WO2023085604A1 - Composition for prevention or treatment of inflammatory diseases containing cartilage tissue-derived extracellular matrix as active ingredient - Google Patents
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- the pre-treated cartilage tissue-derived extracellular matrix is a pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that it comprises extracellular vesicles with enhanced anti-inflammatory properties.
- FIGS. 6A and 6B the expression of IL-1B and IL-6, which are major mediators of inflammation, decreased in all pretreatment groups, as well as COX-2 and p-I ⁇ B- ⁇ as shown in FIGS. 6C and 6D.
- the expression of was significantly decreased compared to the inflammation induction group, and in particular, a more significant decrease was confirmed in the soluble ECM components treatment group after pretreatment.
- M0 Macrophage The group was treated with a concentration of 10 ⁇ g/ml, and ICC analysis for CD86 (M1 marker) and CD163 (M2 marker) was performed.
- M1 marker CD86
- CD163 M2 marker
- 100 ng/ml of LPS and 20 ng/ml of IFN- ⁇ were used to induce differentiation of M1 macrophage
- 20 ng/ml of IL-4 and 20 ng/ml of IL-13 were used for M2 macrophage. were treated with M0 Macrophage for 24 hours to induce differentiation.
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Abstract
The present invention relates to a composition for the prevention or treatment of inflammatory diseases, containing a cartilage tissue-derived extracellular matrix as an active ingredient. More specifically, when the cartilage tissue-derived extracellular matrix or extracellular vesicles in the extracellular matrix were pre-treated with inflammatory cytokine Interleukin-1β and non-steroidal anti-inflammatory drug celecoxib in an osteoarthritis animal model, it was confirmed that an enhanced anti-inflammatory effect was exhibited in the osteoarthritis model, and thus the present invention is intended to provide a composition containing as an active ingredient pre-treated cartilage tissue-derived extracellular matrix or extracellular vesicles, as a therapeutic agent for inflammatory diseases exhibiting an enhanced anti-inflammatory effect.
Description
본 발명은 염증성 사이토카인, 비스테로이드 항염증제 또는 이들의 조합으로 전 처리된 연골조직 유래 세포외기질을 유효성분으로 함유하는 염증성 질환 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating inflammatory diseases, which contains, as an active ingredient, an extracellular matrix derived from cartilage tissue pretreated with an inflammatory cytokine, a non-steroidal anti-inflammatory agent, or a combination thereof.
세포내 다양한 염증 조절 인자들은 유도형 일산화질소 합성효소(inducible nitric oxide synthase; iNOS)나 사이클로-옥시게나제 (cyclo-oxygenase-2; COX-2)에 의해 생체 내 대식세포와 같은 염증 세포들이 활성화되면서 염증 매개인자인 일산화질소(Nitric oxide; NO), 프로스타글란딘(Prostaglandin; PG), 인터루킨-1베타(Interleukin-1 beta; IL-1β), 종양괴사인자-알파 (Tumor necrosis factor-alpha; TNF-α), 사이토카인 (cytokine) 등이 다량 생산된다. 또한, 염증성 사이토카인(cytokine)인 인터루킨-1(IL-1), 종양괴사인자-α(TNF-α) 등을 유도시키게 되는데 이로 인하여 근육, 건, 인대 등과 같은 조직에도 영향을 끼쳐 심한 통증을 일으킨다.Various intracellular inflammatory regulators activate inflammatory cells such as macrophages in vivo by inducible nitric oxide synthase (iNOS) or cyclo-oxygenase-2 (COX-2). inflammatory mediators such as nitric oxide (NO), prostaglandin (PG), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF- α), cytokines, etc. are produced in large quantities. In addition, inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are induced, which also affects tissues such as muscles, tendons, and ligaments, resulting in severe pain. cause
대부분의 염증 질환의 치료제로서 널리 사용되고 있는 제제인 비스테로이드성 소염제(non-steroidal anti-inflammatory drugs, NSAIDS)는 사이클로옥시게나제(cyclooxygenase, COX)라고 하는 아라키돈산(arachidonic acid)으로부터 프로스타글란딘(prostaglandin)의 생합성에 관여하는 효소 활성을 억제함으로써, 항염증 작용을 나타내는데, 주 치료 작용 외에 위장관 장애, 간장애, 신장애 등의 심각한 부작용을 야기하므로 장기간의 사용에 있어서 제약이 따르는 실정이다. 따라서 천연에서 유래한 식물추출물을 이용한 염증성 질환 치료용 조성물에 관한 연구가 진행된 바 있으나, 상기 비스테로이드성 소염제를 대체하여 부작용이 없이 장기간 사용하는데 무리가 없으면서 항염증 효능에 탁월한 새로운 소염 진통제의 개발이 절실하게 요구되고 있다.Non-steroidal anti-inflammatory drugs (NSAIDS), a widely used agent for the treatment of most inflammatory diseases, release prostaglandin from arachidonic acid called cyclooxygenase (COX). By inhibiting the enzyme activity involved in the biosynthesis, it exhibits anti-inflammatory action, but in addition to the main therapeutic action, it causes serious side effects such as gastrointestinal disorders, liver disorders, and renal disorders, so long-term use is limited. Therefore, although studies have been conducted on compositions for treating inflammatory diseases using plant extracts derived from nature, development of new anti-inflammatory analgesics excellent in anti-inflammatory efficacy without side effects and long-term use in place of the non-steroidal anti-inflammatory drugs has been conducted. is desperately needed.
본 발명은 염증성 사이토카인, 비스테로이드 항염증제 또는 이들의 조합으로 전 처리된 연골조직 유래 세포외기질을 유효성분으로 함유하는 조성물을 염증성 질환 예방 또는 치료제로 제공하고자 한다.An object of the present invention is to provide a composition containing, as an active ingredient, a cartilage tissue-derived extracellular matrix pretreated with an inflammatory cytokine, a non-steroidal anti-inflammatory agent, or a combination thereof, as a preventive or therapeutic agent for inflammatory diseases.
본 발명은 염증성 사이토카인, 비스테로이드 항염증제 또는 이들의 조합으로 전 처리된 연골조직 유래 세포외기질을 유효성분으로 함유하는 염증성 질환 예방 또는 치료용 약학조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases containing, as an active ingredient, cartilage tissue-derived extracellular matrix pre-treated with inflammatory cytokines, non-steroidal anti-inflammatory agents, or a combination thereof.
또한, 본 발명은 개체로부터 분리된 연골조직에 염증성 사이토카인, 비스테로이드 항염증제 또는 이들의 조합을 전처리하는 단계; In addition, the present invention comprises the steps of pre-treating an inflammatory cytokine, a non-steroidal anti-inflammatory agent or a combination thereof to the cartilage tissue isolated from the subject;
상기 전 처리된 연골조직을 1 내지 5일간 배양하는 단계; 및 Culturing the pre-treated cartilage tissue for 1 to 5 days; and
상기 배양된 연골조직으로부터 세포외기질을 분리하는 단계를 포함하는 세포외기질 항염증 기능 강화 방법을 제공한다.Provided is a method for enhancing the anti-inflammatory function of the extracellular matrix comprising the step of separating the extracellular matrix from the cultured cartilage tissue.
본 발명에 따르면 염증성 사이토카인 인터루킨-1β(Interleukin-1β)와 비스테로이드 항염제 셀레콕시브 (celecoxib)가 전 처리된 연골조직으로부터 유래된 세포외기질 또는 세포외기질 내 세포외소포를 골관절염 동물모델에서 처리한 경우, 골관절염 모델에서 향상된 항염증 효과를 나타내는 것을 확인함에 따라, 상기 전 처리된 연골조직 유래 세포외기질 또는 세포외소포를 유효성분으로 함유하는 조성물은 강화된 항염증 효과를 나타내는 염증성 질환 치료제로 제공할 수 있다.According to the present invention, the extracellular matrix derived from cartilage tissue pretreated with the inflammatory cytokine Interleukin-1β and the non-steroidal anti-inflammatory drug celecoxib or the extracellular vesicles in the extracellular matrix were prepared in an osteoarthritis animal model. When treated, as it was confirmed that an improved anti-inflammatory effect was exhibited in an osteoarthritis model, the composition containing the pre-treated cartilage tissue-derived extracellular matrix or extracellular vesicles as an active ingredient exhibits an enhanced anti-inflammatory effect. can be provided with
도 1은 전처리 후 연골조직 유래 세포외소포의 제조 및 원리를 도식화한 것이다.Figure 1 is a schematic diagram of the production and principle of cartilage tissue-derived extracellular vesicles after pretreatment.
도 2는 전처리 유효성을 연골세포에서 확인한 결과로, 도 2A는 항염증 인자 유전자 발현 수준을 확인한 결과이며, 도 2B는 항염증 인자 단백질 발현 수준을 확인한 결과이다.Figure 2 is a result confirming the effectiveness of the pretreatment in chondrocytes, Figure 2A is the result of confirming the anti-inflammatory factor gene expression level, Figure 2B is the result of confirming the anti-inflammatory factor protein expression level.
도 3은 전처리 유효성을 연골조직에서 확인한 결과로, 도 3A는 항염증 인자 유전자 발현 수준을 확인한 결과이며, 도 3B는 항염증 인자 단백질 발현 수준을 확인한 결과이며, 도 3C는 항염증 강화 유도의 신호경로를 모식화한 것이다.Figure 3 is the result of confirming the effectiveness of pretreatment in cartilage tissue, Figure 3A is the result of confirming the anti-inflammatory factor gene expression level, Figure 3B is the result of confirming the anti-inflammatory factor protein expression level, Figure 3C is the signal of anti-inflammatory enhancement induction modeled the route.
도 4는 NSAIDs 와 Inflammatory cytokine을 이용한 다양한 전처리 방법을 확인한 결과로, 도 4A는 IL-10 유전자 발현 수준을 확인한 결과이며, 도 4B는 IL-10 단백질 발현 수준을 확인한 결과이다.4 is a result of confirming various pretreatment methods using NSAIDs and inflammatory cytokines, FIG. 4A is the result of confirming the IL-10 gene expression level, and FIG. 4B is the result of confirming the IL-10 protein expression level.
도 5는 전처리 이후 연골조직 성분 중 변화된 항염증 유효성을 확인한 결과로, 도 5A는 연골조직 내 성분 분리 방법의 도식화한 것이며, 도 5B는 분리한 성분을 SDS-PAGE로 분석해 포함된 단백질의 분포를 확인한 결과이다. Figure 5 is a result of confirming the anti-inflammatory effectiveness of cartilage tissue components changed after pretreatment. Figure 5A is a schematic diagram of a method for separating components in cartilage tissue, and Figure 5B shows the distribution of proteins included by analyzing the separated components by SDS-PAGE. This is the result of checking
도 6은 활막세포 염증 모델에 전처리 전후 연골조직 세포외기질을 처리한 뒤 항염증 유효성을 확인한 결과로, 도 6A 및 도 6B는 염증 매개인자의 유전자 발현 수준을 확인한 결과이며, 도 6C는 단백질 발현 수준을 확인한 결과이며, 도 6D는 주요 염증 매개인자인 COX-2의 발현을 정량적으로 분석한 결과이다.Figure 6 is a result of confirming the anti-inflammatory effectiveness after pretreatment of the synovial cell inflammation model before and after treatment with cartilage tissue extracellular matrix, Figures 6A and 6B are the results of confirming the gene expression level of inflammatory mediators, and Figure 6C is protein expression This is the result of confirming the level, and FIG. 6D is the result of quantitatively analyzing the expression of COX-2, a major inflammatory mediator.
도 7는 연골조직 유래 세포외소포 (CEV)의 분리 및 세포외소포의 특성을 확인한 결과로, 도 7A는 연골조직 유래 세포외소포를 분리하는 방법을 모식화한 것이며, 도 7B는 SEM 이미지이며, 도 7C는 크기 분포도를 확인한 결과이며, 도 7D는 PKH 탐지를 붙인 CEV의 세포 침투 후 형광 이미지이며, 도 7E는 CEV의 마커로 활용되는 CD81 및 CD9의 단백질 발현을 확인한 결과이다. Figure 7 is a result of confirming the separation of cartilage tissue-derived extracellular vesicles (CEV) and the characteristics of the extracellular vesicles, Figure 7A is a schematic diagram of a method for isolating cartilage tissue-derived extracellular vesicles, Figure 7B is a SEM image , Figure 7C is the result of confirming the size distribution, Figure 7D is a fluorescence image after cell penetration of CEV with PKH detection attached, Figure 7E is the result of confirming the protein expression of CD81 and CD9 used as markers of CEV.
도 8은 연골조직 유래 세포외소포와 전처리 후 세포외소포 처리에 따른 항염증 유효성을 인간 활액막세포 체외 염증모델에서 확인한 결과로, 도 8A 및 도 8B는 염증성 사이토카인의 유전자 발현 수준을 확인한 결과이며, 도 8C는 주요 염증 매개인자의 단백질 발현 수준을 확인한 결과이며, 도8D는 주요 염증 매개인자인 COX-2의 발현을 정량적으로 분석한 결과이며, 도 8E는 세포 외부로 방출된 염증 매개인자 PGEd의 단백질 정량을 분석한 결과이다.Figure 8 is a result of confirming the anti-inflammatory efficacy according to the treatment of extracellular vesicles after pretreatment with cartilage tissue-derived extracellular vesicles in a human synovial cell in vitro inflammatory model, Figures 8A and 8B are the results of confirming the gene expression level of inflammatory cytokines , Figure 8C is the result of confirming the protein expression level of the main inflammatory mediator, Figure 8D is the result of quantitatively analyzing the expression of the main inflammatory mediator COX-2, Figure 8E is the inflammatory mediator PGEd released outside the cell This is the result of analyzing the protein quantification of
도 9은 연골조직 유래 세포외기질 (CECM) 및 세포외소포 (CEV)의 전처리 후 세포외기질(P_CECM) 및 세포외소포 (P_CEV) 처리에 따른 면역세포 조절능력을 인간 단핵구세포 체외 실험에서 확인한 결과로, 도 9A는 M1 Macrophage 와 M2 Macrophage 마커를 ICC 실험을 통하여 형광이미지로 확인한 결과이며, 도 9B는 확인한 마커들의 발현을 정량적으로 분석한 결과이다.Figure 9 confirms the immune cell regulatory ability according to the extracellular matrix (P_CECM) and extracellular vesicles (P_CEV) treatment after pretreatment of cartilage tissue-derived extracellular matrix (CECM) and extracellular vesicles (CEV) in human monocyte cell in vitro experiments. As a result, FIG. 9A is the result of confirming the M1 Macrophage and M2 Macrophage markers with fluorescence images through an ICC experiment, and FIG. 9B is the result of quantitatively analyzing the expression of the identified markers.
도 10은 연연골조직 유래 세포외기질 (CECM) 및 세포외소포 (CEV)의 전처리 후 세포외기질(P_CECM) 및 세포외소포 (P_CEV) 처리에 따른 항염증 유효성을 골관절염 질환 유도된 랫드 체내 실험에서 확인한 결과로, 도 10A는 Safranin-O, H&E 및 CD68-IHC 염색을 수행하여 연골 보호기능 및 염증 반응 증감을 확인한 결과이며, 도 10B는 염증 반응의 정량적 분석이 가능한 CD68 IHC를 분석한 결과이다.Figure 10 shows the anti-inflammatory efficacy of extracellular matrix (P_CECM) and extracellular vesicles (P_CEV) treatment after pretreatment of cartilage tissue-derived extracellular matrix (CECM) and extracellular vesicles (CEV) in an in vivo experiment in rats induced with osteoarthritis disease As a result of confirmation, FIG. 10A is the result of confirming the cartilage protection function and the increase or decrease of the inflammatory response by performing Safranin-O, H&E, and CD68-IHC staining, and FIG. 10B is the result of CD68 IHC analysis capable of quantitative analysis of the inflammatory response.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 염증성 사이토카인, 비스테로이드 항염증제 또는 이들의 조합으로 전 처리된 연골조직 유래 세포외기질을 유효성분으로 함유하는 염증성 질환 예방 또는 치료용 약학조성물을 제공할 수 있다.The present invention may provide a pharmaceutical composition for preventing or treating inflammatory diseases, which contains, as an active ingredient, cartilage tissue-derived extracellular matrix pre-treated with inflammatory cytokines, non-steroidal anti-inflammatory agents, or a combination thereof.
상기 전 처리된 연골조직 유래 세포외 기질은 항염증성이 강화된 세포외소포를 포함하는 것을 특징으로 하는 염증성 질환 예방 또는 치료용 약학조성물.The pre-treated cartilage tissue-derived extracellular matrix is a pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that it comprises extracellular vesicles with enhanced anti-inflammatory properties.
상기 염증성 사이토카인은 인터루킨-1β, TNF-a 및 인터페론-γ 로 이루어진 군에서 하나 이상 선택되는 것일 수 있다.The inflammatory cytokine may be one or more selected from the group consisting of interleukin-1β, TNF-a, and interferon-γ.
상기 비스테로이드 항염증제는 살리실산(Salicylic Acid), 프로피온산 (Propionic acid), 세레콕시브(celecoxib), 나프록센 (naproxen), 아세트산(acetic acid) 및 페나믹산 (Fenamic acid)으로 이루어진 군에서 하나 이상 선택되는 것일 수 있다.The non-steroidal anti-inflammatory agent is one or more selected from the group consisting of salicylic acid, propionic acid, celecoxib, naproxen, acetic acid and fenamic acid can
상기 염증성 질환은 피부염, 아토피 피부염, 천식, 결막염, 치주염, 비염, 중이염, 홍채염, 인후염, 편도염, 폐렴, 위궤양, 췌장염, 위염, 크론병, 염증성 장질환, 대장염, 치질, 통풍, 강직성 척추염, 루프스, 섬유근통(fibromyalgia), 건선, 류마티스 관절염, 골관절염, 골다공증, 간염, 방광염, 신장염, 쇼그렌 증후군 및 다발성 경화증으로 이루어진 그룹에서 선택되는 어느 하나인 것일 수 있다.The inflammatory diseases include dermatitis, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, gout, ankylosing spondylitis, lupus , It may be any one selected from the group consisting of fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome, and multiple sclerosis.
본 발명의 한 구체예에서, 상기 염증성 질환 예방 또는 치료용 약학조성물은 통상적인 방법에 따라 주사제, 과립제, 산제, 정제, 환제, 캡슐제, 좌제, 겔, 현탁제, 유제, 점적제 또는 액제로 이루어진 군에서 선택된 어느 하나의 제형을 사용할 수 있다.In one embodiment of the present invention, the pharmaceutical composition for preventing or treating inflammatory diseases is prepared in the form of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or solutions according to conventional methods. Any one formulation selected from the group consisting of may be used.
본 발명의 다른 구체예에서, 상기 염증성 질환 예방 또는 치료용 약학조성물은 약학조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제, 붕해제, 감미제, 피복제, 팽창제, 활택제, 향미제, 항산화제, 완충액, 정균제, 희석제, 분산제, 계면활성제, 결합제 및 윤활제로 이루어진 군에서 선택되는 하나 이상의 첨가제를 추가로 포함할 수 있다.In another embodiment of the present invention, the pharmaceutical composition for preventing or treating inflammatory diseases is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, flavoring agent, antioxidant commonly used in the manufacture of pharmaceutical compositions , It may further include one or more additives selected from the group consisting of a buffer, a bacteriostatic agent, a diluent, a dispersing agent, a surfactant, a binder, and a lubricant.
구체적으로 담체, 부형제 및 희석제는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 사용할 수 있으며, 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용할 수 있다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 있으며 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제 등이 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기재로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil may be used, and solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base material of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.
본 발명의 일실시예에 따르면 상기 약학 조성물은 정맥내, 동맥내, 복강내, 근육내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 대상체로 투여할 수 있다.According to one embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject as
상기 돌단풍 추출물의 바람직한 투여량은 대상체의 상태 및 체중, 질환의 종류 및 정도, 약물 형태, 투여경로 및 기간에 따라 달라질 수 있으며 당업자에 의해 적절하게 선택될 수 있다. 본 발명의 일실시예에 따르면 이에 제한되는 것은 아니지만 1일 투여량이 0.01 내지 200 mg/kg, 구체적으로는 0.1 내지 200 mg/kg, 보다 구체적으로는 0.1 내지 100 mg/kg 일 수 있다. 투여는 하루에 한 번 투여할 수도 있고 수회로 나누어 투여할 수도 있으며, 이에 의해 본 발명의 범위가 제한되는 것은 아니다.The preferred dosage of the stone maple extract may vary depending on the condition and weight of the subject, the type and severity of the disease, the type of drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.
본 발명에 있어서, 상기 '대상체'는 인간을 포함하는 포유동물일 수 있으나, 이들 예에 한정되는 것은 아니다.In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.
또한, 본 발명은 개체로부터 분리된 연골조직에 염증성 사이토카인, 비스테로이드 항염증제 또는 이들의 조합을 전처리하는 단계; In addition, the present invention comprises the steps of pre-treating an inflammatory cytokine, a non-steroidal anti-inflammatory agent or a combination thereof to the cartilage tissue isolated from the subject;
상기 전 처리된 연골조직을 1 내지 5일간 배양하는 단계; 및 Culturing the pre-treated cartilage tissue for 1 to 5 days; and
상기 배양된 연골조직으로부터 세포외기질을 분리하는 단계를 포함하는 세포외기질 항염증 기능 강화 방법을 제공할 수 있다.It is possible to provide a method for enhancing the anti-inflammatory function of the extracellular matrix comprising the step of separating the extracellular matrix from the cultured cartilage tissue.
보다 상세하게는 수술용 블레이드를 사용하여 연골조직을 수확한 후 연골 조직은 다시 생리식염수로 세척한 뒤 블레이드를 사용하여 약 5 mm × 5 mm × 2 mm의 크기의 연골 조각으로 만들었다. 연골세포의 전처리 방법과 동일하게 1× PBS로 조직을 세척하고 chondrogenic DMEM 배양액을 처치한 후, 전처리 그룹은 배양액에 0.1 ng/ml의 인터루킨-1β(Interleukin-1β)와 10 μM 셀레콕시브 (celecoxib)를 추가하여 3일 동안 처리하였다.More specifically, after harvesting the cartilage tissue using a surgical blade, the cartilage tissue was washed again with physiological saline and then made into cartilage pieces having a size of about 5 mm × 5 mm × 2 mm using a blade. In the same way as in the pre-treatment of chondrocytes, the tissue was washed with 1× PBS and treated with chondrogenic DMEM culture, and then the pre-treatment group was treated with 0.1 ng/ml of Interleukin-1β and 10 μM celecoxib. ) was added and treated for 3 days.
상기 항염증 기능 강화 방법은 연골조직 유래 세포외기질 또는 세포외소포의 항염증 강화 전처리 방법으로써, 다양한 염증성 사이토카인 및 비스테로이드성 항염증제가 사용될 수 있다. The anti-inflammatory function enhancing method is a pre-treatment method for enhancing anti-inflammatory function of cartilage tissue-derived extracellular matrix or extracellular vesicles, and various inflammatory cytokines and non-steroidal anti-inflammatory drugs may be used.
상기 염증성 사이토카인은 인터루킨-1β, TNF-a 및 인터페론-γ 로 이루어진 군에서 하나 이상 선택되는 것일 수 있으나, 이에 제한되는 것은 아니다.The inflammatory cytokine may be one or more selected from the group consisting of interleukin-1β, TNF-a, and interferon-γ, but is not limited thereto.
상기 비스테로이드 항염증제는 살리실산(Salicylic Acid), 프로피온산 (Propionic acid), 세레콕시브(celecoxib), 나프록센 (naproxen), 아세트산(Acetic Acid) 및 페나믹산 (Fenamic acid)으로 이루어진 군에서 하나 이상 선택되는 것일 수 있으나, 이에 제한되는 것은 아니다.The non-steroidal anti-inflammatory agent is one or more selected from the group consisting of salicylic acid, propionic acid, celecoxib, naproxen, acetic acid and fenamic acid It may be, but is not limited thereto.
상기 항염증 기능 강화 방법을 수득된 세포외기질은 항염증성이 강화된 세포외소포를 포함하는 것일 수 있다.The extracellular matrix obtained by the anti-inflammatory function enhancing method may include extracellular vesicles having enhanced anti-inflammatory properties.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
<실시예 1> 돼지 연골세포 분리 및 전처리 후 생화학적 특성 확인<Example 1> Confirmation of biochemical characteristics after isolation and pretreatment of porcine chondrocytes
1. 돼지 연골 세포의 분리 및 배양1. Isolation and culture of porcine chondrocytes
돼지 연골 조직은 돼지 슬관절에서 수술용 블레이드를 사용하여 수확하였다. 연골 조직을 생리식염수로 세척한 뒤 0.1% Type 2 Collagenase로 16 시간 처리하여 현탁액을 수득하였다. 현탁액을 100 ㎛ 사이즈 나일론 메쉬로 여과한 뒤 600 × g에서 10분간 원심분리하여 상층액을 버리고, 남아있는 세포를 취하여 10% FBS, 1% 페니실린 및 스트렙토마이신(penicillin/streptomycin)을 포함하는 DMEM 배지에서 배양하였다. 매 2~3일 마다 배지를 교체하며, 7~80%의 confluency를 가질 경우 계대를 진행하였다.Porcine cartilage tissue was harvested using a surgical blade from a porcine knee joint. Cartilage tissue was washed with physiological saline and treated with 0.1% Type 2 Collagenase for 16 hours to obtain a suspension. The suspension was filtered through a 100 μm size nylon mesh, centrifuged at 600 × g for 10 minutes, the supernatant was discarded, and the remaining cells were collected in DMEM medium containing 10% FBS, 1% penicillin and streptomycin (penicillin/streptomycin). cultured in. The medium was replaced every 2-3 days, and passages were performed when the confluency was 7-80%.
2. 염증성 사이토카인 및 비스테로이드성 항염증제를 이용한 연골세포의 전처리2. Pretreatment of chondrocytes with inflammatory cytokines and non-steroidal anti-inflammatory drugs
배양 디쉬에 연골 세포를 부착시킨 다음, 6~70%의 confluency를 가질 때까지 배양하였다. 배양배지를 제거하고 1× PBS(phosphate buffered saline)로 세포를 세척한 뒤 FBS가 없는 chondrogenic DMEM 배양액을 처치하였다. Chondrogenic DMEM 배양액은 DMEM에 Insulin-Transferrin-Selenium(ITS) 보충물, 50 μg/ml L-ascorbic acid, 100 nM Dexamethasone, 40 μg/ml L-proline, 1.25 mg/ml Bovine Serum Albumin(BSA), 100 μg/ml Sodium pyruvate, 1% penicillin/streptomycin을 포함하였다. 전처리 그룹은 chondrogenic DMEM 배양액에 0.1 ng/ml 인터루킨-1β(Interleukin-1β)와 10 μM 세레콕시브(celecoxib)를 추가하여 72 시간 동안 처리하였다.After attaching the chondrocytes to the culture dish, they were cultured until they had 6-70% confluency. After removing the culture medium and washing the cells with 1× PBS (phosphate buffered saline), the cells were treated with FBS-free chondrogenic DMEM culture medium. Chondrogenic DMEM cultures were supplemented with Insulin-Transferrin-Selenium (ITS), 50 μg/ml L-ascorbic acid, 100 nM Dexamethasone, 40 μg/ml L-proline, 1.25 mg/ml Bovine Serum Albumin (BSA), 100 μg/ml L-proline in DMEM. μg/ml Sodium pyruvate and 1% penicillin/streptomycin were included. The pretreatment group was treated for 72 hours by adding 0.1 ng/ml interleukin-1β and 10 μM celecoxib to the chondrogenic DMEM culture medium.
3. 전처리 후 연골세포의 생물학적 특성 분석3. Analysis of biological characteristics of chondrocytes after pretreatment
전 처리된 연골세포에서 염증 관련 인자들을 실시간(real-time) RT-PCR을 이용하여 정량 분석하였다. 또한 웨스턴 블랏으로 항염증 인자의 단백질 발현을 평가하였다. 그 결과, 도 2A 및 도 2B와 같이 전처리하지 않은 연골세포에 비해 전 처리된 연골세포에서 항염증 인자로 알려진 인터루킨-10 (IL-10) 및 TGF-β의 발현이 높게 나타나는 것을 확인하였다.Inflammation-related factors in the pre-treated chondrocytes were quantitatively analyzed using real-time RT-PCR. In addition, protein expression of anti-inflammatory factors was evaluated by Western blot. As a result, it was confirmed that the expression of interleukin-10 (IL-10) and TGF-β, known as anti-inflammatory factors, were higher in the pre-treated chondrocytes than in non-pre-treated chondrocytes, as shown in FIGS. 2A and 2B.
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실시예Example
2> 염증성 사이토카인 및 비스테로이드성 항염증제를 이용한 연골조직의 전처리 2> Pre-treatment of cartilage tissue using inflammatory cytokines and non-steroidal anti-inflammatory drugs
1. 돼지 연골 조직 분리 및 염증성 사이토카인과 비스테로이드성 항염증제를 이용한 전처리1. Isolation of porcine cartilage tissue and pretreatment with inflammatory cytokines and non-steroidal anti-inflammatory drugs
상기 연골 조직 분리 방법과 같이 수술용 블레이드를 사용하여 연골조직을 수확하였다. 연골 조직은 다시 생리식염수로 세척한 뒤 블레이드를 사용하여 약 5 mm × 5 mm × 2 mm의 크기의 연골 조각으로 만들었다. 연골세포의 전처리 방법과 동일하게 1× PBS로 조직을 세척하고 chondrogenic DMEM 배양액을 처치한 후, 전처리 그룹은 배양액에 0.1 ng/ml의 인터루킨-1β(Interleukin-1β)와 10 μM 셀레콕시브 (celecoxib)를 처리하여 3일 동안 배양하였다.As in the cartilage tissue separation method, cartilage tissue was harvested using a surgical blade. The cartilage tissue was washed again with physiological saline and then made into pieces of cartilage with a size of about 5 mm × 5 mm × 2 mm using a blade. In the same way as in the pre-treatment of chondrocytes, the tissue was washed with 1× PBS and treated with chondrogenic DMEM culture, and then the pre-treatment group was treated with 0.1 ng/ml of Interleukin-1β and 10 μM celecoxib. ) and cultured for 3 days.
연골조직의 항염증 강화 전처리 방법으로써, 다양한 염증성 사이토카인 및 비스테로이드성 항염증제가 사용될 수 있다. 염증성 사이토카인으로서 인터루킨-1β 또는 TNF-a가 사용될 수 있으며, 비스테로이드성 항염증제로서 살리실산(Salicylic Acid), 프로피온산 (Propionic acid) 또는 Coixb 계열이 사용될 수 있으나 이에 국한된 것은 아니다. As an anti-inflammatory enhancing pretreatment method of cartilage tissue, various inflammatory cytokines and non-steroidal anti-inflammatory drugs can be used. Interleukin-1β or TNF-a may be used as an inflammatory cytokine, and salicylic acid, propionic acid, or Coixb series may be used as a non-steroidal anti-inflammatory agent, but is not limited thereto.
2. 전 처리된 연골조직의 생물학적 특성 분석2. Analysis of biological characteristics of pre-treated cartilage tissue
전 처리된 연골조직에서 웨스턴 블랏으로 항염증 인자의 단백질 발현을 확인하였다. 그 결과 도 3A 및 도 3B와 같이 전 처리된 연골조직은 대조군에 비하여 IL-10, TGF-β와 같은 항염증 인자의 유전자 및 단백질 발현이 높게 나타남을 확인하였다. 전처리 과정에 의한 연골조직 내 세포의 항염증 인자 발현의 작용기전은 도 3C와 같이 염증성 사이토카인은 SMAD 경로를 조절하고, 셀로콕시브와 같은 비스테로이드성 항염증제가 NFkB와 같은 다른 경로를 조절하므로 상기 향상된 항염증 효과와 같은 시너지 효과가 나타나는 것으로 제안될 수 있다.Protein expression of anti-inflammatory factors was confirmed by Western blotting in the pre-treated cartilage tissue. As a result, it was confirmed that the pre-treated cartilage tissue, as shown in FIGS. 3A and 3B, showed higher expression of genes and proteins of anti-inflammatory factors such as IL-10 and TGF-β compared to the control group. The mechanism of action of the expression of anti-inflammatory factors in cells in cartilage tissue by the pretreatment process is as shown in FIG. 3C, since inflammatory cytokines regulate the SMAD pathway and non-steroidal anti-inflammatory agents such as celocoxib regulate other pathways such as NFkB. Synergistic effects such as enhanced anti-inflammatory effects may be suggested.
또한, 인터루킨-1과 셀레콕시브 외 다양한 염증성 사이토카인 및 비스테로이드성 항염증제의 조직 전처리 효능을 RT-PCR 과 웨스턴 블랏을 통하여 평가하였다. 그 결과, 도 4A 및 도 4B와 같이 다양한 염증성 사이토카인 및 비스테로이드성 항염증제 조합에서 대조군에 비해 유의적으로 항염증 인자의 유전자와 단백질 발현이 증가되는 것을 확인하였다. In addition, the tissue pretreatment efficacy of various inflammatory cytokines and non-steroidal anti-inflammatory agents other than interleukin-1 and celecoxib was evaluated through RT-PCR and Western blotting. As a result, as shown in FIGS. 4A and 4B, it was confirmed that the expression of genes and proteins of anti-inflammatory factors was significantly increased compared to the control group in various combinations of inflammatory cytokines and non-steroidal anti-inflammatory agents.
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실시예Example
3> 전 처리된 연골조직의 수용성 및 불용성 인자 분리 후 항염증 특성 분석 3> Analysis of anti-inflammatory properties after separating soluble and insoluble factors from pre-treated cartilage tissue
1. 전 처리된 연골조직의 수용성/불용성 인자 분리1. Separation of soluble/insoluble factors from pre-treated cartilage tissue
전 처리에 따라 연골조직 내 변화되는 성분을 특정하기 위하여 도 5A와 같이 펩신 수용화 처리를 통하여 연골조직의 수용성 신호 분자 (Small molecule)와 불용성 구조 단백질 (Structural molecule)로 분리하여 생물학적 특성을 평가하였다. In order to specify the components changed in the cartilage tissue according to the pretreatment, as shown in FIG. 5A, the biological properties were evaluated by separating the cartilage tissue into soluble signal molecules (small molecules) and insoluble structural proteins (structural molecules) through pepsin solubilization treatment. .
SDS-PAGE 분석 결과, 도 5B와 같이 Structural components에는 콜라겐과 같은 거대 구조 분자가 다량 존재하는 것이 확인되었으며, soluble components에는 상대적으로 작은 사이즈의 단백질들이 다량 존재하고 있음이 확인되었다. As a result of SDS-PAGE analysis, as shown in FIG. 5B, it was confirmed that a large amount of large structural molecules such as collagen were present in the structural components, and a large amount of relatively small-sized proteins were present in the soluble components.
2. Soluble ECM 과 Insoluble ECM components 중 항염증 유효성 확인2. Confirmation of anti-inflammatory efficacy among Soluble ECM and Insoluble ECM components
인간 활액세포(SW982)에 10 ng/ml의 인터루킨-1β(IL-1β)와 25 ng/ml의 종양괴사인자-알파(TNF-α)를 24시간 동안 처리하여 염증을 유도시킨 모델에서 상기에서 분리한 Soluble ECM components와 Insoluble ECM components를 각각 100 μg/ml의 농도로 24 시간 동안 처리하였다. In the model in which inflammation was induced by treating human synovial cells (SW982) with 10 ng/ml of interleukin-1β (IL-1β) and 25 ng/ml of tumor necrosis factor-alpha (TNF-α) for 24 hours, The separated soluble ECM components and insoluble ECM components were treated for 24 hours at a concentration of 100 μg/ml, respectively.
그 결과, 도 6A 및 도 6B 와 같이 염증 주요 매개인자인 IL-1B 및 IL-6 유전자 발현이 모든 전처리군에서 감소하였을 뿐만 아니라, 도 6C 및 도 6D와 같이 COX-2와 p-IκB-α의 발현이 염증 유도군에 비하여 유의적으로 감소하였으며, 특히 전처리 후 soluble ECM components 처치군에서 더욱 유의적인 감소를 확인하였다.As a result, as shown in FIGS. 6A and 6B, the expression of IL-1B and IL-6, which are major mediators of inflammation, decreased in all pretreatment groups, as well as COX-2 and p-IκB-α as shown in FIGS. 6C and 6D. The expression of was significantly decreased compared to the inflammation induction group, and in particular, a more significant decrease was confirmed in the soluble ECM components treatment group after pretreatment.
<실시예 4> 연골조직 유래 세포외소포의 분리 및 생화학적 특성 분석<Example 4> Isolation and biochemical characterization of cartilage tissue-derived extracellular vesicles
1. 연골조직 유래 세포외소포의 분리1. Isolation of cartilage tissue-derived extracellular vesicles
전처리가 끝난 연골조직을 1 × PBS로 세척 후 회수하였다. 이를 - 80℃ 초저온 냉동장치에서 냉각한 후 3일간 동결건조하였다. 건조한 연골 조직의 동일 무게로 소분한 뒤 0.1 mg/ml의 Type 2 Collagenase 용액(in 200 mM NaCl, 5 mM CaCl2, 50 mM Tris buffer)에 2일간 처리하여 현탁액을 얻었다. 현탁액을 고속 원심분리기를 이용하여 500 × g 10분, 2500 × g 20분, 10000 × g 30분 원심분리하여 세포와 세포찌꺼기를 제거한 뒤 순차적으로 0.45 μm 필터튜브, 0.2 μm 필터튜브, 3kD 필터튜브를 이용하여 농축하였다. 농축액을 회수한 뒤 ExoQuick-TC (System Biosciences, USA)를 사용하여 세포외소포를 분리하였으며, Bradford assay를 통하여 동일한 단백질 양으로 소분하고 - 80℃ 초저온 냉동장치에 보관하였다.After the pretreatment, the cartilage tissue was recovered after washing with 1 × PBS. This was cooled in a cryogenic freezer at -80 ° C and freeze-dried for 3 days. After subdividing the same weight of dried cartilage tissue, it was treated with 0.1 mg/ml Type 2 Collagenase solution (in 200 mM NaCl, 5 mM CaCl2, 50 mM Tris buffer) for 2 days to obtain a suspension. The suspension was centrifuged at 500 × g for 10 minutes, 2500 × g for 20 minutes, and 10000 × g for 30 minutes using a high-speed centrifuge to remove cells and cell debris, followed by 0.45 μm filter tube, 0.2 μm filter tube, and 3kD filter tube in sequence. was concentrated using After recovering the concentrate, extracellular vesicles were separated using ExoQuick-TC (System Biosciences, USA), subdivided into equal amounts of protein through Bradford assay, and stored in a cryogenic freezer at -80°C.
2. 세포외소포의 생화학적 특성 분석2. Analysis of biochemical characteristics of extracellular vesicles
전처리한 연골조직으로부터 분리된 세포외소포를 주사전자현미경(scanning electron microscope)을 사용하여 형태 및 사이즈를 분석하였다. 그리고 동적 광 산란 (dynamic light Scattering) 방법을 통하여 입도 분포를 측정하였다 (ELSZ-2000, Otsuka Electronics, Osaka, Japan). The shape and size of the extracellular vesicles isolated from the pretreated cartilage tissue were analyzed using a scanning electron microscope. Then, the particle size distribution was measured using a dynamic light scattering method (ELSZ-2000, Otsuka Electronics, Osaka, Japan).
그 결과, 도 7B 및 도 7C와 같이 세포외소포의 크기는 100 ~ 200 nm size를 가지고 있음을 확인하였다. As a result, it was confirmed that the size of the extracellular vesicles had a size of 100 to 200 nm, as shown in FIGS. 7B and 7C.
또한, 세포외소포의 세포침투능력 확인을 위하여 세포외소포를 PKH-26 labeling한 뒤 SW982에 처리하여 confocal laser scanning을 통하여 확인하였다. 그 결과, 도 7D와 같이 세포외소포가 세포 내 핵 근처에 침투하였음을 확인하였다. 또한, 도 7E와 같이 웨스턴 블랏으로 세포외소포 특이적인 표면항원인 CD9 및 CD81 발현을 확인하였다. In addition, to confirm the cell penetration ability of extracellular vesicles, the extracellular vesicles were labeled with PKH-26, treated with SW982, and confirmed through confocal laser scanning. As a result, as shown in FIG. 7D, it was confirmed that extracellular vesicles penetrated near the nucleus within the cell. In addition, as shown in FIG. 7E, expression of CD9 and CD81, which are surface antigens specific to extracellular vesicles, was confirmed by Western blotting.
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실시예Example
5> 5>
세포외소포의extracellular
전처리 전후 생물학적 특성 및 항염증 기능 유효성 확인 Validation of biological properties and anti-inflammatory function before and after pretreatment
1. One.
연골조직유래cartilage tissue origin
세포외소포extracellular vesicle
및 전처리 후 and after pretreatment
연골조직유래cartilage tissue origin
세포외소포의extracellular
항염증 유효성 확인 Anti-inflammatory validation
연골조직유래 세포외소포와 전처리 후 연골조직유래 세포외소포의 항염증 유효성 수준을 확인하기 위해, 인간 활액막세포 (SW982)에 10 ng/ml의 인터루킨-1β (IL-1β)와 25 ng/ml의 종양괴사인자-알파 (TNF-α)를 이용하여 24시간 동안 염증을 유도한 다음 웨스턴블롯을 수행하여 세포외소포 처리군 및 전처리 후 세포외 소포 처리군을 비교하였다. In order to confirm the anti-inflammatory efficacy level of cartilage tissue-derived extracellular vesicles and cartilage tissue-derived extracellular vesicles after pretreatment, human synovial cells (SW982) were treated with 10 ng/ml of interleukin-1β (IL-1β) and 25 ng/ml Inflammation was induced for 24 hours using tumor necrosis factor-alpha (TNF-α), and Western blotting was performed to compare the extracellular vesicles treated group and the pretreated extracellular vesicles treated group.
그 결과, 도 8A 및 도 8B와 같이 RT-qPCR을 이용하여 유전자 발현을 확인하였을 때 염증성 매개인자인 IL-1β, IL-6는 염증 그룹에서 증가하였으나 전처리된 세포외소포 처리군에서는 감소한 것을 확인할 수 있었다.As a result, when gene expression was confirmed using RT-qPCR as shown in FIGS. 8A and 8B, the inflammatory mediators IL-1β and IL-6 were increased in the inflammatory group but decreased in the pre-treated extracellular vesicles-treated group. could
또한, 도 8C 및 도 8D와 같이 주요 염증매개인자인 COX-2, p-IκB-α 및 phospho p65의 발현이 염증그룹에서는 증가한 반면, 세포외소포 처리군에서는 발현이 모두 감소하는 것을 확인할 수 있었다.In addition, as shown in FIGS. 8C and 8D, the expression of COX-2, p-IκB-α, and phospho p65, which are major inflammatory mediators, increased in the inflammatory group, whereas in the group treated with extracellular vesicles, it was confirmed that the expressions all decreased. .
또한, 각 처리군의 배양액을 회수하여 배양배지 속 염증인자의 발현을 ELISA로 확인하였다. In addition, the culture medium of each treatment group was collected and the expression of inflammatory factors in the culture medium was confirmed by ELISA.
그 결과, 도 8E와 같이 염증 매개인자인 PGE2의 발현이 염증 그룹에서는 증가하였으나 세포외소포 처리군에서는 감소하였다. 상기 결과로부터 연골조직유래 세포외소포가 인간 활액세포의 염증 정도를 감소시키는 항염증 기능이 있음이 확인되었다.As a result, as shown in FIG. 8E, the expression of PGE2, an inflammatory mediator, increased in the inflammatory group but decreased in the extracellular vesicles-treated group. From the above results, it was confirmed that cartilage tissue-derived extracellular vesicles have an anti-inflammatory function that reduces the degree of inflammation of human synovial cells.
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실시예Example
6> 연골조직유래6> Derived from cartilage tissue
세포외기질extracellular matrix
및 and
세포외소포의extracellular
M2 Macrophage 분화 유도능 확인 Confirmation of M2 Macrophage differentiation inducing ability
연골조직유래 세포외기질 (CECM) 및 세포외소포 (CEV)와 전처리 후 연골조직유래 세포외기질(P_CECM) 및 세포외소포 (P_CEV)의 M2 Macrophage 분화 유도 수준을 확인하기 위해, M0 Macrophage에 각 그룹을 10 μg/ml 의 농도로 처치하고 CD86 (M1 marker) 와 CD163 (M2 marker) 에 대한 ICC 분석을 진행하였다. 양성대조군 그룹으로서, M1 Macrophage 분화 유도는 100 ng/ml의 LPS, 와 20 ng/ml의 IFN-γ를 사용하였으며, M2 Macrophage는 20 ng/ml의 IL-4 와 20 ng/ml의 IL-13을 M0 Macrophage에 24시간 처치하여 각각 분화를 유도하였다.To confirm the level of induction of M2 Macrophage differentiation of cartilage tissue-derived extracellular matrix (CECM) and extracellular vesicles (CEV) and cartilage tissue-derived extracellular matrix (P_CECM) and extracellular vesicles (P_CEV) after pretreatment, M0 Macrophage The group was treated with a concentration of 10 μg/ml, and ICC analysis for CD86 (M1 marker) and CD163 (M2 marker) was performed. As a positive control group, 100 ng/ml of LPS and 20 ng/ml of IFN-γ were used to induce differentiation of M1 macrophage, and 20 ng/ml of IL-4 and 20 ng/ml of IL-13 were used for M2 macrophage. were treated with M0 Macrophage for 24 hours to induce differentiation.
그 결과, 도 9A와 같이 ICC 분석에서 M1 및 M2 marker의 발현을 확인하였을 때 전처리된 세포외기질 및 세포외소포 처리군에서는 M2 marker의 발현이 우세한 것을 확인할 수 있었다.As a result, when confirming the expression of the M1 and M2 markers in the ICC analysis as shown in FIG. 9A, it was confirmed that the expression of the M2 marker was dominant in the pretreated extracellular matrix and extracellular vesicles treated groups.
또한, 도 9B와 같이 M1/M2 marker의 비율을 정량적으로 분석하였을 때 전처리 과정에 의하여 세포외기질 및 세포외소포체 모두 유의적으로 M2 marcrophage의 분화 유도가 증가되는 것을 확인 할 수 있었다.In addition, when the ratio of M1 / M2 markers was quantitatively analyzed as shown in FIG. 9B, it was confirmed that the induction of differentiation of M2 marcrophages was significantly increased in both the extracellular matrix and the extracellular vesicles by the pretreatment process.
<<
실시예Example
7> 골관절염 ( 7> Osteoarthritis (
OAOA
)가 유도된 랫 모델에서 ) in the induced rat model
세포외소포의extracellular
항염증 기능 유효성 확인 Validation of anti-inflammatory function
1. 렛 골관절염 질환 모델에서의 연골조직 유래 세포외소포 투여1. Administration of cartilage tissue-derived extracellular vesicles in a rat osteoarthritis disease model
실험에 사용한 동물로는 8주령 수컷 SD-Rat 을 구입하여 일주일 동안 순화기간을 거친 후 9주령에 실험에 사용하였다. 골관절염을 유도하기 위하여 미처리군을 제외한 랫의 왼쪽 다리 관절활액에 Type II collagenase를 3 U/ul 농도로 30 μl를 주입하였다. 처리군은 2주 동안 20 m/min의 속도로 매일 40 분씩 Treadmill running을 강제하였다. 이후 PBS 처리군, 연골조직유래 세포외기질 처리군 (CECM), 전처리후 세포외기질 처리군 (P_CECM), 연골조직유래 세포외소포 처리군 (CEV), 전처리 후 세포외소포 처리군 (P_CEV)은 2 μg/μl 농도로 30 μl를 왼쪽다리 관절활액에 1 주일 간격으로 4 주간 주입하였다. 세포외소포 처치 후 랫을 희생시켜 조직학적 분석을 수행하였다.As the animal used in the experiment, an 8-week-old male SD-Rat was purchased and used for the experiment at 9 weeks of age after a week of acclimatization. To induce osteoarthritis, 30 μl of Type II collagenase at a concentration of 3 U/ul was injected into the synovial fluid of the left leg of rats, except for the untreated group. The treatment group was forced to treadmill running for 40 minutes every day at a speed of 20 m/min for 2 weeks. Then, the PBS treatment group, the cartilage tissue-derived extracellular matrix treatment group (CECM), the pretreatment post-treatment extracellular matrix treatment group (P_CECM), the cartilage tissue-derived extracellular vesicle treatment group (CEV), the pretreatment post-extracellular vesicle treatment group (P_CEV) 30 μl of silver at a concentration of 2 μg/μl was injected into the synovial fluid of the left leg at weekly intervals for 4 weeks. After treatment with extracellular vesicles, rats were sacrificed and histological analysis was performed.
2. 체내 2. body
연골조직유래cartilage tissue origin
세포외소포와with extracellular vesicles
전처리 후 after pretreatment
세포외소포extracellular vesicle
처리군의treatment group
조직학적 분석 histological analysis
관절 내 연골 및 활액막의 염증 정도를 평가하기 위하여, 도 10A와 같이 사프라닌-O 염색, H&E 염색, 단핵구(monocyte) 특이 표면항원 CD68의 면역화학염색(Immunohistochemistry)을 통하여 조직학 평가를 진행하였다.In order to evaluate the degree of inflammation of the cartilage and synovial membrane in the joint, histological evaluation was performed through safranin-O staining, H&E staining, and immunohistochemical staining of the monocyte-specific surface antigen CD68 as shown in FIG. 10A.
그 결과, 도 10A와 같은 사프라닌-O 염색 결과 PBS 처리군에서 연골의 sGAG의 함량이 감소되었으며 초기단계 골관절염의 조직학 형태를 보여주었다. 하지만 세포외기질 및 세포외소포 처리군에서는 미처리군과 유사한 sGAG 함량을 보여주었다. As a result, as a result of safranin-O staining as shown in FIG. 10A, the sGAG content of cartilage was reduced in the PBS-treated group, and the histological morphology of early-stage osteoarthritis was shown. However, the extracellular matrix and extracellular vesicles treated groups showed sGAG content similar to that of the untreated group.
또한, 정량적인 분석을 위하여 염증이 발생할 때 발현되는 단핵구 특이 표면항원인 CD68의 면역화학염색을 진행하였다. In addition, for quantitative analysis, immunochemical staining of CD68, a monocyte-specific surface antigen expressed when inflammation occurs, was performed.
그 결과, 도 10B와 같이 CD68의 발현이 미처리군에 비하여 PBS 처리군에서 증가하며 전처리 후 세포외기질 및 세포외소포 처리군은 PBS 처리군에 비하여 감소를 나타내었다.As a result, as shown in FIG. 10B, the expression of CD68 was increased in the PBS-treated group compared to the untreated group, and after pretreatment, the extracellular matrix and extracellular vesicles-treated group showed a decrease compared to the PBS-treated group.
상기 결과들로부터 전처리 후 세포외기질 및 세포외소포 처리군은 각 전처리 그룹에 비교하여 향상된 연골의 골관절염 감소 효과와 함께 활액막의 염증정도를 감소시키는 효과를 나타내는 것이 확인되었다.From the above results, it was confirmed that the group treated with extracellular matrix and extracellular vesicles showed an effect of reducing the degree of synovial inflammation as well as an improved effect of reducing osteoarthritis of cartilage compared to each pretreatment group.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (9)
- 염증성 사이토카인, 비스테로이드 항염증제 또는 이들의 조합으로 전 처리된 연골조직 유래 세포외기질을 유효성분으로 함유하는 염증성 질환 예방 또는 치료용 약학조성물.A pharmaceutical composition for the prevention or treatment of inflammatory diseases containing, as an active ingredient, an inflammatory cytokine, a nonsteroidal anti-inflammatory agent, or a pre-treated cartilage tissue-derived extracellular matrix with a combination thereof.
- 청구항 1에 있어서, 상기 전 처리된 연골조직 유래 세포외기질은 항염증성이 강화된 세포외소포를 포함하는 것을 특징으로 하는 염증성 질환 예방 또는 치료용 약학조성물.The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 1, wherein the pre-treated cartilage tissue-derived extracellular matrix comprises extracellular vesicles with enhanced anti-inflammatory properties.
- 청구항 1에 있어서, 상기 염증성 사이토카인은 인터루킨-1β, TNF-a 및 인터페론-γ 로 이루어진 군에서 하나 이상 선택되는 것을 특징으로 하는 염증 질환성 예방 또는 치료용 약학조성물.The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 1, wherein the inflammatory cytokine is one or more selected from the group consisting of interleukin-1β, TNF-a and interferon-γ.
- 청구항 1에 있어서, 상기 비스테로이드 항염증제는 살리실산(Salicylic Acid), 프로피온산 (Propionic acid), 세레콕시브(celecoxib), 나프록센 (naproxen), 아세트산(Acetic acid) 및 페나믹산 (Fenamic acid)으로 이루어진 군에서 하나 이상 선택되는 것을 특징으로 하는 염증성 질환 예방 또는 치료용 약학조성물.The method according to claim 1, wherein the non-steroidal anti-inflammatory agent is selected from the group consisting of salicylic acid, propionic acid, celecoxib, naproxen, acetic acid and fenamic acid A pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that one or more are selected.
- 청구항 1에 있어서, 상기 염증성 질환은 피부염, 아토피 피부염, 천식, 결막염, 치주염, 비염, 중이염, 홍채염, 인후염, 편도염, 폐렴, 위궤양, 췌장염, 위염, 크론병, 염증성 장질환, 대장염, 치질, 통풍, 강직성 척추염, 루프스, 섬유근통(fibromyalgia), 건선, 류마티스 관절염, 골관절염, 골다공증, 간염, 방광염, 신장염, 쇼그렌 증후군 및 다발성 경화증으로 이루어진 그룹에서 선택되는 어느 하나인 것을 특징으로 하는 염증성 질환 예방 또는 치료용 약학조성물.The method according to claim 1, wherein the inflammatory disease is dermatitis, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, gout For preventing or treating inflammatory diseases, characterized in that any one selected from the group consisting of ankylosing spondylitis, lupus, fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome and multiple sclerosis pharmaceutical composition.
- 개체로부터 분리된 연골조직에 염증성 사이토카인, 비스테로이드 항염증제 또는 이들의 조합을 전처리하는 단계; Pre-treating the cartilage tissue isolated from the subject with an inflammatory cytokine, a non-steroidal anti-inflammatory agent, or a combination thereof;상기 전 처리된 연골조직을 1 내지 5일간 배양하는 단계; 및 Culturing the pre-treated cartilage tissue for 1 to 5 days; and상기 배양된 연골조직으로부터 세포외기질을 분리하는 단계를 포함하는 세포외기질 항염증 기능 강화 방법.A method for enhancing the extracellular matrix anti-inflammatory function comprising the step of separating the extracellular matrix from the cultured cartilage tissue.
- 청구항 6에 있어서, 상기 염증성 사이토카인은 인터루킨-1β, TNF-a 및 인터페론-γ로 이루어진 군에서 하나 이상 선택되는 것을 특징으로 하는 세포외기질 항염증 기능 강화 방법.The method according to claim 6, wherein the inflammatory cytokine is selected from the group consisting of interleukin-1β, TNF-a and interferon-γ.
- 청구항 6에 있어서, 상기 비스테로이드 항염증제는 살리실산(Salicylic Acid), 프로피온산 (Propionic acid), 세레콕시브(celecoxib), 나프록센 (naproxen), 아세트산(Acetic acid) 및 페나믹산 (Fenamic acid)으로 이루어진 군에서 하나 이상 선택되는 것을 특징으로 하는 세포외기질 항염증 기능 강화 방법.The method according to claim 6, wherein the nonsteroidal anti-inflammatory agent is selected from the group consisting of salicylic acid, propionic acid, celecoxib, naproxen, acetic acid and fenamic acid A method for enhancing the extracellular matrix anti-inflammatory function, characterized in that one or more are selected.
- 청구항 6에 있어서, 상기 세포외기질은 항염증성이 강화된 세포외소포를 포함하는 것을 특징으로 하는 세포외기질 항염증 기능 강화 방법.The method according to claim 6, wherein the extracellular matrix comprises extracellular vesicles with enhanced anti-inflammatory properties.
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