KR20210152094A - Composition for promoting growth of hair or Treating or Improving of Atopic dermatitis - Google Patents
Composition for promoting growth of hair or Treating or Improving of Atopic dermatitis Download PDFInfo
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Abstract
Description
본 발명은 발모 촉진 발모 촉진 또는 아토피 피부염 치료 또는 개선용 조성물에 관한 것이다.The present invention relates to a composition for promoting hair growth, treating or improving atopic dermatitis.
탈모란 모발이 빠져 성기거나 소실된 상태를 말하며, 인간의 머리카락 개수는 약 10만개 내지 15만개 정도로서, 성장기 (anagen), 퇴행기 (catagen) 및 휴지기 (telogen)이라는 고유의 주기를 갖고 성장 및 탈락을 반복하고, 하루 평균 100개 가량의 머리카락이 자연적으로 탈모 된다. 탈모의 기전이 정확히 밝혀진 바는 없으나, 현재까지 연구된 바로는 호르몬 불균형 등 내분비계 이상, 자율신경계 및 혈액순환 장애 등의 순환계 이상으로 인한 과도한 피지생성, 모근의 영양 결핍, 알레르기, 세균 감염, 유전적요인, 정신적 스트레스, 대기오염 또는 음식물 등의 환경적 요인 및 노화 등이 그 원인으로 추정되고 있다. 탈모증은 과거 중 장년층에서만 발생되었으나, 최근에는 20~30대의 젊은 층에서도 빈번하게 발생되고 있다.Hair loss refers to a state in which hair is lost or lost, and the number of human hairs is about 100,000 to 150,000, and has its own cycle of growth (anagen), regression (catagen) and telogen (telogen), and growth and fallout. Repeatedly, an average of 100 hairs per day naturally fall out. Although the exact mechanism of hair loss has not been elucidated, research so far has shown that excessive sebum production due to endocrine system abnormalities such as hormonal imbalance, circulatory system abnormalities such as autonomic nervous system and blood circulation disorders, nutritional deficiency of hair roots, allergies, bacterial infection, heredity Environmental factors such as environmental factors, mental stress, air pollution or food, and aging are presumed to be the causes. Alopecia occurred only in the middle-aged in the past, but recently, it is also frequently occurring in young people in their 20s and 30s.
남성형 탈모증 (Male Pattern Alopecia)는 수년에 걸쳐 점차적으로 발생되고, 머리의 정수리에서 가장 뚜렷하게 시작되어 전두 (forehead) 부위로 진행한다. 여성형 탈모증(Female Pattern Alopecia)의 경우는 모발의 굵기가 가늘어지면서 보다 더 분산된 형태로 발생되며 흔히 폐경기 이후에 나타난다. 탈모증을 완화 또는 치료하기 위해서 많은 연구들이 진행되고 있으며, 특히 모발 성장을 자극하거나 탈모 현상을 완화하기 위한 신 물질 개발을 위해서 화장품 또는 제약 산업 분야에서 오래 전부터 연구가 이루어져 왔고, 이에 따라 여성 호르몬제, 혈류 촉진제 등을 사용하는 약물요법과 모발 이식법 등이 개발된 상태이다. Male pattern alopecia develops gradually over several years, and starts most clearly at the crown of the head and progresses to the forehead area. In the case of female pattern alopecia, it occurs in a more dispersed form as the thickness of the hair becomes thinner and often appears after menopause. Many studies are being conducted to alleviate or treat alopecia, and in particular, research has been made in the cosmetic or pharmaceutical industry for a long time to develop new substances to stimulate hair growth or alleviate the phenomenon of hair loss, and accordingly, female hormones, Drug therapy using blood flow promoters and hair transplantation methods have been developed.
현재, 탈모 치료에 가장 많이 사용되는 약물로 FDA에서 승인을 받은 2,4-디아미노-6-피페리디노피리미딘-3-옥사이드 (일명 '미녹시딜(Minoxidil, MNX)'제제)와 II 형 5α-환원효소의 특정 억제제인 피나스테라이드 (Finasteride)가 상용화 되어있다.Currently, 2,4-diamino-6-piperidinopyrimidine-3-oxide (aka 'Minoxidil (MNX)' formulation) approved by the FDA as the most used drugs for hair loss treatment and type II 5α - Finasteride, a specific inhibitor of reductase, is commercially available.
MNX 제제는 혈관 확장 효과를 통하여 혈류량을 증가시키고 모근에 영양분을 공급하여 모발 성장을 유도하는 약물로서, 특히 가마 부위의 탈모 증상 완화에 효과가 좋은 것으로 알려져 있고, 이를 활성 성분으로 사용하는 의약품은 상품명 로게인 (Rogaine Pharmacia & Upjohn Company의 상표명)으로 시판되고 있다. 로게인은 남성형 탈모증을 앓고 있는 남성의 경우 10%까지 탈모를 감소시키고 모발 성장을 촉진하는 것으로 알려져 있지만, 두피 부위에 외부에서 직접적으로 적용하여야 하며, 장기간에 걸쳐 규칙적으로 사용해야 하고, 가마 부위 이외 부위의 탈모에 대해서는 그다지 좋은 효과를 발휘하지 못한다는 단점이 있다. 피나스테라이드를 활성 성분으로 사용한 의약품은 상품명 프로페시아 (Propecia, Merck & Co., Inc.의 상표명) 로 시판되고 있으며, 이는 경구투여용환제로서 II 형 5α-환원효소의 기능을 억제하여 테스토스테론 (Testosterone)이 디하이드로 테스토스테론(Dihydrotestosterone, DHT)로 전환되는 것을 방지함으로써 탈모를 억제하는 것으로 알려져 있다. 그러나 이 역시 지속적이고 규칙적인 복용을 필요로 하며, 일부 환자들에 대해서는 성욕 감퇴, 발기 부전 등의 부작용을 나타낸다는 문제점이 보도된 바 있다. 따라서 MNX 제제 또는 피나스테라이드 제제가 갖는 문제점을 극복할 수 있으면서도, 기타 천연 식물 추출물 조성물이나 모발 이식법에 비해서 효과 및 효능이 우수하며, 약물 전달력 및 안정성이 우수한 탈모 방지 및 발모 촉진 화합물의 개발이 요구되고 있다.MNX formulation is a drug that induces hair growth by increasing blood flow through the vasodilator effect and supplying nutrients to the hair root, and is known to be particularly effective in relieving hair loss symptoms in the forehead area. It is marketed as Rogaine (trade name of Rogaine Pharmacia & Upjohn Company). Rogaine is known to reduce hair loss by up to 10% and promote hair growth in men suffering from androgenetic alopecia, but it must be applied directly to the scalp from the outside and must be used regularly over a long period of time, and There is a disadvantage that it does not exert a very good effect on hair loss. Pharmaceuticals using finasteride as an active ingredient are marketed under the trade name Propecia (trade name of Propecia, Merck & Co., Inc.), which is a pill for oral administration that inhibits the function of type II 5α-reductase to produce testosterone. It is known to inhibit hair loss by preventing conversion to dihydrotestosterone (DHT). However, this also requires continuous and regular use, and there have been reports of problems with side effects such as decreased libido and erectile dysfunction in some patients. Therefore, while it is possible to overcome the problems of the MNX formulation or the finasteride formulation, the effect and efficacy are excellent compared to other natural plant extract compositions or hair transplantation methods, and the development of a hair loss prevention and hair growth promoting compound with excellent drug delivery and stability is required. have.
최근 유전자를 이용한 탈모 치료 방법 및 줄기세포를 이용한 탈모 치료 방법이 개발되고 있다. 예를 들면, 모낭 줄기세포를 분리, 증식하여, 모낭 세포로 분화시키는 방법 및 대머리 치료용 조성물(한국등록특허 0771171호) 에 관한 연구가 있다.Recently, a method for treating hair loss using genes and a method for treating hair loss using stem cells have been developed. For example, there is a study on a method for isolating, proliferating, and differentiating hair follicle stem cells into hair follicle cells and a composition for treating baldness (Korean Patent No. 0771171).
그러나, 말초혈액단핵구 유래의 면역세포 또는 면역세포치료제를 이용한 우수한 효과의 탈모 방지 또는 발모 촉진 조성물에 대해서는 알려진 바가 없다.However, there is no known information about a composition for preventing hair loss or promoting hair growth using an immune cell or immune cell therapeutic agent derived from peripheral blood mononuclear cells.
또한, 아토피 피부염(atopic dermatitis)은 유소아에서 발생하여 흔히 성인까지 지속되는 심한 소양증과 특징적인 피부 소견을 보이는 만성 염증성 질환이다. 아토피 피부염의 병인으로는 유전적 배경, 면역학적 기전, 환경적 요인 등 다양한 인자가 복합되어 있는 것으로 알려져 있으며, 발병 기전이 매우 복잡하여 여러 가지 가설이 존재한다. 가장 유력한 가설 중 하나는 Th1/Th2 세포의 항상성이 깨지면서 Th2 세포가 과활성되는 것이다. 특정 알레르겐 (allergen)에 의해 과활성화된 Th2 세포는 IL-4 및 IL-31 과 같은 Th2 사이토카인을 분비하고, 분비된 사이토카인은 B 세포의 IgE 분비와 비만세포 (Mast cell)의 탈과립을 유도하여 여러 염증 물질을 방출시킨다.In addition, atopic dermatitis (atopic dermatitis) is a chronic inflammatory disease that occurs in children and often persists into adults with severe pruritus and characteristic skin findings. It is known that various factors such as genetic background, immunological mechanism, and environmental factor are complex as the etiology of atopic dermatitis, and the pathogenesis is very complex, so several hypotheses exist. One of the most promising hypotheses is that Th2 cells become hyperactive when homeostasis of Th1/Th2 cells is disrupted. Th2 cells overactivated by a specific allergen secrete Th2 cytokines such as IL-4 and IL-31, and the secreted cytokines induce IgE secretion by B cells and degranulation of mast cells. It releases several inflammatory substances.
그러나, 아토피 피부염의 치료에 있어 현재까지는 항염증제인 코르티코스테로이드 (Corticosteroid)와 같은 물질을 사용하여 면역세포의 전반적인 활성을 억제시키는 방법을 사용하고 있을 뿐이며, 위 방법은 지속기간이 매우 짧을 뿐만 아니라 심한 부작용을 내포하고 있어 적절한 치료방법으로는 권장되지 않는다. 또한, 최근 개발된 저분자 및 항체 치료제의 경우 한가지의 염증물질에 결합하는 수용체를 차단하는 수준이기 때문에 아토피와 같이 복잡한 염증 신호체계를 매개로 한 만성 피부염을 치료하기에는 한계가 있으며, 다른 경로를 통한 아토피 재발위험을 피하기 쉽지 않다.However, in the treatment of atopic dermatitis, until now, only a method of suppressing the overall activity of immune cells using substances such as anti-inflammatory corticosteroids is used, and the above method has a very short duration and severe side effects. Therefore, it is not recommended as an appropriate treatment method. In addition, in the case of recently developed small molecule and antibody therapeutics, there is a limit to treating chronic dermatitis mediated by a complex inflammatory signaling system such as atopic dermatitis because they block receptors that bind to one inflammatory substance, and atopy through other pathways. It is not easy to avoid the risk of recurrence.
이에, 아토피의 병인에 부합하여 Th1/Th2 면역체계 균형화를 통한 근본적인 치료 접근을 통해 치료효과를 극대화하는 것이 매우 중요하며, 부작용을 유발하지 않는 아토피 피부염 치료제의 개발이 절실하다.Accordingly, it is very important to maximize the therapeutic effect through a fundamental treatment approach through balancing the Th1/Th2 immune system in accordance with the etiology of atopic dermatitis, and the development of a therapeutic agent for atopic dermatitis that does not cause side effects is urgently needed.
이와 관련하여, 세포의 특성과 기능을 포함하고 있어 세포가 지니고 있는 생물학적 기능과 유사하게 나타날 수 있는 세포 배양액이나 세포에서 분비하는 엑소좀을 이용한 연구를 수행하여 그 효과를 확인하였으며, 세포 배양액이나 엑소좀의 경우 동결건조가 가능해 보관이 용이하고 편리하게 피부 등에 도포가 가능하여 화장품 용도로 활용이 가능할 것으로 전망된다.In this regard, the effect was confirmed by conducting a study using cell culture media or exosomes secreted from cells, which contain cell characteristics and functions, which can appear similar to the biological functions possessed by cells. In the case of moth, it can be freeze-dried, so it is easy to store and can be conveniently applied to the skin, so it is expected to be used for cosmetic purposes.
본 발명은 말초혈액단핵구 유래 활성화 림프구의 배양물을 포함하는 발모 촉진 또는 아토피 피부염 치료용 약학 조성물을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a pharmaceutical composition for promoting hair growth or treating atopic dermatitis, comprising a culture of activated lymphocytes derived from peripheral blood mononuclear cells.
말초혈액단핵구 유래 활성화 림프구의 배양물을 포함하는 발모 촉진 또는 아토피 피부염 개선용 화장료 조성물을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a cosmetic composition for promoting hair growth or improving atopic dermatitis comprising a culture of activated lymphocytes derived from peripheral blood mononuclear cells.
1. 말초혈액단핵구 유래 활성화 림프구의 배양물을 포함하는 발모 촉진 또는 아토피 피부염 치료용 약학 조성물.1. A pharmaceutical composition for promoting hair growth or treating atopic dermatitis, comprising a culture of activated lymphocytes derived from peripheral blood mononuclear cells.
2. 위 1에 있어서, 상기 활성화 림프구는 개체의 말초혈액단핵구를 항-CD3 항체, IL-2, IL-12 및 IL-18로 이루어진 군에서 선택되는 하나 이상을 포함하는 세포 배양용 배지에서 배양하여 얻어진 것인, 조성물.2. The method of 1 above, wherein the activated lymphocytes are cultured in a cell culture medium comprising at least one selected from the group consisting of anti-CD3 antibody, IL-2, IL-12, and IL-18. Which is obtained by the composition.
3. 위 2에 있어서, 상기 세포 배양용 배지는 항-CD3 항체, IL-2, IL-12 및 IL-18를 포함하는 것인, 조성물.3. The composition of 2 above, wherein the cell culture medium comprises an anti-CD3 antibody, IL-2, IL-12 and IL-18.
4. 위 1에 있어서, 상기 배양물은, 인간의 말초혈액으로부터 단핵구 및 자가혈장을 각각 분리하여 수득하는 단계; 항-CD3 항체로 세포 배양용기를 코팅하는 단계; 및 상기 단핵구를 상기 배양용기에 접종하여 IL-2, IL-12 및 IL-18로 이루어진 군에서 선택되는 하나 이상을 포함하는 배지에서 배양하는 단계를 포함하는 배양방법으로 얻어진 것인, 조성물.4. The method of 1 above, wherein the culture is obtained by isolating monocytes and autologous plasma from human peripheral blood, respectively; coating the cell culture vessel with an anti-CD3 antibody; And inoculating the monocytes into the culture vessel and culturing in a medium containing at least one selected from the group consisting of IL-2, IL-12 and IL-18, the composition comprising the step of inoculating the monocytes.
5. 위 1에 있어서, 상기 배양물은 상기 활성화 림프구 유래 엑소좀을 포함하는 것인, 조성물.5. The composition of 1 above, wherein the culture includes the activated lymphocyte-derived exosomes.
6. 위 1에 있어서, 상기 배양물은 IRF4(Interferon regulatory factor 4), CD226(Cluster of Differentiation 226), TMIGD2(Transmembrane and immunoglobulin domain-containing protein 2), HAVCR2(Hepatitis A virus cellular receptor 2), TRAT1(T-cell receptor-associated transmembrane adapter 1), SPN(Sialophorin), TRAF2(TNF receptor-associated factor 2), CCDC88B(Coiled-coil domain containing 88B), TYROBP(tyrosine kinase-binding protein), VAV1(Vav Guanine Nucleotide Exchange Factor 1), SLAMF6(SLAM family member 6), ZBTB16(Zinc finger and BTB domain-containing protein 16)로 이루어진 군에서 선택되는 하나 이상의 단백질을 포함하는 것인, 조성물.6. The culture of the above 1, wherein the culture is IRF4 (Interferon regulatory factor 4), CD226 (Cluster of Differentiation 226), TMIGD2 (Transmembrane and immunoglobulin domain-containing protein 2), HAVCR2 (Hepatitis A virus cellular receptor 2), TRAT1 (T-cell receptor-associated transmembrane adapter 1), SPN (Sialophorin), TRAF2 (TNF receptor-associated factor 2), CCDC88B (Coiled-coil domain containing 88B), TYROBP (tyrosine kinase-binding protein), VAV1 (Vav Guanine) Nucleotide Exchange Factor 1), SLAMF6 (SLAM family member 6), ZBTB16 (Zinc finger and BTB domain-containing protein 16), the composition comprising one or more proteins selected from the group consisting of.
7. 말초혈액단핵구 유래 활성화 림프구의 배양물을 포함하는 발모 촉진 또는 아토피 피부염 개선용 화장료 조성물.7. A cosmetic composition for promoting hair growth or improving atopic dermatitis, comprising a culture of activated lymphocytes derived from peripheral blood mononuclear cells.
8. 위 7에 있어서, 상기 활성화 림프구는 개체의 말초혈액단핵구를 항-CD3 항체, IL-2, IL-12 및 IL-18로 이루어진 군에서 선택되는 하나 이상을 포함하는 세포 배양용 배지에서 배양하여 얻어진 것인, 조성물.8. The method of 7 above, wherein the activated lymphocytes are cultured in a cell culture medium comprising one or more selected from the group consisting of an anti-CD3 antibody, IL-2, IL-12 and IL-18 by culturing the peripheral blood mononuclear cells of an individual. Which is obtained by the composition.
9. 위 7에 있어서, 상기 세포 배양용 배지는 항-CD3 항체, IL-2, IL-12 및 IL-18를 포함하는 것인, 조성물.9. The composition of 7 above, wherein the cell culture medium comprises an anti-CD3 antibody, IL-2, IL-12 and IL-18.
10. 위 7에 있어서, 상기 배양물은, 인간의 말초혈액으로부터 단핵구 및 자가혈장을 각각 분리하여 수득하는 단계; 항-CD3 항체로 세포 배양용기를 코팅하는 단계; 및 상기 단핵구를 상기 배양용기에 접종하여 IL-2, IL-12 및 IL-18로 이루어진 군에서 선택되는 하나 이상을 포함하는 배지에서 배양하는 단계를 포함하는 배양방법으로 얻어진 것인, 조성물.10. The method of 7 above, wherein the culture is obtained by isolating monocytes and autologous plasma from human peripheral blood, respectively; coating the cell culture vessel with an anti-CD3 antibody; And inoculating the monocytes into the culture vessel and culturing in a medium containing at least one selected from the group consisting of IL-2, IL-12 and IL-18, the composition comprising the step of inoculating the monocytes.
11. 위 7에 있어서, 상기 배양물은 상기 활성구 림프구 유래 엑소좀을 포함하는 것인, 조성물.11. The composition of the above 7, wherein the culture comprises the active cell lymphocyte-derived exosome.
12. 위 7에 있어서, 상기 배양물은 IRF4(Interferon regulatory factor 4), CD226(Cluster of Differentiation 226), TMIGD2(Transmembrane and immunoglobulin domain-containing protein 2), HAVCR2(Hepatitis A virus cellular receptor 2), TRAT1(T-cell receptor-associated transmembrane adapter 1), SPN(Sialophorin), TRAF2(TNF receptor-associated factor 2), CCDC88B(Coiled-coil domain containing 88B), TYROBP(tyrosine kinase-binding protein), VAV1(Vav Guanine Nucleotide Exchange Factor 1), SLAMF6(SLAM family member 6) 및 ZBTB16(Zinc finger and BTB domain-containing protein 16)로 이루어진 군에서 선택되는 하나 이상의 단백질을 포함하는 것인, 조성물.12. The culture of the above 7, wherein the culture is IRF4 (Interferon regulatory factor 4), CD226 (Cluster of Differentiation 226), TMIGD2 (Transmembrane and immunoglobulin domain-containing protein 2), HAVCR2 (Hepatitis A virus cellular receptor 2), TRAT1 (T-cell receptor-associated transmembrane adapter 1), SPN (Sialophorin), TRAF2 (TNF receptor-associated factor 2), CCDC88B (Coiled-coil domain containing 88B), TYROBP (tyrosine kinase-binding protein), VAV1 (Vav Guanine) A composition comprising one or more proteins selected from the group consisting of Nucleotide Exchange Factor 1), SLAMF6 (SLAM family member 6), and ZBTB16 (Zinc finger and BTB domain-containing protein 16).
본 발명에 따른 말초혈액단핵구 유래 활성화 림프구의 배양물 및 이로부터 분리된 엑소좀을 포함하는 발모 촉진용 조성물로 모유두 세포의 증식을 촉진하고 ALP활성증가, VEGF분비촉진 및 모낭조직 성장과 관련된 mRNA발현을 증가시켜 모낭성장을 촉진시킴으로써 탈모 방지 또는 발모에 탁월한 효능이 있다.A composition for promoting hair growth comprising a culture of peripheral blood mononuclear cell-derived activated lymphocytes and exosomes isolated therefrom according to the present invention, promotes proliferation of dermal papilla cells, increases ALP activity, promotes VEGF secretion, and expression of mRNA related to hair follicle tissue growth It has excellent efficacy in preventing hair loss or hair growth by increasing hair follicle growth.
또한 본 발명에 따른 말초혈액단핵구 유래 활성화 림프구의 배양물 및 이로부터 분리된 엑소좀을 포함하는 아토피 피부염 개선 또는 치료용 조성물은 Th1 세포 면역활성이 증가시키는 것으로, 부작용이 없고, 안전하게 장기간 사용이 가능하여 아토피 피부염의 면역학적 이상을 근본적으로 치료할 수 있을 것으로 기대된다.In addition, the composition for improving or treating atopic dermatitis comprising a culture of peripheral blood mononuclear-derived activated lymphocytes and exosomes isolated therefrom according to the present invention increases Th1 cell immune activity, has no side effects, and can be safely used for a long period of time Therefore, it is expected to be able to fundamentally treat the immunological abnormalities of atopic dermatitis.
도 1은 EBI의 공배양을 이용한 hDPCs 성장 촉진 평가 결과를 나타낸 도이다.
도 2는 EBICM의 hDPCs 성장 촉진 평가 결과를 나타낸 도이다.
도 3은 EBICM을 이용한 인체 모낭조직 성장 평가 결과를 나타낸 도이다.
도 4는 EBICM에 포함된 hDPCs 성장 촉진 인자 탐색을 위한 antibody array 결과를 나타낸 도이다.
도 5는 EBICM에 포함된 hDPCs 성장 촉진 인자 탐색을 위한 antibody array 결과를 Dot blot으로 나타낸 도이다.
도 6은 본 발명의 EBI 배양물 투여에 따른 경피두께 감소를 확인한 것이다.
도 7은 본 발명의 EBI 배양물 투여에 따른 염증 반응 감소 효과를 확인한 것이다.
도 8은 본 발명의 EBI 배양물 투여에 따른 혈액 내 IgE 감소 효과를 확인한 것이다.
도 9는 본 발명의 EBI 배양물 투여에 따른 체중 변화를 확인한 것이다.
도 10은 본 발명의 EBI 배양물 투여에 따른 피부 조직 내 아토피 피부염 관련 사이토카인 및 가려움증(itching) 관련 유전자들의 변화를 확인한 것이다.
도 11 내지 14는 본 발명의 EBI 배양물 내 엑소좀의 피하주사 경로 및 복강 내 투여 경로에 따른 아토피 피부염 치료 효과를 비교한 것이다.
도 15는 HFF(human foreskin fibroblast)에 대비 EBI, EBI 배양물 내 엑소좀에서 발현이 증가하는 단백질체를 분석하여 면역활성 관련 단백질을 선별하는 과정의 순서도이다.
도 16은 HFF 대비 EBI 및 EBI 엑소좀에서 발현이 10배 이상 증가하는 441개 단백질 군 중 면역세포의 생물학적 기능과 연관된 67개 단백질을 나타낸다.
도 17은 Th1 세포, T 세포, NK 세포와 연관된 단백질 중 EBI 엑소좀에서만 Intensity가 확인되는 단백질을 나타낸다.
도 18은 선별된 12개의 단백질에 대한 HFF, EBI, EBI 엑소좀에서의 Intensity를 확인한 것이다.1 is a diagram showing the evaluation results of hDPCs growth promotion using EBI co-culture.
2 is a diagram showing the evaluation results of hDPCs growth promotion of EBICM.
3 is a diagram showing the results of human hair follicle tissue growth evaluation using EBICM.
4 is a diagram showing the results of the antibody array for the detection of hDPCs growth promoting factors included in EBICM.
5 is a diagram showing the results of the antibody array for the detection of hDPCs growth promoting factors included in EBICM as a dot blot.
Figure 6 confirms the decrease in the transdermal thickness according to the administration of the EBI culture of the present invention.
Figure 7 confirms the effect of reducing the inflammatory response according to the administration of the EBI culture of the present invention.
Figure 8 confirms the effect of reducing IgE in the blood according to the administration of the EBI culture of the present invention.
Figure 9 confirms the change in body weight according to the administration of the EBI culture of the present invention.
Figure 10 confirms the change of atopic dermatitis-related cytokines and itch-related genes in the skin tissue according to the administration of the EBI culture of the present invention.
11 to 14 compare the therapeutic effects of atopic dermatitis according to the subcutaneous injection route and the intraperitoneal route of exosomes in the EBI culture of the present invention.
15 is a flowchart of a process for selecting immune activity-related proteins by analyzing a protein whose expression is increased in exosomes in EBI and EBI culture compared to human foreskin fibroblast (HFF).
16 shows 67 proteins associated with the biological function of immune cells among 441 protein groups whose expression in EBI and EBI exosomes is increased 10-fold or more compared to HFF.
17 shows proteins whose intensity is confirmed only in EBI exosomes among proteins associated with Th1 cells, T cells, and NK cells.
Figure 18 is a confirmation of the intensity in the HFF, EBI, EBI exosomes for the selected 12 proteins.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 말초혈액단핵구 유래 활성화 림프구의 배양물을 포함하는 발모 촉진 또는 아토피 피부염 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for promoting hair growth or treating atopic dermatitis, comprising a culture of activated lymphocytes derived from peripheral blood mononuclear cells.
말초혈액단핵구 (Peripheral blood mononuclear cells, 말초혈액단핵세포, PBMCs)는 혈액 내 존재하는 구형 핵을 가진 세포를 의미하며, 이러한 말초혈액단핵구에는 B 세포, T 세포, 대식세포 (macrophage), 수지상 세포 (dendritic cell), 자연살해세포 (NK cell, natural killer cell) 등의 면역세포들이 포함되어 있다. 혈액에서 말초혈액단핵구를 분류하는 방법은 일반적으로 피콜 (Ficoll) 방법을 사용하는데, 피콜은 설탕과 에피클로로히드린을 서로 중합시킨 화합물로 일반적으로 약 40만 분자량을 사용한다. 피콜은 물에 녹이면 저점도에서 고밀도에 이르는 용액으로 변하기 때문에 세포, 바이러스 및 세포 소기관 등을 분리하기 위한 밀도기울기를 형성시키는 물질로 사용한다. 말초혈액단핵구의 경우에는 혈액 속에 포함되어 있는 적혈구, 과립성백혈구 (granulocytes) 및 죽은 세포에 비해 가볍고 혈장 (plasma)보다는 무거우므로 분리가 된다. Peripheral blood mononuclear cells (Peripheral blood mononuclear cells, PBMCs) refer to cells with a globular nucleus existing in the blood, and these peripheral blood mononuclear cells include B cells, T cells, macrophages, dendritic cells ( It contains immune cells such as dendritic cells) and natural killer cells (NK cells). The method of classifying peripheral blood mononuclear cells from blood generally uses the Ficoll method, which is a compound obtained by polymerizing sugar and epichlorohydrin, and generally uses a molecular weight of about 400,000. Ficoll is used as a material to form a density gradient to separate cells, viruses, and organelles, as it changes into a solution ranging from low viscosity to high density when dissolved in water. In the case of peripheral blood mononuclear cells, they are separated because they are lighter than red blood cells, granulocytes and dead cells contained in blood and heavier than plasma.
상기 말초혈액단핵구는 상기 조성물의 적용되는 대상 개체의 자가 혈액으로부터 분리되어 배양된 것일 수 있다. 자가 혈액으로부터 분리된 말초혈액단핵구를 이용할 경우 불필요한 자가면역반응이 배제되어 염증 등의 부작용 없이 효율적으로 발모 촉진 및 아토피 피부염 개선, 치료를 꾀할 수 있다.The peripheral blood mononuclear cells may be isolated and cultured from the autologous blood of the subject to which the composition is applied. In the case of using peripheral blood mononuclear cells isolated from autologous blood, unnecessary autoimmune reactions are excluded, and it is possible to efficiently promote hair growth and improve and treat atopic dermatitis without side effects such as inflammation.
상기 대상 개체는 현재 탈모가 진행되고 있거나, 탈모를 경험한 이력이 있는 동물이거나, 현재 아토피 피부염이 진행되고 있거나, 아토피 피부염을 경험한 이력이 있는 동물로서, 인간을 포함한 포유류일 수 있다.The subject subject may be an animal with a history of experiencing hair loss, or a history of hair loss, or an animal with a history of experiencing atopic dermatitis or atopic dermatitis, including humans.
상기 활성화 림프구는 자연살해세포(NK Cell), 자연살해 T세포(NKT Cell) 또는 T세포 등을 포함할 수 있으나, 이에 제한되는 것은 아니다.The activated lymphocytes may include, but are not limited to, natural killer cells (NK cells), natural killer T cells (NKT cells), or T cells.
자연살해세포는 림프구의 일종으로 특징적인 형태인 거대과립형 림프구(LGL, Large granular lymphocytes)로서, IL-2, IL-12, 인터페론(Interferon) 등의 사이토카인(cytokine)에 반응하며 이에 의해 역가(cytotoxicity), 분비성(secretory), 증식성(proliferative) 기능이 상승한다.Natural killer cells are large granular lymphocytes (LGL), a characteristic type of lymphocyte, and respond to cytokines such as IL-2, IL-12, and interferon, and thereby (cytotoxicity), secretory (secretory), proliferative (proliferative) functions are elevated.
자연살해 T세포는 내재면역의 한 수행원으로서 비교적 근래에 그 기능들이 밝혀지고 있는 T 세포의 한 종류이다. 이름에서도 알 수 있듯이 T 세포의 수용체와 자연살해세포 특이적인 세포표면마커(surface marker)를 발현하고 있다. 자연살해 T세포의 한가지 놀라운 특징은 활성 후 IL-4, IL-10, IL-13, IFN-γ, TNF-α등과 같은 여러 사이토카인을 매우 빠른 시간 안에 분비한다는 점이다. 이러한 특징은 자연살해 T세포가 적응면역반응에 커다란 영향을 끼칠 수 있음을 암시한다.Natural killer T cells are a type of T cell whose functions have been revealed relatively recently as an attendant of innate immunity. As the name suggests, it expresses T-cell receptors and natural killer cell-specific cell surface markers. One surprising feature of NK T cells is that they secrete several cytokines such as IL-4, IL-10, IL-13, IFN-γ, and TNF-α within a very short time after activation. These characteristics suggest that natural killer T cells may have a significant effect on the adaptive immune response.
T 세포는 세포 표면에 항원 수용체(T cell receptor; TCR)를 가지는 세포를 말한다. TCR은 α사슬과 β사슬의 이합체 CD3 항원과 이형 이합체를 형성하고 있다. T세포의 일부(말초혈 T세포의 5% 전후)는 αβ가 아닌, γ사슬과 δ사슬의 이합체로 되어있다. TCR은 CD3 항원 (γ, δ, ε, ζ, ζ또는η)과 복합체를 형성하고 있는데, CD3 항원은 TCR이 항원을 인식하면 그 신호를 세포 내에 전달하는 역할을 맡는다.T cells refer to cells having an antigen receptor (TCR) on the cell surface. TCR forms a heterodimer with the α-chain and β-chain dimer CD3 antigen. Some T cells (around 5% of peripheral blood T cells) are not αβ but a dimer of γ chain and δ chain. TCR forms a complex with CD3 antigen (γ, δ, ε, ζ, ζ or η), and CD3 antigen is responsible for transmitting a signal to the cell when the TCR recognizes the antigen.
본 발명의 배양물은 말초혈액단핵구를 배양한 배양 배지를 의미하는 것으로서, 상기 배양 배지에서 활성화 림프구를 포함하거나, 이를 분리해낸 것일 수도 있다. The culture of the present invention refers to a culture medium in which peripheral blood mononuclear cells are cultured, and may contain activated lymphocytes in the culture medium or may be isolated.
본 발명의 일 실시예에 있어서, 배양물은 활성화 림프구의 분비물질을 더 포함하는 것일 수도 있다.In one embodiment of the present invention, the culture may further include a secretory material of activated lymphocytes.
또한, 상기 배양물은 활성화 림프구 유래 엑소좀을 포함하는 것일 수 있다. 상기 엑소좀은 배양물을 원심분리하여 수득한 것일 수 있다. 상기 배양방법을 거친 세포로부터 분비된 엑소좀은 발모 촉진 또는 아토피 피부염 개선 또는 치료 효능이 우수하다.In addition, the culture may include activated lymphocyte-derived exosomes. The exosomes may be obtained by centrifuging the culture. The exosomes secreted from the cells through the culture method are excellent in promoting hair growth or improving or treating atopic dermatitis.
본 발명의 상기 활성화 림프구의 배양물은 말초혈액으로부터 분리된 단핵구를 활성화, 증식, 배양하여 얻어진 것일 수 있으며, 구체적으로는 말초혈액에서 분리된 단핵구를 항-CD3 항체, IL-2, IL-12, IL-18, FBS 또는 L-glutamine을 포함하는 배지에서 배양하여 얻어진 것일 수 있으나, 이에 제한되는 것은 아니다.The culture of activated lymphocytes of the present invention may be obtained by activating, proliferating, and culturing monocytes isolated from peripheral blood. Specifically, monocytes isolated from peripheral blood are treated with anti-CD3 antibody, IL-2, IL-12. , may be obtained by culturing in a medium containing IL-18, FBS or L-glutamine, but is not limited thereto.
본 발명의 일 실시예에 있어서, 활성화 림프구의 배양물은 인간의 말초혈액으로부터 단핵구 및 자가혈장을 각각 분리하여 수득하는 단계; 항-CD3 항체로 세포 배양용기를 코팅하는 단계; 및 상기 단핵구를 상기 배양용기에 접종하여 IL-2, IL-12 및 IL-18로 이루어진 군에서 선택되는 하나 이상을 포함하는 배지에서 배양하는 단계를 포함하는 배양방법으로 얻어질 수 있다.In one embodiment of the present invention, the culture of activated lymphocytes is obtained by separating monocytes and autologous plasma from human peripheral blood, respectively; coating the cell culture vessel with an anti-CD3 antibody; and inoculating the monocytes into the culture vessel and culturing in a medium containing at least one selected from the group consisting of IL-2, IL-12 and IL-18.
상기 말초혈액단핵구는 자가말초혈액으로부터 분리된 것일 수 있다.The peripheral blood mononuclear cells may be isolated from autologous peripheral blood.
필요에 따라 개체의 말초혈액을 원심분리하여 상층의 혈장층을 제거하여 단핵구층을 분리하여 배양할 수 있다.If necessary, the monocyte layer may be separated and cultured by centrifuging the individual's peripheral blood to remove the upper plasma layer.
본 발명의 활성화 림프구의 배양물은 개체의 말초혈액단핵구를 배양하여 얻을 수 있다. 보다 구체적으로 본 발명의 상기 활성화 림프구의 배양물은 말초혈액단핵구를 항-CD3 항체, IL-2, IL-12 및 IL-18로 이루어진 군에서 선택되는 하나 이상을 포함하는 세포 배양용 배지에서 배양하여 얻을 수 있다.The culture of activated lymphocytes of the present invention can be obtained by culturing peripheral blood mononuclear cells of an individual. More specifically, in the culture of the activated lymphocytes of the present invention, peripheral blood mononuclear cells are cultured in a cell culture medium comprising at least one selected from the group consisting of anti-CD3 antibody, IL-2, IL-12 and IL-18. can be obtained by
본 발명의 일 실시예에 있어서, 말초혈액단핵구의 배양 방법이 항 CD-3항체를 포함하는 경우 항-CD3 항체는 세포 배양용 배지에 코팅된 것일 수 있다. 본 발명의 일 실시예에 따라 항-CD3 항체가 세포 배양용 배지에 코팅된 경우, 상기 세포 배양용 배지는 IL-2, IL-12 및 IL-18로 이루어진 군에서 선택되는 하나 이상을 추가로 포함할 수 있다.In one embodiment of the present invention, when the method for culturing peripheral blood mononuclear cells includes an anti-CD-3 antibody, the anti-CD3 antibody may be coated on a cell culture medium. According to an embodiment of the present invention, when the anti-CD3 antibody is coated on a cell culture medium, the cell culture medium further comprises at least one selected from the group consisting of IL-2, IL-12 and IL-18. may include
본 발명의 일 실시예에 있어서, 상기 세포 배양용 배지는 항-CD3 항체, IL-2, IL-12 및 IL-18를 포함할 수 있다. 보다 구체적으로 항-CD3 항체는 세포 배양용 배지에 코팅되고, 상기 세포 배양용 배지에 IL-2, IL-12 및 IL-18가 추가로 포함될 수 있다.In one embodiment of the present invention, the cell culture medium may include an anti-CD3 antibody, IL-2, IL-12 and IL-18. More specifically, the anti-CD3 antibody is coated on a cell culture medium, and IL-2, IL-12 and IL-18 may be further included in the cell culture medium.
상기 항-CD3 항체는 T 세포 수용체(TCR)과 결합하여 항원인식복합체를 형성하는 분자군인 CD3 항원에 특이적으로 결합하는 항체로, CD3 분자는 TCR과 결합하여 항원인식신호를 세포 내에 전달하는 역할을 담당한다. 본 발명에서 사용 가능한 항-CD3 항체는 CD3에 결합하는 특성을 가지는 항체라면 제한 없이 이용 가능하다. 항-CD3 항체는 0.1~5 ㎍/㎖의 범위로 포함될 수 있으며, 바람직하게는 0.5~2 ㎍/㎖, 보다 바람직하게는 0.8~1.5 ㎍/㎖의 범위로 포함될 수 있으나, 이에 제한되는 것은 아니다.The anti-CD3 antibody is an antibody that specifically binds to the CD3 antigen, a group of molecules that bind to the T cell receptor (TCR) to form an antigen recognition complex. is responsible for The anti-CD3 antibody usable in the present invention can be used without limitation as long as it has a CD3 binding property. The anti-CD3 antibody may be included in the range of 0.1-5 μg/ml, preferably in the range of 0.5-2 μg/ml, more preferably in the range of 0.8-1.5 μg/ml, but is not limited thereto. .
인터루킨은 림프구나 단핵구 및 대식세포 등 면역담당 세포가 생산하는 단백질성 생물활성물질의 총칭으로 본 발명은 인터루킨류의 사이토카인으로서 IL-2, IL-12 및 IL-18로 이루어진 군에서 선택되는 하나 이상을 포함할 수 있다. IL-2는 100~2000 IU/㎖의 범위로 포함될 수 있으며, 바람직하게는 500~1500 IU/㎖, 보다 바람직하게는 800~1200 IU/㎖를 포함할 수 있으나, 이에 제한되는 것은 아니다. IL-12는 0.5~10 ng/㎖의 범위로 포함될 수 있으며, 바람직하게는 1~8 ng/㎖, 보다 바람직하게는 2~6 ng/㎖ 포함될 수 있으나, 이에 제한되는 것은 아니다. IL-18은 1~100 ng/㎖의 범위로 포함될 수 있으며, 바람직하게는 10~80 ng/㎖, 보다 바람직하게는 20~60 ng/㎖의 범위로 포함될 수 있으나, 이에 제한되는 것은 아니다. Interleukin is a generic term for proteinaceous biologically active substances produced by immune cells such as lymphocytes, monocytes and macrophages. may include more than one. IL-2 may be included in the range of 100-2000 IU/mL, preferably 500-1500 IU/mL, more preferably 800-1200 IU/mL, but is not limited thereto. IL-12 may be included in the range of 0.5-10 ng/mL, preferably 1-8 ng/mL, more preferably 2-6 ng/mL, but is not limited thereto. IL-18 may be included in the range of 1 ~ 100 ng / ㎖, preferably 10 ~ 80 ng / ㎖, more preferably may be included in the range of 20 ~ 60 ng / ㎖, but is not limited thereto.
본 발명에 있어서, 이용 가능한 인터루킨류는 이에 한정되는 것은 아니며, 본 발명에 속하는 기술분야에서 통상의 지식을 가진 자에게 다른 인터루킨류도 본 발명의 목적에 부합하는 한 제한 없이 이용 가능하다.In the present invention, the available interleukins are not limited thereto, and other interleukins can be used by those of ordinary skill in the art to which the present invention pertains without limitation as long as they meet the purpose of the present invention.
본 발명의 방법은 세포 배양용 배지에 FBS 또는 L-glutamine을 더 포함할 수 있다. 상기 FBS의 농도는 특별히 한정되지 않으며, 예를 들면, 총 배지 부피의 1~20%일 수 있고, 바람직하게는 8~15%일 수 있다. 상기 L-glutamine의 농도는 특별히 한정되지 않으며, 예를 들면 총 배지 부피의 0.1~5%일 수 있고, 바람직하게는 0.5~2%일 수 있다.The method of the present invention may further include FBS or L-glutamine in the cell culture medium. The concentration of the FBS is not particularly limited, and for example, may be 1 to 20% of the total medium volume, preferably 8 to 15%. The concentration of L-glutamine is not particularly limited, and may be, for example, 0.1 to 5% of the total medium volume, preferably 0.5 to 2%.
본 발명의 배지는 그 외 림프구 배양을 위해 통상적으로 사용되는 성분을 더 포함할 수 있다. 예를 들면, 글라이신(Glycine), L-아르기닌(L-Arginine), L-아스파라긴(L-Asparagine), L-아스파르트산(L-Aspartic acid), L-시스틴 2HCl(LCystine 2HCl), L-글루탐산(L-Glutamic Acid), L-히스티딘(L-Histidine), L-히드록시프롤린(L-Hydroxyproline), L-이소류신(LIsoleucine), L-류신(L-Leucine), L-라이신 하이드로클로라이드(L-Lysine hydrochloride), L-메티오닌(L-Methionine), L-페닐알라닌(L-Phenylalanine), L-프롤린(L-Proline), L-세린(L-Serine), L-트레오닌(L-Threonine), L-트립토판(LTryptophan), L-타이로신 이나트륨 2수화물(L-Tyrosine disodium salt dihydrate), L-발린(L-Valine), 비오틴(Biotin), 염화콜린(Choline chloride), D- 판토텐산칼슘(D-Calcium pantothenate), 엽산(Folic Acid), 나이아신아마이드(Niacinamide), 파라-아미노벤조산(Para-Aminobenzoic Acid), 피리독신 하이드로클로라이드(Pyridoxine hydrochloride), 리보플라빈(Riboflavin), 티아민 하이드로클로라이드(Thiamine hydrochloride), 비타민 B12(Vitamin B12), i-이노시톨(i-Inositol), 질산칼슘(Calcium nitrate), 황산마그네슘(Magnesium Sulfate), 염화칼륨(Potassium Chloride), 중탄산나트륨(Sodium Bicarbonate), 염화나트륨(Sodium Chloride), 제일인산나트륨 수화물(Sodium Phosphate dibasic anhydrous), D-글루코스(DGlucose), 글루타티온(Glutathione), HEPES, 페놀레드(Phenol Red) 등을 포함할 수 있으나, 이에 제한되는 것은 아니다.The medium of the present invention may further include components commonly used for culturing other lymphocytes. For example, Glycine, L-Arginine, L-Asparagine, L-Aspartic acid, L-Cystine 2HCl (LCystine 2HCl), L-Glutamic acid (L-Glutamic Acid), L-Histidine, L-Hydroxyproline, L-Isoleucine, L-Leucine, L-Lysine Hydrochloride (L -Lysine hydrochloride), L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine disodium salt dihydrate, L-Valine, Biotin, Choline chloride, D-Calcium Pantothenate (D) -Calcium pantothenate), Folic Acid, Niacinamide, Para-Aminobenzoic Acid, Pyridoxine hydrochloride, Riboflavin, Thiamine hydrochloride, Vitamins B12 (Vitamin B12), i-Inositol, Calcium nitrate, Magnesium Sulfate, Potassium Chloride, Sodium Bicarbonate, Sodium Chloride, Monobasic Phosphate sodium hydrate (Sodium Phosphate dibasic anhydrous), D-glucose (DGlucose), Glutathione (Glutathione), HEPES, Phenol Red (Phenol Red), etc. However, the present invention is not limited thereto.
또한, 여기에 혈청 또는 혈장과 림프구의 증식을 지지하는 추가의 증식인자를 첨가하여 배양한 것일 수도 있다. 배지에 첨가하는 혈청 또는 혈장의 종류는 특별히 한정되지 않아, 시판의 각종 동물 유래의 것을 사용할 수 있지만, 인간 유래로서 본인 유래의 것이 더욱 바람직하다. 예를 들어, 말초혈액단핵구로부터 림프구를 증식시키는 사이토카인의 조합이나, 림프구 증식을 자극하는 렉틴류 등을 첨가하는 등 당업자에게 알려져 있는 방법을 사용할 수 있다.In addition, it may be cultured by adding serum or plasma and an additional growth factor that supports the proliferation of lymphocytes. The type of serum or plasma to be added to the medium is not particularly limited, and commercially available ones derived from various animals can be used, but those derived from humans are more preferable. For example, methods known to those skilled in the art can be used, such as adding a combination of cytokines that proliferate lymphocytes from peripheral blood mononuclear cells, lectins that stimulate lymphocyte proliferation, and the like.
또한, 배지는 혈청 또는 혈장과 림프구의 증식을 지지하는 추가의 증식인자를 더 포함하는 것일 수 있고, 혈청 또는 혈장 자체를 포함하는 것일 수 있다. 배지에 첨가하는 혈청 또는 혈장의 종류는 특별히 한정되지 않아, 시판의 각종 동물 유래의 것을 사용할 수 있지만, 인간 유래로서 본인 유래의 것이 바람직하며, 예를 들면 human AB serum 또는 auto plasma일 수 있다.In addition, the medium may further include serum or plasma and an additional growth factor supporting the proliferation of lymphocytes, and may include serum or plasma itself. The type of serum or plasma added to the medium is not particularly limited, and various commercially available animal-derived ones can be used, but human-derived ones are preferred, and for example, human AB serum or auto plasma can be used.
필요에 따라 상기 배양은 여러 단계로 수행될 수 있다. 예를 들어 1단계 또는 2단계 이상으로 수행될 수 있다.If necessary, the culturing may be performed in several stages. For example, it may be performed in one step or two or more steps.
2단계 이상으로 수행되는 경우, 예를 들면 1단계에서 배양된 림프구를 분리해 내고, 2단계에서 이를 새로운 배지로 옮겨 배양할 수 있다. 2단계 이상으로 수행되는 경우, 1단계 배지는 항-CD3 항체, FBS, L-glutamine, IL-2, IL-12 및 IL-18을 포함하고, 2단계 배지는 FBS, L-glutamine, IL-2, IL-12 및 IL-18을 포함할 수 있다. 보다 구체적으로 1단계 배지는 항-CD3 항체 및 IL-2, IL-12, IL-18을 포함하고, 2단계 배지는 IL-2, IL-12, IL-18을 포함하되, 항-CD3 항체를 포함하지 않을 수 있다.When carried out in two or more steps, for example, the lymphocytes cultured in
항-CD3항체, FBS, L-glutamine, IL-2, IL-12 및 IL-18은 전술한 범위 내의 농도로 포함될 수 있으며, 앞서 예시한 배지를 사용할 수 있다.The anti-CD3 antibody, FBS, L-glutamine, IL-2, IL-12 and IL-18 may be included at a concentration within the above-mentioned range, and the medium exemplified above may be used.
배양은 일반적인 세포배양방법, 예를 들면 CO2 인큐베이터 내에서 행해질 수 있다. CO2 농도는 예를 들면 1 내지 10%, 구체적으로는 3 내지 7% 일 수 있고, 온도는 30 내지 40℃, 구체적으로 35 내지 38℃일 수 있으나, 이에 제한되는 것은 아니다.Cultivation can be carried out in a general cell culture method, for example, in a CO 2 incubator. CO 2 concentration may be, for example, 1 to 10%, specifically 3 to 7%, the temperature may be 30 to 40 ℃, specifically 35 to 38 ℃, but is not limited thereto.
배양은 림프구가 충분히 활성, 증식할 때까지 수행될 수 있으며 예를 들면 3일 내지 20일, 구체적으로 8일 내지 16일간 수행될 수 있으나, 이에 제한되는 것은 아니다.Cultivation may be performed until lymphocytes are sufficiently active and proliferate, for example, 3 to 20 days, specifically 8 to 16 days, but is not limited thereto.
배양 효율 개선을 위해, 배양 동안 세포 수 증가에 맞추어 배지를 추가해주는 것이 바람직하다. 배지 추가의 주기는 배양물의 열화 방지를 위해 예를 들면 1 내지 10일, 구체적으로 1 내지 7일에 1회씩 추가될 수 있으나, 이에 제한되는 것은 아니다.In order to improve the culture efficiency, it is preferable to add a medium according to the increase in the number of cells during culture. The cycle of adding the medium may be added, for example, once every 1 to 10 days, specifically 1 to 7 days, in order to prevent deterioration of the culture, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명에 따른 배양물의 발모 촉진 효과를 입증하기 위해, 인간 모유두 세포(hDPCs)등을 포함하는 배지를 형성하고, 이에 본 발명에 따른 배양물을 첨가하여 실험용 배양배지를 제조하였다. In one embodiment of the present invention, in order to prove the hair growth promoting effect of the culture according to the present invention, a medium containing human dermal papilla cells (hDPCs), etc. is formed, and the culture according to the present invention is added thereto to culture the experiment Medium was prepared.
본 발명의 일 실시예에 있어서, 실험용 배양배지 총 부피에 대해 활성화 림프구의 배양물은 1~20 % 포함된 것일 수 있고, 바람직하게는 2~15 % 포함된 것일 수 있다. 본 발명의 일 실시예에 따라 활성화 림프구의 배양배지가 5~10 부피% 포함된 경우, 모유두 세포가 상대적으로 높은 증식능을 나타낼 수 있다.In one embodiment of the present invention, the culture of activated lymphocytes may contain 1 to 20%, preferably 2 to 15%, with respect to the total volume of the experimental culture medium. According to an embodiment of the present invention, when the culture medium of activated lymphocytes is contained in 5 to 10% by volume, the dermal papilla cells may exhibit a relatively high proliferative capacity.
본 발명의 말초혈액단핵구 유래 활성화 림프구의 배양물은 모유두 세포에서 모발성장 관련 유전자의 발현을 증가시킬 수 있다. 상기 모발성장 관련 유전자는 ALPL, BMP4, CCND1, CTNNB1, SHH, SOX2, VEGFA, VCAN 및 HEY1으로 이루어진 군에서 선택되는 하나 이상을 포함할 수 있으나, 이에 제한되지 않는다.The culture of activated lymphocytes derived from peripheral blood mononuclear cells of the present invention can increase the expression of hair growth-related genes in dermal papilla cells. The hair growth-related gene may include one or more selected from the group consisting of ALPL, BMP4, CCND1, CTNNB1, SHH, SOX2, VEGFA, VCAN and HEY1, but is not limited thereto.
ALPL은 알칼리 포스파타아제(alkaline phosphatase) 를 코딩하는 유전자로, 알칼리 포스파타아제 활성은 제한된 간엽 조직과 상피 영역에서 발현하며, 그것의 위치 및 발현은 모낭주기 (hair cycle) 도중에 변화하게 된다. 모유두 세포에서의 활성은 초기 성장기에서 증가하고, 중기 성장기에서 최대 수준에 도달한다. 중기 성장기 이후 모유두 세포의 하단부 영역 (inferior segment)에서 수가 감소하며, 퇴행 기간 동안 낮은 수준으로 유지된다.ALPL is a gene encoding alkaline phosphatase. Alkaline phosphatase activity is expressed in restricted mesenchymal tissue and epithelial regions, and its location and expression are changed during the hair cycle. Activity in dermal papilla cells increases in the early anagen phase and reaches a maximum level in the metaphase. After the metaphase, the number decreases in the inferior segment of the dermal papilla cells and remains at a low level during the regression period.
BMP4은 β-catenin 타겟 유전자로, BMP의 발현은 모낭조직 형성에서 모구 형성과 모발주기 동안 β-catenin 발현에 영향을 미친다.BMP4 is a β-catenin target gene, and the expression of BMP affects β-catenin expression during hair follicle formation and hair cycle in hair follicle tissue formation.
CCDN1 및 CTNNB1은 β-catenin의 타겟 유전자로, 이 유전자에 의해 코딩 되는 단백질은 cyclin 계열에 속하며, 세포 주기 전반에 걸쳐 영향을 미친다.CCDN1 and CTNNB1 are target genes for β-catenin, and the proteins encoded by these genes belong to the cyclin family and have effects throughout the cell cycle.
SHH는 인체 모유두 세포에서 발현하며, 모유두 세포의 특성 유지 및 모낭조직 형성, 성장에 관여되어 있는 것으로 밝혀져 있다.SHH is expressed in human dermal papilla cells, and it has been found to be involved in maintaining the characteristics of dermal papilla cells and in the formation and growth of hair follicles.
SOX2는 중배엽줄기세포의 마커이며, 모낭조직 형성 (morphogenesis) 시에 발현이 증가한다.SOX2 is a marker of mesenchymal stem cells, and its expression is increased during morphogenesis.
VEGFA는 혈관 내피 성장 인자이며, 모낭성장 촉진에 긍정적으로 작용하고, 간엽조직-상피 영역 상호작용(mesenchymal-epithelial interaction)에 관여하는 것으로 밝혀져 있다.VEGFA is a vascular endothelial growth factor, and it has been shown to act positively in promoting hair follicle growth, and to be involved in mesenchymal-epithelial interaction.
VCAN은 모낭성장 촉진에 작용하고, 간엽조직-상피 영역 상호작용 (mesenchymal-epithelial interaction)에 관여하는 것으로 밝혀져 있다. 모유두 세포 전도능을 측정하는 마커로 사용되기도 한다.It has been found that VCAN acts to promote hair follicle growth and is involved in mesenchymal-epithelial interaction. It is also used as a marker to measure dermal papilla cell conductance.
HEY1은 β-catenin의 타겟 유전자로, 성장기 모낭조직의 모유두 세포에서 발현이 상승하는 것으로 밝혀져 있다.HEY1 is a target gene of β-catenin, and its expression has been found to be elevated in dermal papilla cells of hair follicle tissue during growth phase.
본 발명의 말초혈액단핵구 유래 활성화 림프구는 다양한 성장인자 및 사이토카인을 분비하여 발모 촉진 기능을 수행할 수 있다. 상기 성장인자 및 사이토카인은 GM-CSF, IGFBP-2, PDGF-AA, IL-5, IL-8, IL-13, IFN-gamma 및 RANTES로 이루어진 군에서 하나 이상 일 수 있으나, 이에 제한되지 않는다.The activated lymphocytes derived from peripheral blood mononuclear cells of the present invention can secrete various growth factors and cytokines to perform a hair growth promoting function. The growth factors and cytokines may be one or more from the group consisting of GM-CSF, IGFBP-2, PDGF-AA, IL-5, IL-8, IL-13, IFN-gamma and RANTES, but is not limited thereto. .
종래에는 탈모나 아토피 피부염의 개선 또는 치료를 위해서는 면역 반응을 억제하는 방식을 채택하여 왔다. 하지만, 오히려 본 발명은 면역활성 또는 Th1세포의 활성을 촉진하여 발모를 촉진하거나 아토피 피부염을 개선 또는 치료할 수 있음을 확인하고 안출된 것이다. 본 발명에 따른 활성화 림프구의 배양물은 면역활성 또는 Th1세포의 활성을 촉진할 수 있다. Conventionally, in order to improve or treat hair loss or atopic dermatitis, a method of suppressing an immune response has been adopted. However, rather, the present invention has been devised after confirming that it can promote immune activity or Th1 cell activity to promote hair growth or improve or treat atopic dermatitis. The culture of activated lymphocytes according to the present invention can promote immune activity or Th1 cell activity.
본 발명의 말초혈액단핵구 유래 활성화 림프구의 배양물 및 이로부터 분리된 엑소좀은 면역활성 또는 Th1세포의 활성 촉진에 관여하는 단백질을 포함할 수 있다. 상기 단백질은 IRF4(Interferon regulatory factor 4), CD226(Cluster of Differentiation 226), TMIGD2(Transmembrane and immunoglobulin domain-containing protein 2), HAVCR2(Hepatitis A virus cellular receptor 2), TRAT1(T-cell receptor-associated transmembrane adapter 1), SPN(Sialophorin), TRAF2(TNF receptor-associated factor 2), CCDC88B(Coiled-coil domain containing 88B), TYROBP(tyrosine kinase-binding protein), VAV1(Vav Guanine Nucleotide Exchange Factor 1), SLAMF6(SLAM family member 6), ZBTB16(Zinc finger and BTB domain-containing protein 16)로 이루어진 군에서 선택되는 하나 이상의 단백질을 포함할 수 있으나, 이에 제한되지 않는다. The culture of peripheral blood mononuclear-derived activated lymphocytes and the exosomes isolated therefrom of the present invention may contain proteins involved in promoting immune activity or activation of Th1 cells. The protein is Interferon regulatory factor 4 (IRF4), Cluster of Differentiation 226 (CD226), Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2), Hepatitis A virus cellular receptor 2 (HAVCR2), T-cell receptor-associated transmembrane (TRAT1). adapter 1), SPN (Sialophorin), TRAF2 (TNF receptor-associated factor 2), CCDC88B (Coiled-coil domain containing 88B), TYROBP (tyrosine kinase-binding protein), VAV1 (Vav Guanine Nucleotide Exchange Factor 1), SLAMF6 ( It may include one or more proteins selected from the group consisting of SLAM family member 6) and ZBTB16 (Zinc finger and BTB domain-containing protein 16), but is not limited thereto.
본 발명의 약학 조성물은 약학적으로 허용되는 담체를 더 포함하며, 이는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약학 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다. 본 발명의 약학 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 질병 증상의 정도, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 목적하는 치료에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. 한편, 본 발명의 약학 조성물의 투여량은 이에 한정되는 것이 아니며 1일 당 0.01-2000 mg/kg(체중)일 수 있다. The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier, which is commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil; The present invention is not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, age, weight, sex, disease severity, food, administration time, administration route, excretion rate, and response sensitivity of the patient. However, an ordinarily skilled physician can readily determine and prescribe a dosage effective for the desired treatment. On the other hand, the dosage of the pharmaceutical composition of the present invention is not limited thereto, and may be 0.01-2000 mg/kg (body weight) per day.
본 발명의 약학 조성물은 경구 또는 비경구로 투여할 수 있고, 비경구로 투여되는 경우, 정맥내 주입, 피하주입, 근육 주입, 복강 주입, 경피 투여 등으로 투여할 수 있다. 본 발명의 약학 조성물은 적용되는 질환의 종류에 따라, 투여경로가 결정되는 것이 바람직하다.The pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like. The pharmaceutical composition of the present invention is preferably administered according to the type of disease to be applied.
본 발명의 약학 조성물은 당해 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. Alternatively, it may be prepared by being introduced into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.
본 발명은 상기 말초혈액단핵구 유래 활성화 림프구의 배양물을 포함하는 발모 촉진 또는 아토피 피부염 개선용 화장료 조성물을 제공한다.The present invention provides a cosmetic composition for promoting hair growth or improving atopic dermatitis, comprising the culture of activated lymphocytes derived from peripheral blood mononuclear cells.
상기 배양물에 관하여는 전술한 바와 같다.The culture is the same as described above.
상기 화장료 조성물은 유연화장수, 수렴화장수, 영양화장수, 영양크림, 마사지크림, 에센스, 아이크림, 아이에센스, 클렌징크림, 클렌징폼, 클렌징 워터, 팩, 파우더, 바디로션, 바디크림, 바디오일, 바디에센스, 메이크업 베이스, 파운데이션, 염모제, 샴푸, 린스 또는 바디 세정제 등으로 제형화 될 수 있으나, 이에 한정되는 것은 아니다.The cosmetic composition is a softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil, body It may be formulated as an essence, makeup base, foundation, hair dye, shampoo, conditioner or body cleaner, but is not limited thereto.
화장료 조성물로 사용시, 피부 외용제 또는 화장료의 제형에 맞게 물질을 더 첨가할 수 있다. 예를 들어, 이에 한정되는 것은 아니나 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있고, 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있으며, 특히, 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다. 또한 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되는데, 바람직하게는 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르일 수 있으나 이에 한정되는 것은 아니다. 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소 결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있으며, 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있으나 이에 한정되는 것은 아니다.When used as a cosmetic composition, a substance may be further added according to the formulation of an external preparation for skin or cosmetics. For example, but not limited thereto, when the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or Zinc oxide and the like may be used, and when the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally propellants such as chlorofluorohydrocarbons, propane/butane or dimethyl ether. In addition, when the formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, preferably water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-Butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan may be used, but is not limited thereto. When the formulation is a suspension, as a carrier component, water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, Aluminum metahydroxide, bentonite, agar or tracanth may be used, and when the formulation is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivatives, methyltaurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester may be, but is not limited thereto.
본 발명에서 사용되는 "발모 촉진" 및 "탈모 방지"는 당업계에서 이용되는 다른 용어 양모 또는 육모 촉진을 포함하는 의미이다.As used herein, "promoting hair growth" and "prevention of hair loss" are other terms used in the art to include other terms used in the art to promote hair growth or hair growth.
본 발명의 조성물이 발모 촉진 용도로 사용되는 경우, 적용될 수 있는 부위는 두피뿐만 아니라 발모를 필요로 하는 신체 부위라면 어디나 적용할 수 있다. 예를 들면, 외상으로 인한 흉터로 모발 또는 털이 손상된 부위 또는 단순 미용효과를 목적으로 하는 넓은 이마 또는 M형 이마, 속눈썹 또는 눈썹 및 무모증의 상태 호전에도 사용할 수 있다.When the composition of the present invention is used for promoting hair growth, it can be applied to any part of the body that requires hair growth as well as the scalp. For example, it can be used to improve the condition of hair or hair damaged by trauma or a wide forehead or M-shaped forehead, eyelashes or eyebrows for the purpose of simple cosmetic effect, and alopecia.
또한, 본 발명의 조성물이 아토피 피부염 개선 또는 치료용도로 사용되는 경우, 아토피 피부염이 발병된 환부라면 어디나 적용될 수 있으며, 예를 들어, 아토피 피부염으로 인해 수포가 올라온 부위, 아토피 피부염으로 인한 흉터로 손상된 피부 부위의 상태 호전에도 사용할 수 있다. In addition, when the composition of the present invention is used for improving or treating atopic dermatitis, it can be applied to any affected area where atopic dermatitis has occurred, for example, a site where blisters have risen due to atopic dermatitis, damaged by scars due to atopic dermatitis It can also be used to improve the condition of the skin area.
상술한 바와 같이, 본 발명에 따른 말초혈액단핵구 유래 활성화 림프구의 배양물 및 이로부터 분리된 엑소좀은 발모를 촉진하거나 탈모를 예방 내지 치료하고, 아토피 피부염을 개선하거나 치료할 수 있는 의약(외)품 개발의 원료로 유용하게 사용될 수 있다.As described above, the culture of peripheral blood mononuclear cell-derived activated lymphocytes and the exosomes isolated therefrom according to the present invention promote hair growth, prevent or treat hair loss, and improve or treat atopic dermatitis. It can be usefully used as a raw material for development.
이하, 본 발명을 구체적으로 설명하기 위해 실시예를 들어 상세하게 설명하기로 한다.Hereinafter, examples will be given to describe the present invention in detail.
실험물질test substance
1. 체외증강 면역세포(EBI) 및 이를 배양한 배지의 준비1. Preparation of in vitro enhanced immune cells (EBI) and cultured medium
1-1. 단핵구 세포의 분리1-1. Isolation of monocytes
혈액이 담긴 채혈관을 2000 rpm, 20℃에서 5분간 원심분리 하였다. 혈장과 단핵구층을 각각 새로운 50㎖ 튜브에 수집하였다. 혈장은 56℃ 수조에서 30분간 보존하여 불활성화 처리를 한 뒤, 2000 rpm, 20℃에서 5분간 원심 분리한 후, 상층액만 수집하여 이후 배양에 사용하였다.The blood collection vessel was centrifuged at 2000 rpm and 20° C. for 5 minutes. Plasma and monocyte layers were each collected in a new 50 ml tube. Plasma was preserved in a water bath at 56°C for 30 minutes, inactivated, and centrifuged at 2000 rpm and 20°C for 5 minutes, and only the supernatant was collected and used for subsequent culture.
단핵구층은 ALyS505N-0 배지를 동일 양으로 가하여 희석시킨 후, 피콜이 담긴 새로운 15㎖ 튜브에 배지와 희석된 혈구를 피콜과 섞이지 않도록 천천히 올려주었다. 그 후 400 x g, 20℃에서 30분간 원심 분리하여 중간의 PBMC 층은 새로운 50㎖ 튜브에 수집하였다. 수집한 PBMC 양의 3배 가 되게 멸균생리식염수를 채운 후, 2000 rpm, 20℃에서 5분간 원심분리하여 상층액을 제거하였다. 멸균생리식염수로 가라앉은 펠렛을 재부유시키고 동일 조건으로 원심 분리하여 상층액을 제거하였다. 그 후, RBC lysis 버퍼로 부유시킨 후, 37℃ 수조에서 10분간 보존하였다. 멸균생리식염수를 이용하여 동일한 세포 세척과정을 1회 더 실시한 후, ALyS505N-0 배지에 부유시켜 배양을 위한 초기 세포로 사용하였다.The mononuclear layer was diluted by adding the same amount of ALyS505N-0 medium, and then slowly placed in a new 15ml tube containing Ficoll so that the medium and diluted blood cells were not mixed with Ficoll. After that, centrifugation was performed at 400 x g, 20 °C for 30 minutes, and the intermediate PBMC layer was collected in a new 50 ml tube. After filling with sterile physiological saline to 3 times the amount of collected PBMC, centrifugation was performed at 2000 rpm and 20° C. for 5 minutes to remove the supernatant. The pellet submerged in sterile physiological saline was resuspended and centrifuged under the same conditions to remove the supernatant. Thereafter, after floating in RBC lysis buffer, it was stored in a water bath at 37° C. for 10 minutes. After performing the same cell washing process once more using sterile physiological saline, the cells were suspended in ALyS505N-0 medium and used as initial cells for culture.
1-2. CD3 활성화 유도1-2. CD3 activation induction
T25 플라스크에 1X HBSS 에 용해된 CD3 항체 1 ㎍/㎖을 가하고, 4℃에서 하룻밤 동안 인큐베이션하여 바닥면에 CD3 코팅층을 형성시켰다. CD3 코팅된 T25 플라스크에 PBMC 1x107 개, FBS (10%), Lglutamine(1%), IL-2 (1,000IU/㎖), IL-12 (3ng/㎖), IL-18(30ng/㎖)를 첨가하고, ALyS505N-0 배지를 총 5 ㎖이 되도록 넣어준 후, 37℃, 5% CO2 조건으로 40분 동안 배양하였다. 배양이 끝난 후 셀 스크래퍼로 플라스크 바닥을 긁어내어 새로운 T25 플라스크에 옮기고 이후 배양에 사용하였다.1 μg/ml of CD3 antibody dissolved in 1X HBSS was added to a T25 flask, and incubated at 4° C. overnight to form a CD3 coating layer on the bottom surface. In CD3-coated T25 flasks, 7 PBMCs 1x10, FBS (10%), Lglutamine (1%), IL-2 (1,000IU/ml), IL-12 (3ng/ml), IL-18 (30ng/ml) was added, and ALyS505N-0 medium was added to make a total of 5 ml, and then incubated for 40 minutes at 37° C. and 5% CO 2 conditions. After incubation, the bottom of the flask was scraped with a cell scraper, transferred to a new T25 flask, and used for subsequent culture.
1-3. 말초혈액단핵구의 배양1-3. Culture of peripheral blood mononuclear cells
플라스크 배양flask culture
체외증강 면역세포 (Ex-vivo Boosted Immune cell for Human, EBI)의 배양에는 플라스크 (flask) 및 백 (bag)을 사용하였다. 플라스크를 이용한 배양 시, 상기 실시예 1-2에서 제조한 T25 플라스크에서 PBMC 배양 3일 째에 FBS (10%), L-glutamine (1%), IL-2 (1,000 IU/㎖), IL-12 (3ng/㎖), IL-18 (30ng/㎖) 및 ALyS505N-0 배지를 첨가하였다(총 부피 10㎖). 이 후, 1 내지 2일 마다 세포 성장속도에 따라 동량 혹은 2 내지 3배의 배양물을 첨가해주었으며, 이 때 사용하는 배양물은 FBS (10%), L-glutamine (1%), IL-2 (1000 IU/㎖), IL-12/IL-18이 첨가된 ALyS505N-0 배지를 사용하였다Flasks and bags were used for culturing Ex-vivo Boosted Immune cells for Human (EBI). When cultured using a flask, on the 3rd day of PBMC culture in the T25 flask prepared in Example 1-2, FBS (10%), L-glutamine (1%), IL-2 (1,000 IU/ml), IL- 12 (3ng/ml), IL-18 (30ng/ml) and ALyS505N-0 medium were added (total volume 10ml). After that, the same amount or 2 to 3 times the culture was added according to the cell growth rate every 1 to 2 days, and the culture used at this time was FBS (10%), L-glutamine (1%), IL- 2 (1000 IU/ml), ALyS505N-0 medium supplemented with IL-12/IL-18 was used.
백 배양back culture
배양 8일째, ALyS505N-0 1L가 들어 있는 백에 IL-2 (1,000 IU/㎖)을 넣고 배양물과 잘 섞이도록 마사지 해준 후, 백의 1/3 지점을 클램프로 고정한 후, T175 플라스크에서 긁어낸 세포들을 백으로 옮겨 주었다. 배양 10일째, 세포 클러스터 (cluster)가 풀어지도록 백을 마사지해준 후 백의 2/3 지점을 클램프로 고정하고 1/3 지점의 클램프를 제거한 뒤, 배지와 잘 섞이도록 마사지해주었다. 배양 12일째, 배양 10일 째와 동일한 방법으로 진행하였다.On the 8th day of culture, put IL-2 (1,000 IU/ml) in a bag containing 1L of ALyS505N-0, massage it to mix well with the culture,
실험방법Experimental method
1. 인간모유두 세포(hDPCs: human dermal papilla cells)의 배양1. Culture of human dermal papilla cells (hDPCs)
인간 모유두 세포 (hDPCs: human dermal papilla cells)는 primary 세포로서 Cefobio (Seoul, Korea)에서 구입하여, 10% Fetal Bovine Serum (FBS, 소 태아 혈청)과 1 % penicillin이 포함된 Dulbecco’s modified Eagle’s medium (DMEM)에서 배양하였으며, passage 3 또는 4의 세포를 사용하였다.Human dermal papilla cells (hDPCs) were purchased from Cefobio (Seoul, Korea) as primary cells, and Dulbecco's modified Eagle's medium (DMEM) containing 10% Fetal Bovine Serum (FBS, fetal bovine serum) and 1% penicillin. ), and cells from
2. 세포 증식 분석2. Cell proliferation assay
세포를 6-well plate에 1 x 105 cells/well의 밀도로 배양하고 각 테스트 조건 하에서 120 시간 동안 연속적으로 배양하였다. EBI 또는 배양배지 (Conditioned media, CM)와 동시 배양 후, WST-8 분석 (Dojindo; Rockville, MD, USA)을 사용하여 세포 증식능력을 측정하였다. WST-8 용액 (100 μL)을 DMEM 1 ㎖에 세포에 첨가하고 37 ℃에서 1시간 동안 배양하였다. SpectraMax 190 마이크로 플레이트 판독기 (Molecular Devices, Sunnyvale, CA, USA)를 사용하여 450 nm에서 흡광도를 측정하였다.Cells were cultured in a 6-well plate at a density of 1 x 10 5 cells/well and continuously cultured for 120 hours under each test condition. After co-culture with EBI or culture medium (Conditioned media, CM), cell proliferation ability was measured using WST-8 assay (Dojindo; Rockville, MD, USA). WST-8 solution (100 μL) was added to the cells in 1 ml of DMEM and incubated at 37° C. for 1 hour. Absorbance was measured at 450 nm using a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
3. Alkaline phosphatase (ALP) 활성측정3. Alkaline phosphatase (ALP) activity measurement
hDPCs를 6-well plate에 1 x 105 cells/well의 밀도로 배양하고 각 테스트 조건 하에서 120 시간 동안 연속적으로 배양하였다. CM을 농도 별로 72 시간 동안 동시 배양 후, hDPCs의 alkaline phosphatase 활성을 ALP assay kit을 사용하여 측정하였다. 1Xphosphate-buffered saline (PBS)로 세척한 후, 세포를 1% (v/v) Triton X-100 (Sigma) 및 2 mM MgSO4 를 함유하는 0.1 M NaNO3-Na2CO3 완충액에서 용해시켰다. 이어서 6mM p-nitrophenyl phosphate를 각 well에 첨가하고 37 ℃에서 30분 동안 배양하였다. 마지막으로, 1.5 M NaOH (Sigma)를 첨가하여 enzyme-substrate 반응을 정지시켰다. SpectraMax 190 마이크로 플레이트 판독기 (Molecular Devices, Sunnyvale, CA, USA)를 사용하여 405 nm의 파장에서 광학 밀도 (OD) 판독을 수행하였다.hDPCs were cultured in a 6-well plate at a density of 1 x 10 5 cells/well and continuously cultured for 120 hours under each test condition. After co-culture of CMs for 72 hours at each concentration, alkaline phosphatase activity of hDPCs was measured using an ALP assay kit. After washing with 1Xphosphate-buffered saline (PBS), cells were lysed in 0.1 M NaNO3-Na2CO3 buffer containing 1% (v/v) Triton X-100 (Sigma) and 2 mM MgSO4. Then, 6mM p-nitrophenyl phosphate was added to each well and incubated at 37°C for 30 minutes. Finally, 1.5 M NaOH (Sigma) was added to stop the enzyme-substrate reaction. Optical density (OD) readings were performed at a wavelength of 405 nm using a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
4. 모낭 조직 배양법 (organ culture)4. Hair follicle tissue culture method (organ culture)
인체에서 분리된 모낭조직은 주변 구성물을 모두 분리하여 Earle’s balanced salts solution (EBSS; Sigma)에 보관하였다. Anagen phase follicle은 주의하여 손상을 주지 않게 stereomicroscope (Olympus, Tokyo, Japan)를 사용하여 분리하였다. 30개 이상의 조직을 분리하여 실험에 사용하였다. 분리된 모낭을 Williams medium E (Gibco, Grand Island, NY, USA)에 2 mM L-glutamine (Gibco, NY, USA), 10 ㎍/㎖ insulin (Sigma, St. Louis MO, USA), 50 nM hydrocortisone (Sigma, St. Louis MO, USA), 100 unit/㎖ penicillin, 100 ㎍/㎖ streptomycin를 첨가하여 37℃, 5% CO2 95% air의 조건에서 배양하였다. Minoxidil (10 ㎍/㎖, MNX, Sigma, St. Louis MO, USA) 를 배양 시스템에서의 양성 대조군으로 사용하였다. DP controller (Olympus, Tokyo, Japan)를 이용하여 길이를 측정하여 분석하였다.Hair follicle tissue isolated from the human body was stored in Earle's balanced salts solution (EBSS; Sigma) after all surrounding components were separated. Anagen phase follicles were separated using a stereomicroscope (Olympus, Tokyo, Japan) carefully to avoid damage. More than 30 tissues were isolated and used for experiments. The isolated hair follicles were placed in Williams medium E (Gibco, Grand Island, NY, USA) with 2 mM L-glutamine (Gibco, NY, USA), 10 μg/ml insulin (Sigma, St. Louis MO, USA), 50 nM hydrocortisone. (Sigma, St. Louis MO, USA), 100 unit/ml penicillin, and 100 μg/ml streptomycin were added and cultured at 37° C., 5% CO 2 95% air. Minoxidil (10 μg/ml, MNX, Sigma, St. Louis MO, USA) was used as a positive control in the culture system. The length was measured and analyzed using a DP controller (Olympus, Tokyo, Japan).
5. 단백질 발현 분석 (Western blot analysis)5. Protein expression analysis (Western blot analysis)
세포를 50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% 데옥시콜릭산 (deoxycholic acid), 1 mM PMSF를 포함하는 RIPA 완충액으로 용해하고 아이스에서 15분 동안 프로테아제 억제제 (Roche Applied Science, Indianapolis, IN)를 섞어서 4 ℃에서 10분 동안 20,000×g로 원심 분리하였다. 상층액을 10분 동안 재-원심 분리하였고, BCA protein assay kit (Bio-Rad, Hercules, CA, USA)를 사용하여 단백질 농도를 측정하였다. 단백질 (20 ㎍)을 SDS-PAGE (polyacrylamide gel electrophoresis)로 분리하였으며, PVDF (polyvinylidene fluoride) 멤브레인 (Millipore Corp, Bedford, MA)으로 흡착시켰다. 멤브레인을 실온에서 2시간 동안 0.1% Tween-20 및 5%의 skim milk (non-fat milk)가 포함된 PBS에서 반응시키고, 1시간 동안 0.2% BSA (in PBS)로 희석된 1차 항체를 사용하여 배양하였다. 3회 세척한 후, 멤브레인을 2차 HRP가 부착된 anti-rabbit 또는 HRP가 부착된 2차 항체 (Amersham, Arlington Heights, IL)로 반응하고, 상기 밴드를 ECL Advance kit (Amersham)로 시각화 하였다. 로딩 대조군 (β-actin) 발현양을 이용하여 표준화하였다.Cells were lysed with RIPA buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% deoxycholic acid, 1 mM PMSF and placed on ice for 15 min. The mixture was mixed with a protease inhibitor (Roche Applied Science, Indianapolis, IN) and centrifuged at 20,000×g for 10 minutes at 4°C. The supernatant was re-centrifuged for 10 minutes, and the protein concentration was measured using a BCA protein assay kit (Bio-Rad, Hercules, CA, USA). Proteins (20 μg) were separated by SDS-PAGE (polyacrylamide gel electrophoresis), and adsorbed on a polyvinylidene fluoride (PVDF) membrane (Millipore Corp, Bedford, Mass.). The membrane was reacted in PBS containing 0.1% Tween-20 and 5% skim milk (non-fat milk) for 2 hours at room temperature, and primary antibody diluted with 0.2% BSA (in PBS) for 1 hour was used. and cultured. After washing three times, the membrane was reacted with a secondary HRP-attached anti-rabbit or HRP-attached secondary antibody (Amersham, Arlington Heights, IL), and the band was visualized with an ECL Advance kit (Amersham). It was normalized using the loading control (β-actin) expression level.
6. 역전사 양적 중합 효소 연쇄 반응 (qRT-PCR)6. Reverse Transcription Quantitative Polymerase Chain Reaction (qRT-PCR)
총 RNA는 TRIzol (Invitrogen, CA, USA)을 사용하여 추출하였다. 전체 RNA 주형으로부터의 제 1 가닥 cDNA 합성은 PrimeScriptTM RT 마스터 믹스 (Takara, Tokyo, Japan)로 수행하였다. 얻어진 cDNA를 qPCR 2X PreMIX SYBR (Enzynomics, Seoul, Korea) 및 CFX-96 thermocycler (Bio-Rad, Hercules, CA, USA)를 사용하여 RT- PCR에 적용하였다. 모든 유전자를 증폭하는 데 사용된 PCR 조건은 95 ℃에서 10분, 10초 동안 95 ℃, 15초 동안 60 ℃, 20초 동안 72 ℃의 40주기였다. 발현 데이터는 정량화 방법인 ΔCt를 사용하여 사이클 임계치 (Ct) 값으로 계산하였다. GAPDH는 정상화에 사용되었다.Total RNA was extracted using TRIzol (Invitrogen, CA, USA). First strand cDNA synthesis from total RNA template was performed with PrimeScript™ RT Master Mix (Takara, Tokyo, Japan). The obtained cDNA was subjected to RT-PCR using qPCR 2X PreMIX SYBR (Enzynomics, Seoul, Korea) and CFX-96 thermocycler (Bio-Rad, Hercules, CA, USA). The PCR conditions used to amplify all genes were 40 cycles of 95 °C for 10 min, 95 °C for 10 sec, 60 °C for 15 sec, and 72 °C for 20 sec. Expression data were calculated as cycle threshold (Ct) values using the quantification method, ΔCt. GAPDH was used for normalization.
7. Ex-vivo boosted immune cell conditioned media (EBICM) 포함물질 정성평가7. Qualitative evaluation of substances containing ex-vivo boosted immune cell conditioned media (EBICM)
EBICM에 포함된 다양한 분비인자를 분석을 위해 Ray biotech cytokine array kit(Human Growth Factor Array C1, Human Cytokine Array C1)을 이용하여 실험을 수행하였다. Cytokine array 실험을 위해 kit에서 제공되는 cytokine membrane은 실온에서 1시간 동안 blocking 한 후 Con sup과 EBICM 1 ㎖을 각각 loading한 후 4 ℃에서 하루 동안 반응한다. 다음 날 membrane은 washing 과정을 거친 후 kit에서 제공되는 primary antibody를 4 ℃에서 하루 동안 반응한 후 다음 날 secondary antibody를 상온에서 1시간 동안 반응한 후 ECL kit를 이용하여 실험 결과를 확인하였다.Experiments were performed using Ray biotech cytokine array kit (Human Growth Factor Array C1, Human Cytokine Array C1) to analyze various secretory factors included in EBICM. For the cytokine array experiment, the cytokine membrane provided in the kit is blocked at room temperature for 1 hour, and after loading 1 ml of Consup and EBICM, respectively, react at 4 ° C for one day. The next day, after the membrane was washed, the primary antibody provided in the kit was reacted at 4 °C for one day, and the next day, the secondary antibody was reacted at room temperature for 1 hour, and then the experimental results were confirmed using the ECL kit.
8. 전사체 시퀀싱 (RNA Sequencing)8. Transcript Sequencing (RNA Sequencing)
전사체 시퀀싱은 전사체의 발현을 측정하는 매우 예민하고 정확한 방법이다. 전사체 시퀀싱은 샘플 내에 존재하는 모든 전사체 (RNA)에 대한 종합적인 지식을 제공해줄 뿐만 아니라 풍부한 각각의 전사체를 수량화 한다. 게다가, 이중가닥 전사체 (Stranded RNA) 시퀀싱은 연구자들이 total RNA, mRNA, and miRNA 염기서열과 상보적인 염기서열 (Sense vs anti-sense)의 방향성 정보를 발견하도록 한다. 전사체의 발현을 측정하는 매우 예민하고 정확한 수단은 치료법에 대응하여 다른 환경적인 조건과 다른 연구설계의 넓은 범위 하에 질병 상태에서 나타나는 이전에 발견되지 않은 변화들에 대한 질병가시성을 제공한다. RNA-Seq 는 ‘테라젠이텍스’사에 의뢰하여 실시하였다. 12시간 배양된 각각 샘플에서 RNA 를 추출하여 실험을 실시하였다.Transcriptome sequencing is a very sensitive and accurate method for measuring transcript expression. Transcript sequencing not only provides a comprehensive knowledge of all transcripts (RNA) present in a sample, but also quantifies the abundance of each transcript. In addition, stranded RNA sequencing allows researchers to discover directional information on total RNA, mRNA, and miRNA sequences and complementary sequences (sense vs anti-sense). A highly sensitive and accurate means of measuring transcript expression provides disease visibility into previously undiscovered changes in disease state under different environmental conditions and a wide range of different study designs in response to therapy. RNA-Seq was performed by requesting ‘Theragen Etex’. An experiment was performed by extracting RNA from each sample incubated for 12 hours.
9. EBI 배양물의 엑소좀(exosome)단백질체 분석9. Analysis of exosome proteomic bodies of EBI culture
Human foreskin fibroblast (HFF)대조군 세포 및 EBI 활성면역세포를 exosome-depleted plasma가 포함된 배지에서 배양한 후 배양액을 수집하여 3,000 rpm, 5분 동안 원심분리하여 세포 찌꺼기를 제거하였다. 100,0000 rpm, 1시간 30분 조건으로 초원심분리기를 통해 바닥에 가라앉은 엑소좀을 얻고 PBS용매로 재현탁하여 엑소좀을 분리한다. 단백질체 분석을 Cell pellet 또는 생체 시료를 고농도 SDS 버퍼에서 용해하였다. 100-200 ㎍의 단백질을 Microcon filter에 넣은 후 SDS를 제거하기 위해 원심분리를 이용하여 8M UREA 버퍼를 통해 버퍼교환을 진행하였다. urea를 제거하기 위해, ABC 버퍼로 여러번 버퍼교환을 진행하였다. 트립신을 1:100 의 비율로 첨가하여 트립신화를 밤새 수행하고 Tryptic peptide를 용출하였다. 추가로 ABC 및 NaCl을 첨가하여 최종 용출을 하였다. 정량의 경우 시료를 트립신을 통해 단백질가수분해 시킨 후에 펩타이드들을 Triple Quadrupole LC-MS/MS 를 사용하여 MRM 분석을 진행하였다. After culturing human foreskin fibroblast (HFF) control cells and EBI-activated immune cells in a medium containing exosome-depleted plasma, the culture medium was collected and centrifuged at 3,000 rpm for 5 minutes to remove cell debris. The exosomes that have sunk to the bottom are obtained through an ultracentrifuge at 100,0000 rpm for 1 hour and 30 minutes, and the exosomes are separated by resuspending in PBS solvent. For proteomic analysis, cell pellets or biological samples were dissolved in high concentration SDS buffer. After 100-200 μg of protein was put into a microcon filter, the buffer was exchanged through 8M UREA buffer using centrifugation to remove SDS. To remove urea, buffer exchange was performed several times with ABC buffer. Trypsinization was performed overnight by adding trypsin in a ratio of 1:100, and Tryptic peptide was eluted. Further, ABC and NaCl were added for final elution. For quantification, after proteolysis of the sample through trypsin, the peptides were analyzed for MRM using triple quadrupole LC-MS/MS.
10. 아토피성 피부염 오발부민(OVA) 감작 아토피 마우스 모델10. Atopic Dermatitis Ovalbumin (OVA) Sensitization Atopic Mouse Model
7주령의 암컷 Balb/c 마우스에 ovalbumin (OVA, grade V, Sigma, St. Louis, MO, USA) 20 ㎍과 immunologic adjuvant인 알루미늄수산화물 aluminum hydroxide (Alum, Sigma) 1 mg을 saline에 희석하여 일주일에 한번씩 2주 간 복강에 투여하여 아토피피부염 감작을 유도하였다. OVA 복강투여 2주 후 OVA 100 ㎍을 제모한 등 피부에 1 × 1 cm patch로 1주 감작 후 2주 휴식을 3회 반복시켜 국소부위에 피부염을 유도하였다. 각 실험이 끝난 후 혈액을 채혈하고, 등 피부를 적출하였다.To 7-week-old female Balb/c mice, 20 μg of ovalbumin (OVA, grade V, Sigma, St. Louis, MO, USA) and 1 mg of aluminum hydroxide (Alum, Sigma), an immunologic adjuvant, were diluted in saline for a week. Once administered intraperitoneally for 2 weeks, atopic dermatitis sensitization was induced. After 2 weeks of intraperitoneal administration of OVA, 100 μg of OVA was removed and sensitized with a 1 × 1 cm patch on the back skin for 1 week, followed by a 2
11. 통계 처리11. Statistical processing
시험 결과의 분석은 대조군과 시험군의 비교는 통계프로그램인 SPSS로 Student’s t-test 방법을 통해 p 값을 측정하고 p < 0.05 인 경우에 각 군 간에 유의한 차이가 있는 것으로 판정하였다. (p < 0.05: *; p < 0.01: **; p < 0.001: ***)For the analysis of the test results, the comparison of the control group and the test group measured the p value through Student's t-test method with SPSS, a statistical program, and when p < 0.05, it was determined that there was a significant difference between each group. (p < 0.05: *; p < 0.01: **; p < 0.001: ***)
실험결과Experiment result
1. EBI의 공배양을 이용한 hDPCs 성장 촉진 평가1. Evaluation of hDPCs growth promotion using co-culture of EBI
1-1. WST-8 assay, 농도 별 세포 공배양 이후 세포 생존율1-1. WST-8 assay, cell viability after cell co-culture by concentration
총 3종 (Batch 1-3) 농도별 EBI (T1, 1 x 104 cells; T2, 2 x 104 cells; T3, 3 x104 cells; T4, 1 x 105 cells; T5, 1 x 106 cells)와 공 배양 48 시간 이후에 평가한 결과, 각 배치 별 일정 농도에서 (Batch 1, T5; Batch 2, T3~T5; Batch 3, T2~T5) hDPCs의 성장을 촉진하는 것으로 관찰되었으며, 통계적으로 유의하였다. (도 1A)EBI by concentration of 3 types (Batch 1-3) (T1, 1 x 10 4 cells; T2, 2 x 10 4 cells; T3, 3 x 10 4 cells; T4, 1 x 10 5 cells; T5, 1 x 10 6 cells) and after 48 hours of co-culture, it was observed to promote the growth of hDPCs at a constant concentration for each batch (
1-2. Western blot, 모발성장 관련 기전 단백질 발현 분석1-2. Western blot, hair growth-related mechanism protein expression analysis
Immunoblotting assay를 이용하여 hDPCs (1 x 105 cells, lower chamber)에서 EBI (Batch No. 014-05(지엔에스바이오㈜), 1 x 105 cells, upper chamber) 와 공배양 (6 well, trans-well plate, Corning 3412 Permeable Supports, 6-Cluster Plate) 이후 특정 단백질의 발현을 비교함으로써, EBI가 모발성장에 긍정적인 효과를 나타내는 기전에 대해서 탐색하고자 하였다. 그룹의 형성은 48시간의 공배양 (EBI #1, #2) 이후, 공배양 하지 않은 hDPCs (hDPCs only #1, #2)군과 관련 단백질의 발현량을 비교 평가하였다.Using a Immunoblotting assay hDPCs (1 x 10 5 cells, lower chamber) at the EBI (Batch No. 014-05 (S. Jian Bio ㈜), 1 x 10 5 cells , upper chamber) and the co-culture (6 well, trans- Well plate, Corning 3412 Permeable Supports, 6-Cluster Plate) after comparing the expression of specific proteins, we tried to explore the mechanism by which EBI exerts a positive effect on hair growth. Group formation was evaluated by comparing the expression levels of related proteins with the non-cocultured hDPCs (hDPCs only #1, #2) group after 48 hours of co-culture (
도 1B-C에서 보이는 바와 같이, 공 배양을 실시한 2군에서 그렇지 않은 군에 비하여 AKT, GSK3β 인산화가 증가하였고, 특히 β-catenin의 발현이 증가하였다. 추가적으로, β-catenin의 타겟 단백질인 CyclinD1 발현이 증가하는 것을 확인하였다. 결론적으로, hDPCs와 EBIH의 공배양을 통한 세포 증식과 성장기 관련 결과들은 모발성장 positive regulator인 β-catenin 발현 증가와 관련성이 있음을 확인할 수 있었다.As shown in FIG. 1B-C , AKT and GSK3β phosphorylation increased in
1-3. qRT-PCR, 모발 성장 관련 mRNA 발현비교1-3. qRT-PCR, hair growth-related mRNA expression comparison
EBI의 공배양은 hDPCs에서 모발성장 관련 mRNA수준을 높게 조절하였으며, transcription이 증가된 유전자들은 도 1D에 나타내었다.Co-culture of EBI highly regulated hair growth-related mRNA levels in hDPCs, and genes with increased transcription are shown in FIG. 1D .
2. EBICM을 이용한 hDPCs 성장 촉진 평가2. Evaluation of hDPCs growth promotion using EBICM
2-1. 모유두세포 관찰 및 세포 생존율 측정2-1. Observation of dermal papilla cells and measurement of cell viability
농도 별 EBICM을 이용하여 hDPCs 배양 이후 48h (5%, p < 0.05 vs. Con; 10%, p < 0.01 vs. Con), 72h (10%, p < 0.05 vs. Con), 120h (10%, p < 0.05 vs. Con) 이후에 평가한 결과, 일정 농도에서 hDPCs의 성장을 촉진하는 것으로 관찰되었으며, 통계적으로 유의하였다. (도 2A, 도 2B)48h (5%, p < 0.05 vs. Con; 10%, p < 0.01 vs. Con), 72h (10%, p <0.05 vs. Con), 120h (10%, p < 0.05 vs. Con) as a result of subsequent evaluation, it was observed that the growth of hDPCs was promoted at a certain concentration, and it was statistically significant. (Fig. 2A, Fig. 2B)
2-2. ALP 활성측정2-2. ALP activity measurement
농도 별 EBICM을 이용하여 hDPCs 배양 이후 48h (5%, p < 0.05 vs. Con; 10%, p < 0.01 vs. Con), 72h (5%, p < 0.01 vs. Con, 10%, p < 0.01 vs. Con) 이후에 평가한 결과, 일정 농도에서 hDPCs의 ALP activity를 상향 조절하는 것으로 관찰되었으며, 통계적으로 유의하였다. (도 2C)48 h (5%, p < 0.05 vs. Con; 10%, p < 0.01 vs. Con), 72 h (5%, p < 0.01 vs. Con, 10%, p < 0.01) after hDPCs culture using EBICM by concentration vs. Con), it was observed to upregulate the ALP activity of hDPCs at a certain concentration, and was statistically significant. (Fig. 2C)
2-3. ELISA를 이용한 hDPCs에서 분비된 VEGF분비량 평가2-3. Evaluation of VEGF secretion from hDPCs using ELISA
농도 별 EBICM을 이용하여 hDPCs 배양 이후 72h (2.5%, p < 0.01 vs. Con; 5%, p < 0.01 vs. Con; 10%, p < 0.01 vs. Con) 이후에 평가한 결과, 일정 농도에서 hDPCs의 VEGF 분비 촉진을 상향 조절하는 것으로 관찰되었으며, 통계적으로 유의하였다. (도 2D)As a result of evaluation after 72 h (2.5%, p < 0.01 vs. Con; 5%, p < 0.01 vs. Con; 10%, p < 0.01 vs. Con) after hDPCs culture using EBICM by concentration, at a certain concentration It was observed to upregulate the promotion of VEGF secretion in hDPCs, and it was statistically significant. (Fig. 2D)
2-4. qRT-PCR, 모발 성장 관련 mRNA 발현비교2-4. qRT-PCR, hair growth-related mRNA expression comparison
농도 별 EBICM을 이용하여 hDPCs 배양 이후 24h 이후에 평가한 결과, hDPCs의 성장과 모낭조직 성장에 긍정적으로 평가되는 gene의 발현이 증가하는 것으로 평가되었으며, 통계적으로 유의하였다. (도 2E, ALPL, p < 0.01; CTNNB1, p < 0.01; SHH, p < 0.05; SOX2, p < 0.01; VEGFA, p < 0.01; VCAN, p < 0.01; HEY1, p < 0.01; vs Con)As a result of evaluation 24 h after hDPCs culture using EBICM by concentration, it was evaluated that the expression of genes positively evaluated for hDPCs growth and hair follicle growth increased, and it was statistically significant. (Figure 2E, ALPL, p < 0.01; CTNNB1, p < 0.01; SHH, p < 0.05; SOX2, p < 0.01; VEGFA, p < 0.01; VCAN, p < 0.01; HEY1, p < 0.01; vs Con)
3. EBICM을 이용한 인체 모낭조직 성장 평가3. Evaluation of human hair follicle tissue growth using EBICM
인체에서 분리된 모낭조직에 농도별 EBICM을 처리하여 5일 이후 모간(hair shaft)의 길이변화를 관찰하고, 상대적 길이 변화를 평가하였다.Hair follicle tissue isolated from the human body was treated with EBICM for each concentration, and the change in the length of the hair shaft was observed after 5 days, and the relative length change was evaluated.
농도 별 EBICM 처리 후, 120 시간까지 인체 모낭조직에서 성장한 모간의 모습은 도 3A와 같다. 도 3A 조직학적 관찰에서, 특이적으로 농도 별 조직 내 hDPCs의 형태 차이가 있음을 확인하였다. 10% 처리군에서 hDPCs의 모습은 대조군에 비하여 모유두 세포의 구성 비가 높았으며, 모낭조직 하단부에 모구(hair bulb)에서 관찰된 모습이 성장기 모낭조직의 모습에서 발견할 수 있는 특징과 비슷한 경향성임을 확인하였다.The appearance of hair shafts grown in human hair follicle tissue up to 120 hours after EBICM treatment for each concentration is shown in FIG. 3A. In the histological observation of FIG. 3A , it was confirmed that there was a specific difference in the morphology of hDPCs in the tissue for each concentration. The appearance of hDPCs in the 10% treatment group was higher than that of the control group, and it was confirmed that the appearance observed in the hair bulb at the lower part of the hair follicle tissue had a tendency similar to the characteristic found in the appearance of the hair follicle tissue in the growth phase. did
EBICM 10%에서 일부 가장 월등한 모간 (hair shaft)의 길이 성장이 관찰되었다. 단, 일부 모낭조직에서 성장하였으며, 네 개의 모간 각각 17.6%, 13.2%, 9.7%, -0.9%의 변화를 보였다. 결과적으로 n수가 적고 통계적으로 유의하지는 않았지만 75%의 모간에서 약 10% 이상의 길이 성장을 보였다(도 3B)Some superior hair shaft length growth was observed with EBICM 10%. However, it grew in some hair follicle tissues, and the four hair shafts showed changes of 17.6%, 13.2%, 9.7%, and -0.9%, respectively. As a result, although the number of n was small and not statistically significant, 75% of the hair shafts showed a length growth of about 10% or more (Fig. 3B).
4. EBICM에 포함된 hDPCs 성장 촉진 인자 탐색을 위한 분석4. Analysis for the detection of hDPCs growth-promoting factors included in EBICM
인체 성장 인자/사이토카인 antibody array kit (dot blot array)를 사용하여 EBICM로부터 분비된 성장인자 및 사이토 카인의 프로파일을 조사하였다.The profiles of growth factors and cytokines secreted from EBICM were investigated using a human growth factor/cytokine antibody array kit (dot blot array).
그 결과, EBICM에서 GM-CSF (11.25), IGFBP-2 (6.92), PDGF-AA (3.81), IL-5 (2.91), IL-8 (2.61), IL-13 (5.13), IFN-γ (1.63), RANTES (6.4) 등이 Media only (p < 0.01)와 비교하여 유의적으로 높게 발현하는 것을 확인하였다(도 4). Dot blot의 결과와 내용은 도 5에서 나타내었다.As a result, in EBICM, GM-CSF (11.25), IGFBP-2 (6.92), PDGF-AA (3.81), IL-5 (2.91), IL-8 (2.61), IL-13 (5.13), IFN-γ (1.63), RANTES (6.4), etc. were confirmed to be significantly higher than that of Media only (p < 0.01) ( FIG. 4 ). The results and contents of the dot blot are shown in FIG. 5 .
5. RNA-seq 통합 분석5. RNA-seq Integration Assay
관련된 유전자 전사를 더 잘 이해하기 위해 hDPCs 에서 모발성장 가능성에 대한 RNA 발현을 비교 평가하였다 [16]. 일반 배양상태와 10% EBICM 에서 배양한 hDPCs 의 12 시간 배양 후 mRNA 전사내용을 확인할 결과. 총 739 개 (EBICM, 385 상승, 30 단독 상승; 381 저하, 15 단독 저하)의 차별적으로 발현된 유전자 (DEGs)가 RNA-seq 에 의하여 분석되었다. To better understand the gene transcription involved, we compared and evaluated RNA expression for hair growth potential in hDPCs [16]. Results of confirming the mRNA transcript content of hDPCs cultured in normal culture condition and 10% EBICM for 12 hours. A total of 739 differentially expressed genes (DEGs) (EBICM, 385 elevated, 30 alone; 381 down, 15 alone) were analyzed by RNA-seq.
그룹 간 상반된 전사인자 중 세포증식 양성조절과 관련된 전사인자는 아래 표 1과 같다.Among the transcription factors conflicting between groups, the transcription factors related to the positive regulation of cell proliferation are shown in Table 1 below.
[표 1][Table 1]
그룹 간 상반된 전사인자 중 항 세포 사멸 음성조절과 관련된 전사인자는 아래 표 2와 같다.Among the transcription factors contradictory between groups, the transcription factors related to the negative regulation of anti-apoptosis are shown in Table 2 below.
[표 2][Table 2]
그룹 간 상반된 전사인자 중 세포증식 관련된 전사인자는 아래 표 3과 같다.Among the transcription factors conflicting between groups, the transcription factors related to cell proliferation are shown in Table 3 below.
[표 3][Table 3]
그룹 간 상반된 전사인자 중 성장인자 활성 관련된 전사인자는 아래 표 4와 같다.Among the transcription factors conflicting between groups, the transcription factors related to growth factor activity are shown in Table 4 below.
[표 4][Table 4]
6. 정상대조군(normal), PBMC 투여군과 EBI 배양물 투여군 간의 아토피 피부염 치료 효과 비교6. Comparison of the treatment effect of atopic dermatitis between the normal control group, the PBMC-administered group, and the EBI culture-administered group
혈액에서 분리한 PBMC를 대조군으로 사용하여 아토피 마우스 모델에 투여하고, EBI 배양물은 배양된 EBI에서 배양물을 획득한 후 3000 rpm, 5분 원심분리하여 세포 찌거기를 제거하여 사용하였다. EBI 배양물의 경우 매일 아토피 마우스 피부병변 부위에 도포하는 방식으로 처리하였다. 도포 후 마우스 피부병변 부위에 도포한 샘플이 잘 머무를 수 있게 마우스를 고정시킨 상태에서 10분 이상 유지하였다.PBMCs isolated from blood were used as a control and administered to atopic mouse models, and the EBI culture was used to remove cell debris by centrifugation at 3000 rpm for 5 minutes after obtaining a culture from the cultured EBI. In the case of EBI culture, it was treated by applying it to the skin lesion site of atopic mice every day. After application, the mouse was maintained in a fixed state for more than 10 minutes so that the sample applied to the mouse skin lesion could stay well.
(1) EBI 배양물 투여에 따른 경피 두께 감소 효과 확인(1) Confirmation of the effect of reducing the transdermal thickness according to the administration of EBI culture
도 6을 참조하면, 아토피 피부염에서 흔히 나타나는 경피두께의 증가가 EBI 배양물을 국소도포한 결과, PBMC 투여군과 비교하여 현저히 감소함을 알 수 있었다.Referring to FIG. 6 , it can be seen that the increase in transdermal thickness, which is common in atopic dermatitis, is significantly reduced compared to the PBMC-administered group as a result of topical application of the EBI culture.
(2) 염증반응 감소 효과 확인(2) Confirmation of inflammatory response reduction effect
비만세포의 증가는 아토피 피부염에서 흔하게 나타나고, 아토피를 악화시키는 요인으로 알려져 있는데, 도 7을 참조하면, PBMC 투여군에서는 비만세포의 수가 매우 증가하였지만, EBI 배양물 국소 도포 시 비만세포의 수가 현저히 감소함을 알 수 있었다.The increase in mast cells is common in atopic dermatitis and is known to be a factor exacerbating atopic dermatitis. Referring to FIG. 7 , the number of mast cells greatly increased in the PBMC-administered group, but the number of mast cells significantly decreased when applied locally to the EBI culture. And it was found.
(3) 혈액 내 IgE 감소 확인(3) Confirmation of IgE reduction in blood
아토피 병변부위에 EBI 배양물을 도포했을 때 혈액 내 IgE 수치가 매우 감소하였다(도 8).When the EBI culture was applied to the atopic lesion site, the IgE level in the blood was greatly reduced ( FIG. 8 ).
(4) EBI 배양물 투여에 따른 체중 변화 확인(4) Confirmation of body weight change according to EBI culture administration
정상대조군, 아토피 마우스 모델에서 PBMC 투여군, EBI 배양물 투여군 모두에서 눈에 띄는 체중의 변화는 없었다(도 9). There was no noticeable change in body weight in both the PBMC-administered group and the EBI culture-administered group in the normal control group and the atopic mouse model (FIG. 9).
(5) 피부 조직 내 아토피피부염 관련 사이토카인 및 가려움증(itching) 관련 유전자의 발현 분석(5) Expression analysis of atopic dermatitis-related cytokines and itch-related genes in skin tissue
피부의 조직 내 아토피 피부염 관련 사이토카인, 염증, 가려움증(itching) 관련 유전자들의 발현 변화를 확인한 결과(도 10), EBI 배양물 처리시 관련 유전자의 발현이 현저히 감소함을 확인하였다.As a result of confirming the expression changes of atopic dermatitis-related cytokines, inflammation, and itch-related genes in the skin tissue ( FIG. 10 ), it was confirmed that the expression of the related genes was significantly reduced during EBI culture treatment.
7. 아토피 유도 마우스 모델에서 EBI 엑소좀의 치료 효능 확인7. Confirmation of therapeutic efficacy of EBI exosomes in atopic induced mouse model
EBI 세포로부터 배양물을 얻어 초원심분리를 통해 분리된 EBI 엑소좀을 PBS에 재현탁시킨 후 동일한 양을 처리하기 위해 BCA단백질 정량을 통해 농도를 결정하였다. 고농축된 엑소좀의 표면마커 평가 및 크기를 측정하여 엑소좀 특성을 평가한 후 엑소좀을 아토피 마우스 모델에 2가지 경로를 통해 투여하였다. 인슐린 주사기로 엑소좀 100 ㎕를 취하고 아토피 마우스의 병변 피부아래 엑소좀(50 ㎍/100 ㎕)을 주 3회씩 3주 동안 9회 피하 주사하였다. 동일양의 정제된 엑소좀을 다른 아토피 유도 마우스 복강 내에 동일한 횟수로 투여하여 아토피 개선 효과를 비교하였다.After obtaining a culture from EBI cells and resuspending EBI exosomes isolated through ultracentrifugation in PBS, the concentration was determined through BCA protein quantification to process the same amount. After evaluating the characteristics of the exosomes by measuring the surface marker evaluation and size of the highly concentrated exosomes, the exosomes were administered to the atopic mouse model through two routes. 100 μl of exosomes were taken with an insulin syringe, and exosomes (50 μg/100 μl) under the skin of the lesion of atopic mice were subcutaneously injected 3 times a week, 9 times for 3 weeks. The atopy improvement effect was compared by administering the same amount of purified exosomes the same number of times into the abdominal cavity of other atopic-induced mice.
그 결과는 도 11 내지 도14에 나타내었다.The results are shown in FIGS. 11 to 14 .
엑소좀을 아토피 마우스의 복강 내 투여한 경우는 PBS 투여군과 비교하여 피부개선 효과가 보이지 않았으나, 피하주사한 경우는 경피두께가 현저히 감소됨을 알 수 있었다(도 11 및 12).In the case of intraperitoneal administration of exosomes to atopic mice, compared to the PBS-administered group, the skin improvement effect was not seen, but when subcutaneously injected, it was found that the transdermal thickness was significantly reduced ( FIGS. 11 and 12 ).
또한, 엑소좀 피부 주사군에서는 비만세포의 침윤이 감소되어 정상에 가까운 모습을 나타내었고(도 13), 정상 대조군과 유사한 수치까지 경피 수분 손실량이 감소하는 것으로 평가되었다(도 14).In addition, in the exosome skin injection group, the infiltration of mast cells was reduced, showing a close-to-normal appearance (FIG. 13), and it was evaluated that the amount of transdermal water loss decreased to a value similar to that of the normal control group (FIG. 14).
8. EBI 배양물의 엑소좀(exosome)단백질체 분석 결과 8. Result of analysis of exosome proteome of EBI culture
HFF(human foreskin fibroblast)와 비교하여 EBI 배양물 내 엑소좀에서 발현이 증가하는 단백질체들을 도 15에 따라 선별하였다.In comparison with HFF (human foreskin fibroblast), proteomic bodies with increased expression in exosomes in EBI culture were selected according to FIG. 15 .
구체적으로, 총 7434개의 단백질 유전자 중에서 HFF에서 Intensity가 확인되지 않는 단백질 1462개를 우선 선별하였으며, EBI와 EBI_exo에서 Intensity가 확인되는 단백질 441개를 선별하였다. ShyniGO v0.61을 이용하여 Biological Process 중 Immune System Process에 해당하는 단백질을 209개 선별하였고, 그 중 면역세포(NK세포 또는 T세포)의 생물학적 기능과 연관된 67개의 단백질을 선별하였다(도 16). 이후, Uniprot를 이용하여 T cell과 NK cell의 기능과 연관 있는 Biological Process 중 Immune System Process가 확인되는 Protein을 24개 선별하였다.Specifically, among a total of 7434 protein genes, 1462 proteins whose intensity was not confirmed in HFF were first selected, and 441 proteins whose intensity was confirmed in EBI and EBI_exo were selected. ShyniGO v0.61 was used to select 209 proteins corresponding to the Immune System Process among Biological Processes, and among them, 67 proteins related to the biological function of immune cells (NK cells or T cells) were selected (FIG. 16). Then, using Uniprot, 24 proteins in which the Immune System Process was confirmed among the Biological Processes related to the functions of T cells and NK cells were selected.
Th1 세포, T 세포, NK 세포 연관 단백질 중 EBI 엑소좀에서만 Intensity가 확인되는 단백질들은 IL2RA, IL12RB2, TNFAIP3, CD40LG;TNFSF5, TNFRSF4, TNFSF8, TNFSF10, TNFSF4, TNFSF9이었다(도 17).Among the proteins associated with Th1 cells, T cells, and NK cells, the proteins whose intensity was confirmed only in EBI exosomes were IL2RA, IL12RB2, TNFAIP3, CD40LG; TNFSF5, TNFRSF4, TNFSF8, TNFSF10, TNFSF4, and TNFSF9 (FIG. 17).
또한, 선별된 24개의 단백질 중 면역 활성, Th1세포 활성과 관련된 단백질을 선별한 결과 12개의 단백질이 선별되었으며, 이들은 세포 자극을 통한 재생 유도 및 면역 조절 장애에 대한 면역균형을 유도하는 데 중요한 물질임을 알 수 있었다. 선별된 12개의 단백질에 대한 HFF, EBI, EBI 엑소좀에서의 Intensity를 도 18에 나타내었다.In addition, as a result of screening proteins related to immune activity and Th1 cell activity among the selected 24 proteins, 12 proteins were selected. Could know. Intensities in HFF, EBI, and EBI exosomes for the selected 12 proteins are shown in FIG. 18 .
Claims (12)
A pharmaceutical composition for promoting hair growth or treating atopic dermatitis, comprising a culture of activated lymphocytes derived from peripheral blood mononuclear cells.
The method according to claim 1, wherein the activated lymphocytes are obtained by culturing the peripheral blood mononuclear cells of an individual in a cell culture medium comprising at least one selected from the group consisting of anti-CD3 antibody, IL-2, IL-12 and IL-18. The composition.
The composition of claim 2, wherein the cell culture medium comprises an anti-CD3 antibody, IL-2, IL-12 and IL-18.
인간의 말초혈액으로부터 단핵구 및 자가혈장을 각각 분리하여 수득하는 단계;
항-CD3 항체로 세포 배양용기를 코팅하는 단계; 및
상기 단핵구를 상기 배양용기에 접종하여 IL-2, IL-12 및 IL-18로 이루어진 군에서 선택되는 하나 이상을 포함하는 배지에서 배양하는 단계를 포함하는 배양방법으로 얻어진 것인, 조성물.
The method according to claim 1, wherein the culture,
Separating and obtaining monocytes and autologous plasma from human peripheral blood, respectively;
coating the cell culture vessel with an anti-CD3 antibody; and
Inoculating the monocytes into the culture vessel and culturing in a medium containing one or more selected from the group consisting of IL-2, IL-12 and IL-18, the composition comprising the step of inoculating the culture vessel.
The composition of claim 1, wherein the culture comprises the activated lymphocyte-derived exosome.
The method according to claim 1, wherein the culture is IRF4 (Interferon regulatory factor 4), CD226 (Cluster of Differentiation 226), TMIGD2 (Transmembrane and immunoglobulin domain-containing protein 2), HAVCR2 (Hepatitis A virus cellular receptor 2), TRAT1 (T -cell receptor-associated transmembrane adapter 1), SPN (Sialophorin), TRAF2 (TNF receptor-associated factor 2), CCDC88B (Coiled-coil domain containing 88B), TYROBP (tyrosine kinase-binding protein), VAV1 (Vav Guanine Nucleotide Exchange) Factor 1), the composition comprising one or more proteins selected from the group consisting of SLAMF6 (SLAM family member 6) and ZBTB16 (Zinc finger and BTB domain-containing protein 16).
A cosmetic composition for promoting hair growth or improving atopic dermatitis, comprising a culture of activated lymphocytes derived from peripheral blood mononuclear cells.
The method according to claim 7, wherein the activated lymphocytes are obtained by culturing the peripheral blood mononuclear cells of an individual in a cell culture medium comprising at least one selected from the group consisting of anti-CD3 antibody, IL-2, IL-12 and IL-18. The composition.
The composition of claim 7, wherein the cell culture medium comprises an anti-CD3 antibody, IL-2, IL-12 and IL-18.
인간의 말초혈액으로부터 단핵구 및 자가혈장을 각각 분리하여 수득하는 단계;
항-CD3 항체로 세포 배양용기를 코팅하는 단계; 및
상기 단핵구를 상기 배양용기에 접종하여 IL-2, IL-12 및 IL-18로 이루어진 군에서 선택되는 하나 이상을 포함하는 배지에서 배양하는 단계를 포함하는 배양방법으로 얻어진 것인, 조성물.
The method according to claim 7, wherein the culture,
Separating and obtaining monocytes and autologous plasma from human peripheral blood, respectively;
coating the cell culture vessel with an anti-CD3 antibody; and
Inoculating the monocytes into the culture vessel and culturing in a medium containing one or more selected from the group consisting of IL-2, IL-12 and IL-18, the composition comprising the step of inoculating the culture vessel.
The composition of claim 7, wherein the culture comprises the activated lymphocyte-derived exosomes.
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