좀을 분리하기 위하여 엑소좀-휴먼 CD63 분리/검출 키트(ThermoFisher Scientific에서 구입)를 제조사의 방법에 따라 사용하였고, PE-마우스 항-인간 CD63 (PE-Mouse anti-human CD63)(BD에서 구입) 및 PE-마우스 항-인간 CD81 (PE-mouse anti-human CD81)(BD에서 구입)을 사용하여 마커를 염색한 후, 유세포분석기 (ACEA Biosciences)를 이용하여 분석하였다.Figure 4E confirmed the presence of CD63 and CD81 markers as a result of analysis using a flow cytometer for the exosomes isolated in accordance with the method of one embodiment of the present invention. To isolate exosomes positive for CD63, an exosome-human CD63 separation / detection kit (purchased from ThermoFisher Scientific) was used according to the manufacturer's method, and PE-mouse anti-human CD63 (PE-Mouse anti markers were stained using -human CD63) (purchased from BD) and PE-mouse anti-human CD81 (purchased from BD) and analyzed using a flow cytometer (ACEA Biosciences). It was.
상기 결과들을 종합하면, 본 발명은 접선흐름여과를 이용한 제조과정에서 트레할로오스를 첨가하여 고순도이면서 입자크기 분포가 균일한 엑소좀을 높은 수율로 경제적이면서 효율적으로 분리 및 정제할 수 있음을 확인할 수 있었다. 또한, 본 발명의 일 구체예의 분리방법의 공정들은 스케일-업이 가능하고 GMP에도 적합함을 알 수 있었다.Summarizing the above results, the present invention confirms that exosomes with high purity and uniform particle size distribution can be efficiently and efficiently separated and purified in high yield by adding trehalose in the manufacturing process using tangential flow filtration. Could. In addition, it can be seen that the processes of the separation method of one embodiment of the present invention are scale-up and suitable for GMP.
실시예 4: 엑소좀 처리에 따른 세포 독성 측정Example 4 Measurement of Cytotoxicity According to Exosome Treatment
인체 피부 섬유아세포인 HS68 세포에서 본 발명의 일 구체예의 분리 방법에 따라 수득된 엑소좀의 독성을 평가하기 위해 세포에 농도별로 엑소좀을 처리하고 세포의 증식률을 확인하였다. HS68 세포를 10% FBS를 포함한 DMEM에 현탁시킨 후 80 내지 90%의 밀집도(confluency)를 갖도록 분주하고 37℃, 5% CO2 인큐베이터에서 24시간 배양하였다. 24시간 후, 배양액을 제거하고 실시예 2에서 준비된 엑소좀을 농도 별로 처리하여 24 내지 72시간 동안 배양하면서 세포 생존율을 평가하였다. 세포 생존율을 WST-1 시약(WST-1 reagent)(Takara에서 구입), MTT 시약(Sigma에서 구입), 셀타이터-글로 시약(CellTiter-Glo reagent)(Promega에서 구입), 또는 아라마르 블루 시약(alamarBlue reagent)(ThermoFisher Scientific에서 구입)과 마이크로플레이트 리더(microplate reader)(Molecular Devices에서 구입)를 이용하여 측정하였다. In order to evaluate the toxicity of exosomes obtained according to the isolation method of an embodiment of the present invention in HS68 cells, which are human skin fibroblasts, cells were treated with exosomes at different concentrations, and cell proliferation rates were confirmed. HS68 cells were suspended in DMEM containing 10% FBS and then aliquoted to have a confluency of 80-90% and incubated in 37 ° C., 5% CO 2 incubator for 24 hours. After 24 hours, the culture solution was removed and the exosomes prepared in Example 2 were treated for each concentration, and cultured for 24 to 72 hours to evaluate cell viability. Cell viability was determined by WST-1 reagent (purchased from Takara), MTT reagent (purchased from Sigma), CellTiter-Glo reagent (purchased from Promega), or Aramamar blue reagent ( Measurements were performed using alamarBlue reagent (purchased from ThermoFisher Scientific) and a microplate reader (purchased from Molecular Devices).
비교군은 엑소좀이 처리되지 않은 일반 세포배양배지에서 배양된 세포수를 기준으로 하였고, 시험된 농도 범위 내에서 본 발명의 엑소좀에 의한 세포 독성이 나타나지 않음을 확인하였다(도 7).The comparison group was based on the number of cells cultured in the normal cell culture medium not treated with exosomes, it was confirmed that no cytotoxicity by the exosomes of the present invention within the concentration range tested (Fig. 7).
실시예 5: 마크로파지 세포주를 이용한 염증 반응 측정Example 5 Inflammation Response Measurement Using Macrophage Cell Line
RAW 264.7 세포를 10% FBS를 포함한 DMEM 배지에 현탁시키고 이를 멀티웰 플레이트(multiwell plate)의 각 웰에 80 내지 90%의 밀집도(confluency)를 갖도록 분주하였다. 다음 날 LPS가 포함된 새로운 무혈청 배지에 희석한 본 발명의 엑소좀 (실시예 2에서 준비된 엑소좀)을 적정 농도로 1~24시간 동안 처리하여 배양하였다. 배양이 끝난 배양상층액을 취하고 배양액 내에 존재하는 NO 및 염증성 사이토카인을 측정하여 염증 반응을 확인하였다. 배양액 내의 염증 반응은 NO 검출키트(detection kit)(인트론바이오 혹은 프로메가에서 구입)를 이용하여 측정하였다. ELISA 키트 (R&D system에서 구입) 제조사의 매뉴얼대로 수행하여 LPS만을 처리한 군과 본 발명의 엑소좀이 함께 처리된 군의 염증성 사이토카인 TNF-α 양을 확인하였다. 양성대조군으로 덱사메타손(dexamethasone) (Sigma에서 구입)을 처리하였다. 또한, 위와 같이 처리된 RAW 264.7 세포로부터 얻은 총 RNA로부터 cDNA를 제조하였고 리얼타임 PCR 방법을 이용하여 TNF-α 및 IL-6의 mRNA 변화량을 측정하였다. 상기 유전자들을 정량하기 위한 표준 유전자로서 GAPDH 유전자를 사용하였다. 리얼타임 PCR에 사용한 프라이머의 종류와 서열은 하기의 표 1과 같다.RAW 264.7 cells were suspended in DMEM medium containing 10% FBS and aliquoted into 80-90% confluency in each well of a multiwell plate. Next day, the exosomes of the present invention (exosomes prepared in Example 2) diluted in fresh serum-free medium containing LPS were incubated for 1 to 24 hours at an appropriate concentration. After culturing, the culture supernatant was taken, and the inflammatory response was confirmed by measuring NO and inflammatory cytokines present in the culture. Inflammatory responses in the culture were measured using a NO detection kit (purchased from Intron Bio or Promega). ELISA kit (purchased from the R & D system) was performed according to the manufacturer's manual to determine the amount of inflammatory cytokine TNF-α of the group treated with LPS only and the group treated with the exosome of the present invention. Positive control was treated with dexamethasone (purchased from Sigma). In addition, cDNA was prepared from total RNA obtained from the above treated RAW 264.7 cells, and mRNA changes of TNF-α and IL-6 were measured using real-time PCR. The GAPDH gene was used as a standard gene for quantifying the genes. The kind and sequence of primers used for real-time PCR are shown in Table 1 below.
궤양성 대장염 환자에서 친염증성 사이토카인인 TNF-α 및 IL-6의 수치가 증가하는 것이 알려져 있으며, 환자의 생검에서 TNF-α의 발현이 증가하고 생검 및 혈청에서 IL-6의 레벨이 증가하는 것이 보고된 바가 있다(Hindawi Publishing Corporation, Ulcers Volume 2011. Review Article, Cytokine Networks in Ulcerative Colitis, pp. 1-5). 이 밖에도 다양한 문헌에서 확인되는 바와 같이 TNF-α 및 IL-6은 궤양성 대장염 발병과 밀접한 관련이 있다. 따라서, 염증반응에 관여하는 대식세포에서 TNF-α 및 IL-6의 생성량이나 발현량 변화를 분석하면 본 발명의 엑소좀이 궤양성 대장염이나 이에 관여하는 염증 반응을 억제, 감소 또는 완화시키는 시험관내 효능을 평가할 수 있다. Increased levels of proinflammatory cytokines, TNF-α and IL-6, are known in patients with ulcerative colitis, and increased expression of TNF-α in patients' biopsies and increased levels of IL-6 in biopsies and serum. (Hindawi Publishing Corporation, Ulcers Volume 2011. Review Article, Cytokine Networks in Ulcerative Colitis, pp. 1-5). In addition, TNF-α and IL-6 are closely related to the development of ulcerative colitis, as confirmed by various documents. Therefore, in vitro analysis of exosomes of the present invention inhibits, decreases or alleviates ulcerative colitis or the inflammatory response involved therein by analyzing changes in the amount or expression of TNF-α and IL-6 in macrophages involved in the inflammatory response. Efficacy can be assessed.
이와 관련하여 도 8에 도시된 바와 같이, 생쥐의 대식세포인 RAW 264.7 세포에 대해 LPS와 함께 본 발명의 엑소좀을 처리한 경우 LPS에 의해 유도되는 염증성 사이토카인인 TNF-α 및 IL-6의 mRNA의 발현량이 감소하였다. 다음으로, 도 9에 도시된 바와 같이, 생쥐의 대식세포인 RAW 264.7 세포에 LPS 존재 하에서 본 발명의 엑소좀을 처리한 결과 LPS에 의해 유도되는 염증 반응인 NO 생성을 농도의존적으로 감소시킴을 확인하였다. 또한, 도 10에 도시된 바와 같이, 생쥐의 대식세포인 RAW 264.7 세포에 LPS 존재 하에서 본 발명의 엑소좀을 처리한 결과 LPS에 의해 유도되는 염증성 사이토카인인 TNF-α 생성을 감소시킴을 확인하였다. In this regard, as shown in Figure 8, the treatment of the exosome of the present invention with LPS to RAW 264.7 cells, which are macrophages of mice, of the inflammatory cytokines TNF-α and IL-6 induced by LPS The amount of mRNA decreased. Next, as shown in Figure 9, as a result of treating the exosome of the present invention in the presence of LPS in macrophage RAW 264.7 cells of the mouse it was confirmed that the concentration of the inflammatory response NO induced by LPS reduced concentration-dependently It was. In addition, as shown in Figure 10, the treatment of the exosome of the present invention in the presence of LPS in macrophage RAW 264.7 cells of the mouse was confirmed to reduce the TNF-α production of inflammatory cytokines induced by LPS .
이러한 결과들은 본 발명의 엑소좀이 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료를 위해 유용한 기능적 활성, 즉 궤양성 대장염의 원인이 되는 사이토카인인 TNF-α 및 IL-6의 생성량 및/또는 발현량을 감소시키는 활성을 가지고 있는 것을 보여주고 있다. These results indicate that the exosomes of the present invention are useful for the prevention, inhibition, alleviation, amelioration or treatment of ulcerative colitis, i.e. the amount of TNF-α and IL-6, the cytokines responsible for ulcerative colitis, and / or Or it has been shown to have an activity to reduce the amount of expression.
따라서, 본 발명의 엑소좀을 유효성분으로 함유하는 조성물은 장내의 염증 반응을 감소시켜 염증 반응에 기인한 궤양성 대장염의 예방, 억제, 완화, 개선 또는 치료에 유용할 것으로 기대된다.Therefore, the composition containing the exosome of the present invention as an active ingredient is expected to be useful in the prevention, inhibition, alleviation, improvement or treatment of ulcerative colitis due to the inflammatory response by reducing the intestinal inflammatory response.
실시예 6: 분리 방법에 따른 NO 형성 감소 효과의 비교Example 6 Comparison of NO Formation Reduction Effects According to Separation Methods
분리 방법에 따른 엑소좀의 NO 형성 감소 효과를 비교하기 위하여 본 발명의 일 구체예의 TFF 분리 정제에 의해 얻어진 엑소좀 이외에 종래의 침전법에 의해 분리된 엑소좀을 준비하였다. 침전법은 제조사(System Biosciences)의 프로토콜에 따라 시행하였다. 종래의 침전법에 의해 분리된 엑소좀(도 11A 참조)은 본 발명의 일 구체예의 TFF 방법에 의해 분리 정제된 엑소좀(도 11B 참조)과 비교하여 입자크기 분포의 균일도가 낮고 다양한 크기를 갖는 것을 확인하였다. 또한, 도 11C에 도시한 바와 같이 본 발명의 일 구체예의 TFF 방법에 의해 분리 정제된 엑소좀은 종래의 침전법에 의해 얻어진 엑소좀에 비해 훨씬 높은 수준으로 NO 형성을 억제함을 확인하였다. 이러한 결과들은 본 발명의 일 구체예에 따라 분리 정제된 엑소좀이 종래 방법에 따라 분리된 엑소좀에 비해 입자크기 분포의 균일도와 NO 형성 억제 측면에서 우수하다는 것을 보여주고 있다.In order to compare the NO formation reduction effect of the exosomes according to the separation method, in addition to the exosomes obtained by the TFF separation and purification of one embodiment of the present invention was prepared exosomes separated by the conventional precipitation method. Precipitation was performed according to the protocol of the manufacturer (System Biosciences). Exosomes separated by the conventional precipitation method (see FIG. 11A) have low uniformity in particle size distribution and have various sizes as compared with the exosomes separated by the TFF method (see FIG. 11B) according to one embodiment of the present invention. It was confirmed. In addition, as shown in FIG. 11C, the exosomes separated and purified by the TFF method of one embodiment of the present invention were confirmed to inhibit NO formation at a much higher level than the exosomes obtained by the conventional precipitation method. These results show that the exosomes separated and purified according to one embodiment of the present invention are superior in terms of uniformity of particle size distribution and inhibition of NO formation compared to exosomes separated according to the conventional method.
따라서, 본 발명의 일 구체예의 분리 방법에 따라 수득된 엑소좀은 종래의 분리 방법에 따라 수득된 엑소좀과 비교하여 성능 또는 기능적 활성(예를 들어, 입자크기 분포의 균일성, NO 생성 억제, 염증 반응 감소 등)이 훨씬 우수하고, 이와 같이 기능적 활성이 우수한 지방줄기세포 유래의 엑소좀을 유효성분으로 함유하는 본 발명의 조성물은 궤양성 대장염을 예방, 억제, 완화, 개선 또는 치료하는 효과 면에서 종래기술 보다 훨씬 우수하다고 할 수 있다.Thus, the exosomes obtained according to the separation method of one embodiment of the present invention have a performance or functional activity (eg, uniformity of particle size distribution, inhibition of NO production, It is much better to reduce the inflammatory response, etc., and the composition of the present invention containing an exosome derived from adipose stem cells with excellent functional activity as an active ingredient is effective in preventing, inhibiting, alleviating, improving or treating ulcerative colitis. It can be said that much better than in the prior art.
실시예 7: 궤양성 대장염 유발 동물모델Example 7: Ulcerative Colitis-Induced Animal Model
궤양성 대장염 치료 효과를 확인하기 위한 동물 모델을 확립하기 위해 DSS(dextran sodium sulfate)를 사용한다. DSS를 섭취한 마우스는 인간의 궤양성 대장염과 유사한 증세를 나타낸다(Cooper HS, Merthy SN, Shs RS, Sedergran DJ. Clinicopsthologic study of dextran sulfate sodium experimental murine solitis. 1993. Laboratory Investigation. 69:238-249). Dextran sodium sulfate (DSS) is used to establish an animal model for confirming the effect of treating ulcerative colitis. Mice fed DSS show symptoms similar to human ulcerative colitis (Cooper HS, Merthy SN, Shs RS, Sedergran DJ.Clinicopsthologic study of dextran sulfate sodium experimental murine solitis. 1993. Laboratory Investigation. 69: 238-249) .
수컷 C57BL/6NCrljOri 생쥐(6주령, (주)오리엔트바이오에서 구입)를 구매하여 7일 동안의 적응기간을 통해 본 실험에 사용하였다. 적응기간을 거친 생쥐들은 아래와 같이 3군으로 분류하였다. 적응기간 중 건강한 것으로 판정된 생쥐들의 체중을 측정하고 순위화된 체중에 따라 각 군의 평균 체중이 최대한 균일하게 분포하도록 무작위법으로 분배하였다. 하기 (2) 및 (3)의 실험군에 대해 궤양성 대장염 유발 개시일(0일째)부터 2% DSS와 음수를 실험 종료시까지 자유롭게 섭취하도록 하여 궤양성 대장염을 유발시켰다.Male C57BL / 6NCrljOri mice (6 weeks old, purchased from Orient Bio Co., Ltd.) were purchased and used for this experiment through an adaptation period of 7 days. Mice that had undergone acclimation were classified into three groups as follows. The weights of the mice determined to be healthy during the adaptation period were measured and distributed randomly so that the average weight of each group was distributed as evenly as possible according to the ranked weight. Ulcerative colitis was induced by freely ingesting 2% DSS and negative water from the start date of ulcerative colitis induction (day 0) for the experimental groups of (2) and (3) below.
(1) 정상 대조군: 궤양성 대장염이 없는 정상 대조군;(1) normal control: normal control without ulcerative colitis;
(2) UC 유발 대조군(궤양성 대장염 유발 대조군): DSS로 궤양성 대장염을 유발한 음성 대조군;(2) UC-induced control (ulcerative colitis-induced control): negative control that induced ulcerative colitis with DSS;
(3) 엑소좀 투여군: DSS로 궤양성 대장염 유발 개시 후 실시예 2에서 준비된 엑소좀을 개체 당 7.5 μg의 용량으로 하루에 2회씩 3일간 (DSS에 의한 궤양성 대장염 유발 개시일로부터 2일째, 4일째 및 6일째) 혈관 내 투여(IV: intravenous injection)한 실험군.(3) Exosome-administered group: Exosome prepared in Example 2 after initiation of ulcerative colitis induced by DSS twice a day at a dose of 7.5 μg per individual for 3 days (2 days from the start of ulcerative colitis induced by DSS, 4
상기 각 군의 생쥐들의 체중을 DSS 투여 전과 투여 후 매일 1회 측정하였다. 투여 및 관찰기간 동안 사망을 포함한 일반증상의 종류, 증상 발현일 및 증상의 정도를 1일 1회 관찰하고 개체별로 기록하였다. 예를 들어, 개체별 체중, 대변 형태, 대변 내 출혈 등을 관찰하여 각각 스코어를 부여하고 개체 사망 후에는 부검하여 비장 및 대장의 무게와 길이를 측정하였다.Body weights of the mice in each group were measured before and after DSS once daily. During the administration and observation period, the type of general symptoms including the death, the date of onset of symptoms, and the degree of symptoms were observed once a day and recorded by individual. For example, each body weight, stool type, intra-femoral bleeding, and the like were scored, and after the death of the individual, the autopsy was performed to measure the weight and length of the spleen and colon.
궤양성 대장염 유발 대조군의 경우 궤양성 대장염 유발 개시 후 8일째 사망율이 70%인 반면에, DSS로 궤양성 대장염 유발 개시 후 본 발명의 엑소좀을 투여한 군에서는 사망율이 20%임을 알 수 있었다(도 12). 따라서, 본 발명의 엑소좀은 궤양성 대장염에 기인하는 개체 사망율을 감소시키고 개체 사망을 지연하는 효과가 있다고 할 수 있다.In the ulcerative colitis-induced control group, the mortality rate was 70% on the 8th day after the initiation of ulcerative colitis, whereas in the group administered with the exosome of the present invention after initiation of ulcerative colitis by DSS, the mortality rate was 20% 12). Therefore, it can be said that the exosome of the present invention has an effect of reducing the mortality rate and delaying the mortality caused by ulcerative colitis.
실시예 8: THP-1 단핵구를 이용한 항염증 효과 확인Example 8: Confirmation of anti-inflammatory effect using THP-1 monocytes
THP-1 단핵구(THP-1 human monocyte)를 대식세포로 분화시킨 후 본 발명의 엑소좀의 항염증 효과를 다음과 같이 확인하였다. After differentiating THP-1 monocytes into macrophages, the anti-inflammatory effects of the exosomes of the present invention were confirmed as follows.
THP-1 단핵구를, 10% FBS(Fetal Bovine Serum), 1% 페니실린-스트렙토마이신 및 100nM 포르볼 12-미리스테이트 13-아세테이트(Phorbol 12-myristate 13-acetate)(Sigma에서 구입)가 첨가된 RPMI 1640(Roswell Park Memorial Institute 1640; ThermoFisher Scientific에서 구입) 배지에 현탁시킨 후 12웰 플레이트에 6×105 세포/mL의 밀도로 접종하고 37℃, 5% CO2 배양기에서 48시간 배양하여 THP-1 유래 대식세포(THP-1 derived macrophage)로 분화를 유도하였다. 이후 분화된 세포를 PBS로 1회 세척하고, 포르볼 12-미리스테이트 13-아세테이트가 포함되어 있지 않은 RPMI 1640 배지에서 24시간 동안 배양하여 세포를 안정화하였다. THP-1 monocytes were added RPMI with 10% Fetal Bovine Serum (FBS), 1% penicillin-streptomycin and 100 nM phorbol 12-myristate 13-acetate (purchased from Sigma). Suspended in 1640 (Roswell Park Memorial Institute 1640; purchased from ThermoFisher Scientific) medium, inoculated in a 12 well plate at a density of 6 × 10 5 cells / mL, incubated for 48 hours in a 37 ° C., 5% CO 2 incubator, and THP-1 Differentiation was induced into THP-1 derived macrophage. The differentiated cells were then washed once with PBS and incubated for 24 hours in RPMI 1640 medium without phorbol 12-myristate 13-acetate to stabilize the cells.
동시처리(co-treatment) 실험군의 경우, THP-1 유래 대식세포에 대해 FBS(Fetal Bovine Serum)가 포함되어 있지 않은 RPMI 1640 배지와 100 ng/mL LPS(Sigma에서 구입) 및 20 ng/mL IFN-γ(Peprotech에서 구입)를 처리하여 THP-1 유래 대식세포를 활성화시키고, 또한 양성대조군인 덱사메타손(Sigma에서 구입) 또는 본 발명의 엑소좀(실시예 2에서 준비된 엑소좀)를 처리하여 24시간 배양하였다. 한편, 전처리(pre-treatment) 실험군의 경우, THP-1 유래 대식세포에 대해 FBS(Fetal Bovine Serum)가 포함되어 있지 않은 RPMI 1640 배지와 양성대조군인 덱사메타손(Sigma에서 구입) 또는 본 발명의 엑소좀(실시예 2에서 준비된 엑소좀)를 처리하여 24시간 배양한 다음, 100 ng/mL LPS(Sigma에서 구입) 및 20 ng/mL IFN-γ(Peprotech에서 구입)를 처리하고 4시간 동안 배양하여 THP-1 유래 대식세포를 활성화하였다. For co-treatment groups, RPMI 1640 medium without FBS (Fetal Bovine Serum) and 100 ng / mL LPS (purchased from Sigma) and 20 ng / mL IFN for THP-1 derived macrophages treatment with -γ (purchased from Peprotech) to activate macrophages derived from THP-1 and also treatment of positive control dexamethasone (purchased from Sigma) or exosomes of the present invention (exosomes prepared in Example 2) for 24 hours. Incubated. Meanwhile, in the pre-treatment experimental group, RPMI 1640 medium containing no FBS (Fetal Bovine Serum) for THP-1-derived macrophages and dexamethasone (obtained from Sigma) or the exosome of the present invention. (Exosome prepared in Example 2) treated and incubated for 24 hours, then treated with 100 ng / mL LPS (purchased from Sigma) and 20 ng / mL IFN-γ (purchased from Peprotech) and incubated for 4 hours to THP -1 derived macrophages were activated.
배양이 끝난 THP-1 유래 대식세포(THP-1 derived macrophage)로부터 분리한 RNA로부터 cDNA를 제조하고 리얼타임 PCR 방법을 이용하여 궤양성 대장염의 주요 원인이 되는 친염증성 사이토카인(Pro-inflammatory Cytokine)인 IL-6, TNF-α, IL-12 및 IL-23의 mRNA 발현량을 측정하였다. 상기 유전자들을 정량하기 위한 표준 유전자로서 GAPDH를 사용하였다. PCR에 사용한 프라이머의 종류와 서열은 하기의 표 2와 같다.Pro-inflammatory Cytokine is a major cause of ulcerative colitis by preparing cDNA from RNA isolated from cultured THP-1 derived macrophage and using real-time PCR. MRNA expression levels of IL-6, TNF-α, IL-12 and IL-23 were measured. GAPDH was used as a standard gene for quantifying these genes. The type and sequence of primers used for PCR are shown in Table 2 below.
한편, 앞서 설명한 바와 같이 IL-6 및 TNF-α가 궤양성 대장염의 발병과 밀접한 관련이 있다는 보고 외에 IL-12 및 IL-23도 궤양성 대장염의 발병과 관련이 있는 것으로 알려져 있다. 즉, IL-12는 궤양성 대장염 동물모델의 생검에서 발현이 증가된다는 보고가 있고(World J Gastroenterol 2008 July 21; 14(27): pp.4280-4288), IL-23은 국소적 장관의 염증반응과 관련이 있다고 보고되어 있다(Korean J Gastroenterol Vol.58, No.5, pp.235-244). 따라서, 동물모델이나 염증반응에 관여하는 대식세포에 후보물질 처리 후 IL-12 및 IL-23의 생성량이나 발현량을 분석하면 후보물질의 궤양성 대장염 억제 및 완화에 대한 효능을 평가할 수 있다. Meanwhile, as described above, in addition to the report that IL-6 and TNF-α are closely related to the development of ulcerative colitis, IL-12 and IL-23 are also known to be related to the development of ulcerative colitis. In other words, IL-12 has been reported to increase expression in biopsies of ulcerative colitis animal models (World J Gastroenterol 2008 July 21; 14 (27): pp.4280-4288), and IL-23 is an inflammation of the local intestinal tract. It has been reported to be related to the response (Korean J Gastroenterol Vol. 58, No. 5, pp.235-244). Therefore, by analyzing the production amount and expression level of IL-12 and IL-23 after treating the macrophage cells involved in the animal model or inflammatory response can evaluate the efficacy of the candidate substance to suppress and relieve ulcerative colitis.
도 13에 도시된 바와 같이, THP-1 단핵구를 대식세포로 분화시킨 후 얻은 THP-1 유래 대식세포에 대해, LPS 및 IFN-γ와 함께 본 발명의 엑소좀을 처리(co-treatment)한 경우, 음성대조군에 비해 궤양성 대장염의 원인이 되는 친염증성 사이토카인인 IL-6의 mRNA의 발현량이 감소하였다. 또한, 도 14 내지 도 17에 도시된 바와 같이, THP-1 단핵구를 대식세포로 분화시킨 후 얻은 THP-1 유래 대식세포에 대해, LPS 및 IFN-γ 처리 전에 본 발명의 엑소좀을 전처리(pre-treatment)한 경우, 음성대조군에 비해 궤양성 대장염의 원인이 되는 것으로 알려진 친염증성 사이토카인인 IL-6, TNF-α, IL-12 및 IL-23의 mRNA의 발현량이 감소하였다. 친염증성 사이토카인인 IL-6, TNF-α, IL-12 및 IL-23는 친염증성 M1 대식세포에서는 발현이 증가하고 항염증성 M2 대식세포에서는 발현이 감소한다. 따라서, 상기 결과들은 본 발명의 엑소좀이 염증반응 및 이로 인한 궤양성 대장염을 억제, 완화 및 감소시킬 수 있는 것을 시사하며, 앞서 논의된 동물실험 결과를 강력히 뒷받침하는 결과라고 할 수 있다.As shown in FIG. 13, when THP-1 derived macrophages obtained after differentiating THP-1 monocytes into macrophages, co-treatment of the exosome of the present invention with LPS and IFN-γ , MRNA expression of IL-6, a proinflammatory cytokine that causes ulcerative colitis, was reduced compared to the negative control group. In addition, as shown in Figures 14 to 17, THP-1 derived macrophages obtained after differentiating THP-1 monocytes into macrophages, pre-treated with the exosomes of the present invention before LPS and IFN-γ treatment (pre In the case of -treatment, the expression levels of the proinflammatory cytokines IL-6, TNF-α, IL-12 and IL-23, which are known to cause ulcerative colitis, decreased compared to the negative control group. The proinflammatory cytokines IL-6, TNF-α, IL-12 and IL-23 have increased expression in proinflammatory M1 macrophages and decreased expression in anti-inflammatory M2 macrophages. Therefore, the above results suggest that the exosome of the present invention can inhibit, alleviate, and reduce the inflammatory response and ulcerative colitis caused by it, and can be said to strongly support the animal test results discussed above.
따라서, 본 발명의 엑소좀은 궤양성 대장염의 원인이 되는 염증성 사이토카인인 IL-6, TNF-α, IL-12 및 IL-23에 대해 작용하여 다양한 원인에 기인하는 궤양성 대장염에 대한 광범위한 적용이 가능하고 효과적으로 궤양성 대장염을 예방, 억제 및 완화시킬 수 있을 것으로 기대된다.Accordingly, the exosomes of the present invention act against inflammatory cytokines IL-6, TNF-α, IL-12 and IL-23, which are responsible for ulcerative colitis, and thus have wide application to ulcerative colitis due to various causes. It is expected that this will be possible and effectively prevent, inhibit and alleviate ulcerative colitis.
이상, 본 발명을 상기 실시예를 들어 설명하였으나, 본 발명은 이에 제한되는 것이 아니다. 당업자라면 본 발명의 취지 및 범위를 벗어나지 않고 수정, 변경을 할 수 있으며 이러한 수정과 변경 또한 본 발명에 속하는 것임을 알 수 있을 것이다.As mentioned above, although this invention was demonstrated to the said Example, this invention is not limited to this. Those skilled in the art can make modifications and changes without departing from the spirit and scope of the present invention, and it will be appreciated that such modifications and changes also belong to the present invention.
<110> ExoCoBio Inc. <120> A composition comprising an exosome derived from adipose-derived stem cell as an active ingredient and its application for improving Ulcerative Colitis <130> P17-0038KR <150> KR 10-2018-0017396 <151> 2018-02-13 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha forward primer <400> 1 tctcatcagt tctatggccc agac 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha reverse primer <400> 2 ggcaccacta gttggttgtc tttg 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-6 forward primer <400> 3 gccagagtcc ttcagagaga taca 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-6 reverse primer <400> 4 attggatggt cttggtcctt agcc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 5 gacatcaaga aggtggtgaa gcag 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 6 ccctgttgct gtagccgtat tcat 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-6 (THP-1) forward primer <400> 7 aagccagagc tgtgcagatg agta 24 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6 (THP-1) reverse primer <400> 8 tgtcctgcag ccactggttc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha (THP-1) forward primer <400> 9 ctgcctgctg cactttggag 20 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha (THP-1) reverse primer <400> 10 acatgggcta caggcttgtc act 23 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> IL-12 (THP-1) forward primer <400> 11 ggagcgaatg ggcatctgt 19 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-12 (THP-1) reverse primer <400> 12 tgggtctatt ccgttgtgtc ttta 24 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-23 (THP-1) forward primer <400> 13 agtgccagca gctttcacag a 21 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL-23 (THP-1) reverse primer <400> 14 aatcagaccc tggtggatcc tt 22 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH (THP-1) forward primer <400> 15 ctttggtatc gtggaaggac tc 22 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH (THP-1) reverse primer <400> 16 gtagaggcag ggatgatgtt ct 22 <110> ExoCoBio Inc. <120> A composition comprising an exosome derived from adipose-derived stem cell as an active ingredient and its application for improving Ulcerative Colitis <130> P17-0038KR <150> KR 10-2018-0017396 <151> 2018-02-13 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha forward primer <400> 1 tctcatcagt tctatggccc agac 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha reverse primer <400> 2 ggcaccacta gttggttgtc tttg 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-6 forward primer <400> 3 gccagagtcc ttcagagaga taca 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-6 reverse primer <400> 4 attggatggt cttggtcctt agcc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 5 gacatcaaga aggtggtgaa gcag 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 6 ccctgttgct gtagccgtat tcat 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> IL-6 (THP-1) forward primer <400> 7 aagccagagc tgtgcagatg agta 24 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> IL-6 (THP-1) reverse primer <400> 8 tgtcctgcag ccactggttc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha (THP-1) forward primer <400> 9 ctgcctgctg cactttggag 20 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha (THP-1) reverse primer <400> 10 acatgggcta caggcttgtc act 23 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> IL-12 (THP-1) forward primer <400> 11 ggagcgaatg ggcatctgt 19 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-12 (THP-1) reverse primer <400> 12 tgggtctatt ccgttgtgtc ttta 24 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> IL-23 (THP-1) forward primer <400> 13 agtgccagca gctttcacag a 21 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> IL-23 (THP-1) reverse primer <400> 14 aatcagaccc tggtggatcc tt 22 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH (THP-1) forward primer <400> 15 ctttggtatc gtggaaggac tc 22 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH (THP-1) reverse primer <400> 16 gtagaggcag ggatgatgtt ct 22
Claims (9)
상기 엑소좀은 궤양성 대장염의 원인이 되는 사이토카인인 TNF-α, IL-6, IL-12 및 IL-23의 생성량 또는 발현량을 감소시키는 것을 특징으로 하는, 약학 조성물.The method of claim 1,
The exosomes, characterized in that to reduce the amount of production or expression of cytokines TNF-α, IL-6, IL-12 and IL-23 that causes ulcerative colitis, pharmaceutical composition.
상기 엑소좀은 하기의 단계들을 수행하여 수득되는, 약학 조성물:
(a) 지방줄기세포 배양액에 트레할로오스를 첨가하는 단계, (b) 상기 트레할로오스가 첨가된 지방줄기세포 배양액을 여과하는 단계, (c) 상기 여과된 지방줄기세포 배양액으로부터, TFF(Tangential Flow Filtration)를 이용하여 엑소좀을 분리하는 단계, 및 (d) 탈염과 버퍼교환(diafiltration)에 사용되는 완충용액에 트레할로오스를 첨가하고, 상기 트레할로오스가 첨가된 완충용액을 이용한 TFF(Tangential Flow Filtration)를 이용하여, 상기 분리된 엑소좀에 대한 탈염과 버퍼교환(diafiltration)을 수행하는 단계.The method according to claim 1 or 2,
The exosomes are obtained by performing the following steps:
(a) adding trehalose to the adipose stem cell culture medium, (b) filtering the adipose stem cell culture medium to which the trehalose is added, (c) from the filtered adipose stem cell culture medium, TFF Separating exosomes using tangential flow filtration, and (d) adding trehalose to the buffer solution used for desalting and diafiltration, and adding the trehalose buffer. By using TFF (Tangential Flow Filtration), desalting and diafiltration of the separated exosomes.
상기 탈염과 버퍼교환은 연속적으로 수행하거나 단속적으로 수행하는 것을 특징으로 하는, 약학 조성물.The method of claim 3,
The desalting and buffer exchange are performed continuously or intermittently, the pharmaceutical composition.
시작 부피(starting volume)에 대하여 적어도 4배의 부피를 갖는 완충용액을 이용하여 탈염과 버퍼교환을 수행하는 것을 특징으로 하는, 약학 조성물. The method of claim 3,
Pharmaceutical composition, characterized in that to perform desalting and buffer exchange using a buffer solution having a volume of at least four times the starting volume.
TFF(Tangential Flow Filtration)를 위해 MWCO(molecular weight cutoff) 100,000 Da, 300,000 Da, 500,000 Da 또는 750,000 Da의 TFF 필터, 또는 0.05 μm TFF 필터를 사용하는 것을 특징으로 하는, 약학 조성물. The method of claim 3,
A pharmaceutical composition, characterized by using a molecular weight cutoff (MWCO) 100,000 Da, 300,000 Da, 500,000 Da or 750,000 Da, or 0.05 μm TFF filter for tangential flow filtration (TFF).
상기 (c) 단계는 TFF(Tangential Flow Filtration)를 이용하여 1/100 내지 1/25의 부피까지 농축하는 과정을 더 포함하는, 약학 조성물. The method of claim 3,
The step (c) further comprises the step of concentrating to a volume of 1/100 to 1/25 using TFF (Tangential Flow Filtration).
상기 포유동물은 인간, 개, 고양이, 설치류, 말, 소, 원숭이, 또는 돼지인, 궤양성 대장염을 예방, 억제, 완화, 개선 또는 치료하는 방법.The method of claim 8,
The mammal is a human, dog, cat, rodent, horse, cow, monkey, or pig, the method of preventing, inhibiting, alleviating, ameliorating or treating ulcerative colitis.
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