KR101695140B1 - Pharmaceutical composition for prevention and treatment of bone diseases - Google Patents
Pharmaceutical composition for prevention and treatment of bone diseases Download PDFInfo
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- KR101695140B1 KR101695140B1 KR1020160073685A KR20160073685A KR101695140B1 KR 101695140 B1 KR101695140 B1 KR 101695140B1 KR 1020160073685 A KR1020160073685 A KR 1020160073685A KR 20160073685 A KR20160073685 A KR 20160073685A KR 101695140 B1 KR101695140 B1 KR 101695140B1
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- cartilage
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- extract
- bone
- stem cells
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Abstract
Description
본 발명은 골질환 또는 연골질환의 예방 또는 치료용 약학 조성물에 관한 것으로, 보다 상세하게는 탈단백 소혈액 추출물을 포함하며, 골의 염증 반응 억제, 파골억제, 재생 및 분화 촉진 효과를 나타내는 골질환 또는 연골질환의 예방 또는 치료용 약학 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for preventing or treating bone diseases or cartilage diseases, and more particularly, to a pharmaceutical composition for preventing or treating osteoporosis, Or a pharmaceutical composition for the prevention or treatment of cartilage diseases.
탈단백 송아지 혈액 추출물은 장기추출법에 의해 발육 최성기인 어린 소의 세포내피조직을 부화시킨 후 채혈하여 단백질을 완전히 제거시킨 혈액 추출물이다. 이러한 송아지의 탈단백 혈액 추출물은 유기물질로서 아미노산, 케톤산, 옥시산류, 데옥시리보사이드류, 퓨린류 및 미지의 폴리펩티드를 다량 함유하고 있으며, 무기물로서 나트륨, 칼륨, 칼슘, 철, 코발트, 인, 염화물 및 질소 등의 미량원소를 함유하여 세포의 산소 이용을 증가시키고 괴사조직의 재생을 촉진시켜 각종 피부질환에 있어서 육아조직의 형성과 상피조직의 형성을 촉진시키는 것으로 알려져 있다(비특허문헌 1 및 비특허문헌 2). The deproteinized calf blood extract is a blood extract obtained by hatching the endothelial tissues of young cattle, which is the highest developmental stage, by long term extraction, and collecting blood and completely removing proteins. The deproteinized blood extract of this calf contains a large amount of amino acids, ketonic acids, oxyacids, deoxyribosides, purines and unknown polypeptides as an organic substance, and contains sodium, potassium, calcium, iron, cobalt , Chloride, and nitrogen to increase the oxygen utilization of cells and promote regeneration of necrotic tissue, thereby promoting the formation of granulation tissues and epithelial tissues in various skin diseases (Non-Patent Document 1 And non-patent document 2).
상기 탈단백 송아지 혈액 추출물을 포함하는 제재로서 솔코세릴® (SOLCOSERYL, Solco Basle Ltd., 스위스)이라는 상품명으로 시판되고 있는데, 이는 안과용 겔제로서 상용화되고 있다(특허문헌 1). 상기 추출물을 포함하는 제재로서 다른 상품명인 악토베긴®은 한외여과에 의해 송아지 혈액으로부터 생산되는, 분자량 5kDa 이하인 저분자량 화합물을 포함하는 탈단백 혈액유래 제재로서 당뇨병성 다발성 말초신경병증의 예방 또는 치료에 사용되고 있다(특허문헌 2). It is marketed as SOLCOSERYL (Solco Basle Ltd., Switzerland) as a preparation containing the deproteinized calf blood extract, which has been commercialized as an ophthalmic gel (Patent Document 1). As another medicinal product containing the above extract, Aktobegin® is a deproteinized blood-derived material containing a low molecular weight compound having a molecular weight of 5 kDa or less, produced from calf blood by ultrafiltration, and is used for the prevention or treatment of diabetic multiple peripheral neuropathy (Patent Document 2).
현재까지 소 혈액 유래 탈단백 성분을 유효성분으로 포함하는 조성물의 용도는 골 질환 중 관절염에 한정되어 있어서(특허문헌 3, 특허문헌 4), 보다 다양한 골 질환에 대한 치료 용도의 개발이 필요한 실정이다.To date, the use of a composition comprising a bovine blood-derived deproteinized component as an active ingredient has been limited to arthritis in bone diseases (
본 발명은 상기와 같은 종래의 기술상의 문제점을 해결하기 위해 안출된 것으로, 본 발명의 일 목적은 소혈액 유래 탈단백 조성물을 포함하는 골질환 또는 연골질환의 예방 또는 치료에 사용할 수 있는 조성물 및 그 제조방법을 제공하고자 한다.SUMMARY OF THE INVENTION It is an object of the present invention to provide a composition which can be used for the prevention or treatment of bone diseases or cartilage diseases including a bovine blood-derived deproteinized composition, And to provide a manufacturing method thereof.
본 발명의 다른 목적은 소혈액 유래 탈단백 조성물을 포함하는 골질환 또는 연골질환 개선용 조성물 및 그 제조방법을 제공하고자 한다.Another object of the present invention is to provide a composition for improving bone diseases or cartilage diseases comprising a bovine blood-derived deproteinized composition and a method for producing the same.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당 업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.
상기와 같은 문제점을 해결하고자 본 발명자들은 소혈액 유래 혈액의 탈단백 조성물을 포함하는 조성물의 골 질환 또는 연골질환에 대한 치료 효과, 연골 재생 효과 및 줄기세포에 대한 연골세포로의 분화 효과를 확인하여 본 발명에 이르게 되었다.In order to solve the above-mentioned problems, the inventors of the present invention confirmed the therapeutic effect on the bone diseases or cartilage diseases, the cartilage regeneration effect and the differentiation effect on the chondrocyte for the stem cells of the composition containing the de- Leading to the present invention.
골은 골격계의 안전성을 유지하며 가장 단단한 조직을 이루고, 근육의 지렛대로 사용되고 내부의 장기를 보호할 뿐만 아니라 칼슘, 마그네슘 같은 미네랄을 저장하는 역할을 하는데, 골격에 손상이 일어나 골 질환이 생기면 뼈의 구성 및 밀도가 변화하고 골절이 일어나기 쉬운 상태가 된다. 골질환은 기전에 따라 골괴사증(osteonecrosis), 가관절증(Pseudoarthrosis), 파제트병(Paget’s disease), 골형성 부전증(Osteogenesis imperfecta) 등이 있다. 골괴사증은 허혈성 괴사증, 무혈성 괴사증과 같은 의미로 사용되며 혈액순환의 장애로 산소나 영양공급이 차단되어 골 조직이 사멸되는 통증성 질환으로 모든 골에서 발생되지만 주로 고관절에서 진행되고, 심한 경우 관절염으로 진행되기도 한다. 가관절증은 골절 후의 정복, 고정의 불량, 골절부의 세균 감염증 등이 원인이 되어 골절부의 뼈가 완전히 유합되지 않아 마치 관절처럼 움직이는 상태로, 골절부를 맞추어 단단하게 연결시키고 골이식에 의해 치료된다. 파제트병은 노년기의 골 재형성 과정에서 생기는 대사성 장애로 광범위한 부위의 골격계가 침범되는 국소성 골 질환으로 골 형성이 모자이크 상으로 일어나 섬유화와 혈관신생을 동반하고 기형과 종창이 발생한다. 골형성부전증은 특별한 원인 없이 뼈가 약하여 재채기를 하거나 가볍게 부딪치는 행동만으로도 골절되는 선천성 희귀 유전질환으로 환자에서 골절 증상 이외에 청색 공막이 잘 나타난다. 골질환 중에서 가장 빈번히 발생하는 골다공증은 뼈를 형성하는 무기질과 기질의 양이 과도하게 감소하여 골밀도가 감소된 상태를 나타내는 것으로, 이로 인해 골의 강도가 약해져서 골절이 일어날 가능성이 높으며, 유전, 조기 폐경, 과도한 식이요법, 스테로이드 약제 등이 주요 요인으로 알려져 있다.The goal is to maintain the safety of the skeletal system, to form the most rigid tissue, to leverage the muscles and to protect the internal organs, as well as to store minerals such as calcium and magnesium. The configuration and density change and a fracture tends to occur. Bone disease may be osteonecrosis, pseudoarthrosis, Paget's disease, and osteogenesis imperfecta depending on the mechanism. Osteonecrosis is used in the same sense as ischemic necrosis and avascular necrosis. It is a painful disease in which bone tissue is killed due to interruption of blood circulation due to obstruction of oxygen or nutrition. It occurs in all bones but progresses mainly in the hip joint. In severe cases, arthritis It is also progressing. Knee is treated by bone grafting, connecting the fractured part firmly and moving like a joint, because the bone of the fracture part is not completely united due to congestion after fracture, poor fixation, and bacterial infection of the fracture part. Paget 's disease is a metabolic disorder caused by the bone remodeling process in old age. It is a localized bone disease involving a wide range of skeletal system, resulting in mosaic formation of bone formation, accompanied by fibrosis and angiogenesis, and malformation and swelling. Osteoporosis is a congenital rare genetic disorder in which fractures are caused by only sneezing or light bite without weak bone. The most frequent osteoporosis is osteoporosis, which is a condition in which the amount of minerals and substrates forming the bone is excessively decreased, resulting in a decrease in bone mineral density, which results in a weakening of the bone strength and a high possibility of fracture, , Excessive diet, and steroid medications are known to be major factors.
현재까지 알려진 골 질환의 치료법은 자가골 이식술, 동종골 이식 및 이종골 이식술, Masquelet 수술법, 골 신연술(일라자로프 수술) 등이 사용되고 있으며 줄기세포를 활용한 재생의료 치료 접근법이 최근에 활발하게 개발되고 있다.Currently known bone disease treatments include autogenous bone grafting, allograft grafting and xenograft grafting, masqueting and osteolytic surgery (Illazarov surgery), and regenerative medical therapy approaches using stem cells have been actively developed .
한편, 연골질환은 연골 조직내 발생하는 질환으로 연골 조직은 인체 내에서 독특한 조직으로서 혈관과 신경이 존재하지 않고, 발생하는 과정을 제외하면 대부분 관절에 위치해 있어 관절염의 치료 표적이 되고 있다. 관절염은 관절의 손상을 수반하는 여러 질환을 통칭하는 것으로 원인과 증상에 따라 골 관절염(퇴행성 관절염)과 류마티스 관절염(염증성 관절염)으로 크게 나눌 수 있다. 연골 질환의 치료법으로 생체재료를 이용한 미세골절술, 자가연골세포 이식술, 기질기반 연골세포 이식술이 알려져 있으며 줄기세포를 활용한 재생의료 치료법이 개발되고 있는 중이다. On the other hand, cartilage disease is a disease that occurs in the cartilage tissue. Cartilage tissue is a unique tissue in the human body. It is located in the joints except for the blood vessels and nerves. Arthritis refers to various diseases that involve joint damage, and can be divided into osteoarthritis (degenerative arthritis) and rheumatoid arthritis (inflammatory arthritis) depending on the cause and symptoms. Microfracture using autologous bone, autologous chondrocyte transplantation, and matrix-based chondrocyte transplantation are known as a treatment for cartilage disease, and regenerative medical therapy using stem cells is being developed.
이하, 본원에 기재된 다양한 구현예가 도면을 참조로 기재된다. 하기 설명에서, 본 발명의 완전한 이해를 위해서, 다양한 특이적 상세사항, 예컨대, 특이적 형태, 조성물 및 공정 등이 기재되어 있다. 그러나, 특정의 구현예는 이들 특이적 상세 사항 중 하나 이상 없이, 또는 다른 공지된 방법 및 형태와 함께 실행될 수 있다. 다른 예에서, 공지된 공정 및 제조 기술은 본 발명을 불필요하게 모호하게 하지 않게 하기 위해서, 특정의 상세사항으로 기재되지 않는다. "한 가지 구현예" 또는 "구현예"에 대한 본 명세서 전체를 통한 참조는 구현예와 결부되어 기재된 특별한 특징, 형태, 조성 또는 특성이 본 발명의 하나 이상의 구현예에 포함됨을 의미한다. 따라서, 본 명세서 전체에 걸친 다양한 위치에서 표현된 "한 가지 구현예에서" 또는 "구현예"의 상황은 반드시 본 발명의 동일한 구현예를 나타내지는 않는다. 추가로, 특별한 특징, 형태, 조성, 또는 특성은 하나 이상의 구현예에서 어떠한 적합한 방법으로 조합될 수 있다.Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, the specific embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the invention. In addition, particular features, shapes, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.
본 발명의 일 구현예에서, 소혈액 유래 탈단백 추출물을 유효성분으로 포함하는 골질환 또는 연골질환의 예방 또는 치료용 약학 조성물을 제공한다. 상기 구현예에서, 상기 골질환은 전신 골 질환 또는 국소 골 질환 및 손상 중 어느 하나이며, 상기 전신 골 질환은 골다공증(osteoporosis)이고, 상기 국소 골 질환 및 손상은 골 유합장애(Nonunion/Delayed union), 가관절증(Pseudoarthrosis), 골 괴사(Osteonecrosis) 및 기타 골절(other fractures)로 구성된 군으로부터 선택된 어느 하나이며, 상기 연골질환은 중성 연골질환 또는 연골 손상 중 어느 하나이며, 상기 중성 연골질환은 골 관절염(Osteoarthritis)이고, 상기 연골 손상은 연골부분층결손, 연골전층결손, 골연골결손, 골연골박리염 (Osteochondritis dissecans), 연골연화증(Chondromalacia) 및 반월상 연골 손상(Meniscus injury)으로 구성된 군으로부터 선택된 어느 하나이며, 상기 조성물은 황막세포의 염증 증가를 억제시키는 골질환 또는 연골질환의 예방 또는 치료용 약학 조성물을 제공한다. In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating osteoporosis or cartilage disease comprising an extract of bovine blood derived protein as an active ingredient. In this embodiment, the bone disease is any one of systemic bone disease or local bone disease and injury, the systemic bone disease is osteoporosis, and the local bone disease and damage is a nonunion / Wherein the cartilage disease is any one selected from the group consisting of pseudoarthrosis, osteonecrosis, and other fractures. The cartilage disease is any one of neutral cartilage disease or cartilage damage, Wherein the cartilage damage is selected from the group consisting of a cartilage partial layer defect, an osteochondral defect, an osteochondral defect, an osteochondritis dissecans, a chondromalacia, and a meniscus injury And the composition provides a pharmaceutical composition for preventing or treating osteopathy or cartilage disease that inhibits an increase in inflammation of the cells of the ovary The.
본 발명의 다른 일 구현예에서, 소혈액 유래 탈단백 추출물을 유효성분으로 포함하는 연골세포 재생 또는 분화 촉진용 조성물을 제공한다. 상기 구현예에서, 상기 조성물은 파골인자인 골단백질 분해효소의 발현을 억제하며, 상기 골단백질 분해효소는 MMP-1a, MMP-3, MMP-9 및 MMP-13으로 구성된 군으로부터 선택된 어느 하나인 연골세포 재생 또는 분화 촉진용 조성물을 제공한다. In another embodiment of the present invention, there is provided a composition for promoting chondrocyte regeneration or differentiation comprising an unprotected bovine blood extract as an active ingredient. In this embodiment, the composition inhibits the expression of bone protease, which is an osteoclast factor, and the bone protease is any one selected from the group consisting of MMP-1a, MMP-3, MMP-9 and MMP- There is provided a composition for promoting chondrocyte cell regeneration or differentiation.
본 발명의 다른 일 구현예에서, 소혈액 유래 탈단백 추출물을 유효성분으로 포함하는 골질환 또는 연골질환에 의해 발생하는 통증 완화용 조성물을 제공한다. 상기 구현예에서, 상기 골질환은 전신 골 질환 또는 국소 골 질환 및 손상 중 어느 하나이고 상기 연골질환은 중성 연골질환 또는 연골 손상 중 어느 하나이며, 상기 전신 골 질환은 골다공증(osteoporosis)이고, 상기 국소 골 질환 및 손상은 골 유합장애(Nonunion/Delayed union), 가관절증(Pseudoarthrosis), 골 괴사(Osteonecrosis) 및 기타 골절(other fractures)로 구성된 군으로부터 선택된 어느 하나이며, 상기 중성 연골질환은 골 관절염(Osteoarthritis)이고, 상기 연골 손상은 연골부분층결손, 연골전층결손, 골연골결손, 골연골박리염(Osteochondritis dissecans), 연골연화증(Chondromalacia) 및 반월상 연골 손상(Meniscus injury)으로 구성된 군으로부터 선택된 어느 하나인, 골질환 또는 연골질환에 의해 발생하는 통증 완화용 조성물을 제공한다.In another embodiment of the present invention, there is provided a composition for relieving pain caused by osteopathy or cartilage disease comprising a bovine blood-derived deproteinized extract as an active ingredient. In this embodiment, the bone disease is any one of generalized bone disease, local bone disease and injury, and the cartilage disease is any one of neutral cartilage disease or cartilage damage, the whole body bone disease is osteoporosis, The bone disease and damage are any one selected from the group consisting of Nonunion / Delayed Union, Pseudoarthrosis, Osteonecrosis and other fractures, and the Neutral cartilage disease is osteoarthritis Wherein the cartilage damage is selected from the group consisting of a cartilage partial layer defect, an osteochondral defect, an osteochondral defect, an osteochondritis dissecans, a chondromalacia, and a meniscus injury. The present invention provides a composition for relieving pain caused by bone diseases or cartilage diseases.
본 발명의 또 다른 일 구현예에서, 소혈액 유래 탈단백 추출물을 유효성분으로 포함하는 줄기세포의 연골세포 분화 유도 또는 촉진용 조성물을 제공한다. 상기 구현예에서, 상기 조성물은 줄기세포의 골 조직으로의 이동을 유도하고, 상기 추출물은 연골세포의 SOX9 발현을 증가시키며, 상기 줄기세포는 골수유래 또는 지방조직유래 줄기세포인 줄기세포의 연골세포 분화 유도 또는 촉진용 조성물을 제공한다. In another embodiment of the present invention, there is provided a composition for inducing or promoting chondrocyte differentiation of stem cells comprising a bovine blood-derived deproteinized extract as an active ingredient. In this embodiment, the composition induces migration of stem cells to bone tissue, wherein the extract increases SOX9 expression of chondrocytes, and the stem cells are chondrocytes of bone marrow-derived or adipose tissue-derived stem cells Thereby providing a composition for inducing or promoting differentiation.
본 발명의 또 다른 일 구현예에서, 소혈액 유래 탈단백 추출물을 사용하여 줄기세포로부터 연골세포로의 분화를 유도하는 단계를 포함하는 줄기세포로부터 연골세포 분화 방법을 제공한다. 상기 구현예에서, 상기 줄기세포는 골수유래 또는 지방조직 유래이고, 상기 분화는 SOX9의 발현 증가에 의해 매개되는, 줄기세포로부터 연골세포 분화 방법을 제공한다.In another embodiment of the present invention, there is provided a method of differentiating chondrocytes from stem cells comprising inducing differentiation from stem cells into chondrocytes using a bovine blood-derived deproteinized extract. In this embodiment, the stem cells are derived from bone marrow or adipose tissue, and the differentiation is mediated by increased expression of SOX9, which provides a method of differentiating chondrocytes from stem cells.
본 발명의 또 다른 일 구현예에서, 소혈액 유래 탈단백 추출물을 유효성분으로 포함하는 연골 손상 치료용 약학 조성물을 제공한다.In another embodiment of the present invention, there is provided a pharmaceutical composition for treating cartilage damage comprising an unprotected bovine blood extract as an active ingredient.
본 발명의 또 다른 일 구현예에서, 소혈액 유래 탈단백 추출물을 유효성분으로 포함하는 골질환 또는 연골질환 개선용 식품 조성물을 제공한다. 상기 구현예에서, 상기 골질환은 전신 골 질환 또는 국소 골 질환 및 손상 중 어느 하나이고 상기 연골질환은 중성 연골질환 또는 연골 손상 중 어느 하나이며, 상기 전신 골 질환은 골다공증(osteoporosis)이고, 상기 국소 골 질환 및 손상은 골 유합장애(Nonunion/Delayed union), 가관절증(Pseudoarthrosis), 골 괴사(Osteonecrosis) 및 기타 골절(other fractures)로 구성된 군으로부터 선택된 어느 하나이며, 상기 중성 연골질환은 골 관절염(Osteoarthritis)이고, 상기 연골 손상은 연골부분층결손, 연골전층결손, 골연골결손, 골연골박리염(Osteochondritis dissecans), 연골연화증(Chondromalacia) 및 반월상 연골 손상(Meniscus injury)으로 구성된 군으로부터 선택된 어느 하나인 골질환 또는 연골질환 개선용 식품 조성물을 제공한다.In another embodiment of the present invention, there is provided a food composition for improving a bone disease or a cartilage disease, which comprises a bovine blood-derived deproteinized extract as an active ingredient. In this embodiment, the bone disease is any one of generalized bone disease, local bone disease and injury, and the cartilage disease is any one of neutral cartilage disease or cartilage damage, the whole body bone disease is osteoporosis, The bone disease and damage are any one selected from the group consisting of Nonunion / Delayed Union, Pseudoarthrosis, Osteonecrosis and other fractures, and the Neutral cartilage disease is osteoarthritis Wherein the cartilage damage is selected from the group consisting of a cartilage partial layer defect, an osteochondral defect, an osteochondral defect, an osteochondritis dissecans, a chondromalacia, and a meniscus injury. And a food composition for improving bone diseases or cartilage diseases.
본 발명 내 특별한 정의가 없으면 본 명세서에 사용된 모든 과학적 및 기술적인 용어는 본 발명이 속하는 기술분야에서 당 업자에 의하여 통상적으로 이해되는 것과 동일한 의미를 가진다.Unless defined otherwise within the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs.
본 발명에서 "지질다당체(LPS, LipoPolySaccharide)"는 내독소(endotoxin)의 하나로서 Raw264.7 대식세포에서 TNF-alpha, IL-6, IL-1 beta와 같은 pro-inflammatory 사이토카인을 증가시키며, NO(Nitric Oxide) 및 PGE2(Prostaglandin E2) 등의 염증 매개 물질을 분비한다. 염증상태에서는 COX-2(Cyclooxygenase-2)와 NOS(NO synthase)가 유도되어 과량의 PGE2, NO 등이 생성되며 여러 가지 질병 및 암 발병(Carcinogenesis)이 촉진된다. 특히 NO를 생성하는 효소인 NOS와 다양한 프로스타글란딘(PGs, Prostaglandins)의 생합성을 매개하는 효소인 COX(Cyclooxygenase)가 염증반응을 조절하는 중요한 매개체로 알려져 있다. 이러한 LPS의 수용체인 Toll like receptor는 산화스트레스와도 밀접한 관계가 있는 것으로 밝혀져 있다.In the present invention, "Lipopolysaccharide (LPS)" is one of the endotoxins which increases pro-inflammatory cytokines such as TNF-alpha, IL-6 and IL-1 beta in Raw264.7 macrophages, NO (Nitric Oxide) and PGE2 (Prostaglandin E2). In the inflammatory state, COX-2 (Cyclooxygenase-2) and NOS (NO synthase) are induced and excess PGE2 and NO are produced, and various diseases and carcinogenesis are promoted. In particular, COX (Cyclooxygenase), an enzyme that catalyzes the biosynthesis of various prostaglandins (PGs, Prostaglandins) and NOS, an enzyme that produces NO, is known to be an important mediator for controlling the inflammatory response. The Toll like receptor, a receptor for LPS, has been found to be closely related to oxidative stress.
본 발명에서 약물의 진통효과를 확인하기 위해 복강 내 초산 처치로 유도되는 복부의 수축 반응을 지표로 하는 초산 유도(Acetic acid-induced) 몸부림 테스트(Writhing test)를 실시하였다. 상기 초산 유도-몸부림 테스트는 말초성 통증 억제 물질의 스크리닝에 사용하는 방법이다(비특허문헌 3 내지 5). 초산을 이용한 writhing syndrome 반응을 복부수축반응(abdominal contraction response)라 하여 진통약물 검정에 유용한 방법의 하나로 사용하는데, 상기 반응의 억제여부를 확인하여 진통에 대한 효과를 평가하였다. In the present invention, acetic acid-induced writhing test was performed to check the analgesic effect of the drug, using the abdominal contraction induced by intraperitoneal acetic acid treatment as an index. The acetic acid induction-writhing test is a method used for screening of peripheral pain inhibiting substances (
세포의 증식 또는 세포의 사멸 정도를 정량적으로 측정할 수 있는 기법은 생명과학 분야에서 필수적인 분석 기법 중의 하나이다. 본 발명에서는 흡광염료로서 MTS[3(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium]를 사용하였고, 이를 이용한 분석법에 의해 연골 분화 유도 배지에서 배양한 세포 펠릿 내의 생존한 세포 수를 측정하여 연골생성 분화를 확인하였다. 상기 분석법은 주로 마이크로 플레이트 리더(Microplate reader)를 사용하여 실시하는데, 마이크로플레이트 리더는 마이크로플레이트 웰 내에서 반응한 물질의 정성, 정량 분석에 이용되는 장비로서 적절한 용매, 용해 또는 분산된 시료에 자외선, 가시광선, 적외선 영역 등의 빛을 통과시켜 흡광, 발광, 형광도 등을 측정하여 세포 생리 및 분자생물학, 면역학 분야에서 사용된다. 광원으로부터 유래한 빛은 여러 가지 필터와 단색화 장치를 통해 특정 파장의 빛으로 만들어진 시료에 96개 혹은 그 이상의 경로를 통해 조사되어 각각의 웰에 담긴 시료의 성분 및 농도에 따라 발생한 형광 또는 투과된 빛이 검출기를 통하여 분석될 수 있다. 이와 같은 방법으로 측정된 결과를 바탕으로 세포의 증식 능력과 세포의 생존 능력을 관찰할 수 있다. Quantitative measurement of cell proliferation or cell death is one of the indispensable analytical techniques in life sciences. In the present invention, MTS [3 (4,5-dimethylthiazol-2yl) -5- (3-carboxymethoxy-phenyl) -2- (4-sulfophenyl) -2H-tetrazolium] was used as a light absorbing dye. The number of viable cells in the cell pellet cultured in the cartilage differentiation induction medium was measured to confirm the cartilage differentiation. The assay is performed using a microplate reader. The microplate reader is used for qualitative and quantitative analysis of the reacted substance in a microplate well. The assay is performed using an appropriate solvent, a dissolved or dispersed sample, It is used in the fields of cellular physiology, molecular biology, and immunology by measuring light absorption, luminescence and fluorescence through light such as visible light and infrared light. Light from a light source is irradiated through 96 or more pathways to a sample made of light of a specific wavelength through various filters and monochromators, and the fluorescence or transmitted light generated according to the composition and concentration of the sample contained in each well Can be analyzed through the detector. Based on these results, we can observe cell proliferation and cell viability.
본 발명에서 "셀레콕시브(celecoxib)"는 4-[5-p-톨릴-3-(트리플루오로메틸)-1H-피라졸-1-일]벤젠설폰아미드로도 알려져 있으며 파마시아 코포레이션에서 시판하는 셀레브렉스(등록상표명: Celebrex)의 활성 성분으로서, 치료적 및 예방적으로 유용하며 선택적인 시클로옥시게나아제-2(COX-2) 억제 효과를 지니고 있어서 COX-2 매개 질환들의 치료 또는 예방에 유용하게 사용될 수 있는데, 관절염에 사용하는 것이 일반적이다. 1999년 미국 식품의약품 안전청에서는 가족성 선종성 용종증(Familial adenomatous polyposis)의 치료 및 예방에 셀레콕시브의 사용을 허가하였으나, 셀레콕시브의 용량에 따른 심장 부작용에 기인하여 그 사용은 제한되어 있다. 셀레콕시브는 수성 매질에서 낮은 용해도를 나타내므로 경구 투여시 위장관 내에서 잘 용해되지 않아 셀레콕시브의 절대적인 생체 이용률은 약 30% 정도에 불과하다.The term " celecoxib "in the present invention is also known as 4- [5-p-tolyl-3- (trifluoromethyl) -1H-pyrazol- 1-yl] benzenesulfonamide and commercially available from Pharmacia Corporation (COX-2) inhibitory effect, which is therapeutically and prophylactically useful as an active ingredient of Celebrex (registered trademark: Celebrex), and is useful for the treatment or prevention of COX-2 mediated diseases It is commonly used for arthritis. In 1999, the US Food and Drug Administration approved the use of celecoxib for the treatment and prevention of familial adenomatous polyposis, but its use is limited due to the adverse cardiac effects of celecoxib. Since celecoxib exhibits low solubility in aqueous media, the oral bioavailability of celecoxib is only about 30% since it is not well soluble in the gastrointestinal tract when administered orally.
본 발명에서 "중합효소연쇄반응(PCR, Polymerase Chain Reaction)"은 아주 적은 양의 DNA 만으로도 특정 부위의 DNA 염기서열을 기하급수적으로 증폭시킬 수 있는 간단하고 편리한 방법으로, 증폭하고자 하는 DNA에 특이적으로 결합하여 고온에서도 안정한 Taq DNA 중합효소를 사용하여 변성(denaturation), 어닐링(annealing), 연장(extension)의 과정을 반복적으로 실행함으로써 특정 DNA를 증폭시키게 된다. PCR을 이용한 목적 유전자의 검출은 유전자를 인식하는 프라이머를 이용하여 이를 증폭 및 확인하는 것이다. 상기 "프라이머(primer)"는 짧은 자유 3 말단 수산화기를 갖는 핵산서열로 상보적인 주형(template)과 염기쌍(base pair)을 형성할 수 있고 주형 가닥 복사를 위한 시작 지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응을 위한 시약(DNA 중합효소 또는 역전사 효소) 및 상이한 4가지 dNTP(deoxynucleoside triphosphate)의 존재하에서 DNA 합성을 개시할 수 있다.In the present invention, "PCR (Polymerase Chain Reaction)" is a simple and convenient method for exponentially amplifying a DNA sequence at a specific site using only a small amount of DNA, And DNA amplification is performed by repeatedly performing denaturation, annealing, and extension using a stable Taq DNA polymerase at high temperatures. The detection of the target gene using PCR is performed by amplifying and confirming the gene using a primer recognizing the gene. The term "primer" refers to a nucleic acid sequence having a short free 3-terminal hydroxyl group and a short nucleic acid sequence capable of forming base pairs with a complementary template and serving as a starting point for template strand copying it means. Primers can initiate DNA synthesis in the presence of reagents (DNA polymerase or reverse transcriptase) for polymerisation and four different dNTPs (deoxynucleoside triphosphate) at appropriate buffer solutions and temperatures.
본 발명에서 "리얼타임(real-time) PCR"이란 형광물질을 PCR 기법에 응용한 것으로, 반응 중 검체 내에 존재하는 표적 유전자의 증폭과 함께 형광물질의 발광(emission) 정도를 실시간으로 검출하고 정량 분석하여 표적 유전자의 증폭 유무 및 그 양상을 신속하고 정확하게 분석할 수 있는 방법이다. The term "real-time PCR" used in the present invention refers to the application of a fluorescent substance to a PCR technique. In the reaction, amplification of a target gene existing in a specimen during a reaction is detected, And analyze the amplification of the target gene and its aspects quickly and accurately.
본 발명에서 real-time PCR을 이용한 정량은 일반적으로 증폭반응 개시 시의주형 DNA 양을 정량하기 위한, PCR을 이용한 일련의 반응을 의미한다. 정량적 PCR에는 기준이 되는 표준시료를 사용하는 내부 표준법과 증폭반응에 있어서 경쟁하는 분자를 사용하는 경쟁법이 포함된다. 또한, 각 서열의 분자 수를 정확하고 간편하게 결정하기 위해서 표준시료라고 불리는 기준이 되는 주형 DNA 서열을 이용한 PCR 방법을 사용하고, 반응 중 임의의 시점에서 반응의 진행상황, 즉 증폭의 정도를 확인한다. 따라서, 본 발명에서 "정량"이란 증폭반응 개시 시의 주형 DNA 양을 정량하기 위한 PCR로서, 반응 중 임의의 시점에서 증폭대상의 분자에 대해 증폭을 확인할 수 있는 PCR을 의미한다. 정량을 위해서는 일반적으로 그와 같은 PCR과 반응 개시 시의 주형 DNA 분자 수와 그 증폭의 지표가 되는 신호를 관련짓는 검량선이 조합된다. 이 검량선은 분자 수가 알려진 표준 시료를 이용하여 제작할 수 있다. 이러한 real-time PCR 분석은 시판되고 있는 real-time PCR 기기를 사용하여 수행될 수 있는 바, real-time 기기는, SLAN real time PCR detectionsystem(LG 생명과학, 한국), LightCycler 96/480(Roche, Germany), StepOnePlus™ Real-Time PCR System, ABI 7500 fast/7500/PRISMTM 7000/7700(Applied Biosystems, USA), iCyclerTM/CFX96TM(Bio-Rad, USA), Rotor-GeneTM(Corbett, Australia), OpticonTM(PharmaTech, USA) 등을 들 수 있으나, 이에 한정되는 것은 아니다.In the present invention, quantification using real-time PCR generally means a series of reactions using PCR to quantify the amount of template DNA at the start of the amplification reaction. Quantitative PCR includes internal standard methods using standard reference standards and competitive methods using molecules competing in amplification reactions. In order to accurately and easily determine the number of molecules of each sequence, a PCR method using a template DNA sequence as a standard called a standard sample is used, and the progress of the reaction, that is, the degree of amplification is checked at any time during the reaction . Therefore, in the present invention, "quantitative" means a PCR for quantifying the amount of template DNA at the start of the amplification reaction, and means a PCR capable of confirming the amplification of a molecule to be amplified at any time during the reaction. For quantification, a combination of such a PCR and a calibration curve associating the number of template DNA molecules at the start of the reaction with a signal that is an index of the amplification is combined. This calibration curve can be prepared using standard samples with a known number of molecules. Real-time PCR analysis can be performed using commercial real-time PCR instruments. Real-time instruments include SLAN real time PCR detection system (LG Life Science, Korea), LightCycler 96/480 (Roche, USA), Rotor-Gene (Corbett, Australia), Opticon (USA), StepOnePlusTM Real-Time PCR System, ABI 7500 fast / 7500 / PRISMTM 7000/7700 (Applied Biosystems, USA), iCyclerTM / CFX96 PharmaTech, USA), but the present invention is not limited thereto.
본 발명에서 "연골"은 발생학적으로 경골조직과 같은 중배엽에서 기원한 조직으로, 뼈와 함께 내골격을 형성하며, 연골세포(chondrocytes)가 많은 양의 세포외 기질로 둘러싸인 형태로 이루어져 있는데, 콜라겐을 비롯한 여러 단백질과 글리코사미노글리칸(GAG; glycosaminoglycan)으로 이루어지는 당단백질이 치밀하게 조직을 구성하여 단단하고 탄성을 가지는 연골 조직을 형성하게 된다. 연골은 질기고 탄성이 있어서, 신체 내에서 뼈와 뼈가 마주 닿는 사이에서 관절면을 형성하거나, 저항력과 탄성이 필요한 곳, 물리적 힘을 수용할 수 있어야 하는 곳에 위치하여 기능을 하게 된다. 연골은 한 번 형성이 되면 오랜 수명을 가지고 유지가 되지만, 다른 조직과 달리 혈관이나 신경이 분포되어 있지 않으므로 손상이 생긴 경우 빠른 재생이 일어나기 어렵다. 특히, 관절연골에 생기는 손상은 활동에 심각한 영향을 미치므로 이를 치료하기 위한 방법에 대한 연구가 많이 진행되고 있다. 연골 손상이 심한 경우 연골뿐만 아니라 연골 밑의 골조직까지 손상을 입는 경우도 있다. 이처럼 연골과 뼈는 유기적으로 작용하는 조직이지만 종래의 연골과 뼈의 조직공학적 치료는 이들을 각각 치료하는 방식을 사용하였다. 구체적으로, 기존의 임상적인 연골 재생 수술 방법으로는 (1) 줄기세포의 연골세포로의 분화를 유도하는 방법, (2) 골, 연골 조직, 자가 혹은 동종의 연골조직으로 연골 결손 부위에 이식하는 방법, (3) 연골 결손 부위의 표면에 연골을 유도할 수 있는 조직(연골막, 골막) 혹은 화합물 등을 이식하는 방법, (4) 연골 세포 이식술로 연골 결손 부위에 연골 세포를 이식함으로써 연골 재생을 유도하는 방법 등 연골만을 재생시키는 방법이 사용되고 있다. 연골조직의 분화 초기단계에 필수적인 발현 전사인자로 알려진 SOX 유전자들은 XY 성전환 및 성을 결정하는 데에 관여하는 SRY(Sex-determining Region Y) 관련 HMG(High Mobility Group) 박스 유전자에 속하며, 이들 중 SOX-9은 비대연골세포를 제외한 모든 연골세포에서 발현된다. SOX 유전자들은 연골분화에 관련한 COL2A1(Collagen, type II, alpha 1), COL9A1(Collagen, Type IX, Alpha 1), COL11A2(Collagen, type XI, alpha 2) 및 어글리칸(Agc, Aggrecan)을 활성화시키는 것이 밝혀져 있고, SOX-5 및 SOX-6는 연골 전구세포와 분화된 연골세포에서 SOX-9와 함께 발현되고 COL2A1를 활성화시키기 위해 SOX9와 협력하는 연골세포 분화에 필요한 요소임이 밝혀져 있다. 정상적인 연골세포에서 나타나는 대표적인 3가지의 마커(marker)인, 제 2형 콜라겐 1(Col2 A1), 어그리칸(Agc), Sox9 등의 발현 여부를 확인함으로써 분화 유도된 세포가 정상적인 연골세포라는 것을 확인하는 것이 가능하며, 이와 같은 유전자의 발현은 실시간 중합효소 연쇄반응(Real-time PCR)을 이용하여 확인하는 것이 가능하다.In the present invention, "cartilage" is embryonic tissue derived from mesoderm such as tibial tissue. It forms an endoskeleton with bone, and chondrocytes are surrounded by a large amount of extracellular matrix. Collagen And a glycoprotein composed of various proteins and glycosaminoglycan (GAG) form dense tissue to form hard and elastic cartilage tissue. The cartilage is tough and elastic, functioning in a place where it must be able to accommodate physical forces, where it needs to form a joint surface between the bones and bones in the body, where resistance and elasticity are needed. Once formed, cartilage is maintained with a long life span, but unlike other tissues, blood vessels and nerves are not distributed, and it is difficult for quick regeneration to occur when damaged. Particularly, damage to articular cartilage has serious effects on the activity, and therefore, researches on methods for treating it have been carried out. In the case of severe cartilage damage, not only the cartilage but also the bone tissue under the cartilage may be damaged. Although the cartilage and bone are organically functioning tissues, conventional tissue engineering treatments of the cartilage and bone treat them separately. Specifically, conventional clinical cartilage regeneration methods include (1) inducing differentiation of stem cells into cartilage cells, (2) bone, cartilage tissue, autologous or allogeneic cartilage tissue, (Cartilage, periosteum) or a compound capable of inducing cartilage on the surface of cartilage deficient area, (4) cartilage regeneration by transplanting cartilage cells in a cartilage defect site by cartilage cell transplantation, And a method of regenerating only the cartilage is used. SOX genes, which are known to be essential transcription factors in the early stage of differentiation of cartilage tissue, belong to the HMY (High Mobility Group) box gene related to SRY (Sex-determining Region Y) involved in determining XY transcription and sex, -9 is expressed in all cartilage cells except hypertrophic chondrocytes. The SOX genes activate COL2A1 (Collagen, type II, alpha 1), COL9A1 (Collagen, Type IX, Alpha 1), COL11A2 (Collagen, type XI, alpha 2) and aggrecan (Agc, Aggrecan) And SOX-5 and SOX-6 are expressed together with SOX-9 in chondrocyte precursor cells and differentiated chondrocytes and are found to be necessary elements for chondrocyte differentiation cooperating with SOX9 to activate COL2A1. It was confirmed that the differentiated cells were normal cartilage cells by confirming the expression of
본 발명에서 "줄기세포"는 다양한 조직으로 분화할 수 있는 능력을 가진 세포, 즉 '미분화세포'로서 자기 재생을 통해 오랫동안 분열을 할 수 있으며, 특정 환경 하에서는 다양한 종류의 세포로 분화할 수 있는 세포이다. 줄기세포는 기원이 되는 조직에 따라 배아 줄기세포와 성체 줄기세포로 나누어진다. 이를 이용한 연구적인 측면에서 성체 줄기세포의 잠재능력은 배아 줄기세포보다 단점이 있으나, 윤리적인 문제가 적어 성체 줄기세포를 대상으로 많은 연구가 진행되고 있다.In the present invention, "stem cells" are cells capable of differentiating into various tissues, that is, undifferentiated cells, which can divide for a long period of time through self-renewal, and cells capable of differentiating into various types of cells to be. Stem cells are divided into embryonic stem cells and adult stem cells depending on the originating tissue. Although the potential of adult stem cells is more disadvantageous than that of embryonic stem cells, research on adult stem cells has been under way.
본 발명에서 "연골질환"은 연골, 연골 조직 및/또는 관절조직(활막, 관절포, 연골하골 등)이 기계적 자극이나 염증 반응에 의해 상해됨으로써 발생하는 질환을 말하며, 연골손상 질환을 포함한다. 이러한 연골 손상 질환에는 퇴행성 관절염, 류마티스성 관절염, 무릎연골 연화증, 골절, 근육조직의 손상, 족저근막염, 상완골외과염, 석회화근염, 골절의 불유합 또는 외상에 의한 관절손상이 있으나, 이에 한정되는 것은 아니다. The term "cartilage disease" in the present invention refers to a disease caused by injury to cartilage, cartilage tissue and / or joint tissue (synovial membrane, arthroscopic capsule, cartilaginous bone, etc.) by mechanical stimulation or inflammatory reaction, and includes cartilage damage disease. Such cartilage injuries include, but are not limited to, degenerative arthritis, rheumatoid arthritis, knee cartilage dystrophy, fractures, muscle tissue damage, plantar fasciitis, humerus ulcer, calcified myositis, nonunion of the fracture, or traumatic joint damage.
손상된 연골을 원래의 연골과 조직학적, 생체역학적으로 유사한 조직으로 재생시키는 것은 연골 손상을 예방하고 치료한다는 관점에서 매우 중요하다. 이러한 관점에서 기존 연골 치료방법의 한계를 극복하기 위해 많은 연구 개발이 진행되고 있는 가운데, 고도의 생체기능을 실현하기 위해서 장기나 조직의 실질세포를 이용하는 인공 생체조직의 개발에 대한 필요성이 대두되었다. 효과적인 연골 치료제의 개발을 위해, 증식 및 배양된 연골세포를 이용하여 자연 연골과 유사한 조직을 형성하면서도 기계적 강도와 일정한 형태를 유지하여 조직 재생을 효과적으로 유도할 수 있어야 한다. 연골 결손부를 골막조직으로 덮고 주위 연골에 잘 봉합한 후, 체외 배양한 연골세포를 연골 결손부에 이식하는 방법으로 비교적 자연 연골에 가까운 재생 연골조직을 얻을 수 있음이 보고된 후, 이와 같은 방법을 실제 임상에 적용한 사례가 미국과 북유럽을 중심으로 꾸준히 증가하고 있다(비특허문헌 6). 자가 연골세포 이식법에 의한 연골조직의 재생이 최근 시도되어 임상적으로 매우 효과적인 수술법으로 알려지고 있으나 연골의 광범위한 손상 부위에는 사용할 수 없다는 점, 관절 연골의 손상 위치, 환자의 나이 등에 따른 제한점을 갖고 있다. 이와 같은 문제점을 해결하기 위해 연골의 조직 공학적 재생 방법이 등장하였다. 인공연골의 제작에 관한 연구가 이미 외국에서 활발하게 수행 중이며, 줄기세포에서 분화된 연골세포의 조직공학적 관절연골 제작은 중심 요인들인 연골세포화된 줄기세포 및 생분해성 지지체가 필수적이고, 이상적인 연골세포는 증식능력이 좋아야 하며, 연골세포의 특이 표현형인 콜라겐 타입(collagen type) II를 유지하는 세포이다.Regeneration of injured cartilage with the original cartilage and histologically and biomechanically similar tissues is very important in terms of preventing and treating cartilage damage. In this respect, in order to overcome the limitations of existing cartilage treatment methods, many research and development are proceeding, and in order to realize a high degree of biological function, the necessity of development of an artificial biotissue using parenchymal cells of organs has emerged. In order to develop an effective cartilage therapeutic agent, it is necessary to maintain the mechanical strength and uniform shape while inducing tissue regeneration effectively by forming a tissue similar to natural cartilage by using proliferated and cultured chondrocytes. After the cartilage defect is covered with the periosteal tissue, the cartilage is sutured to the surrounding cartilage, and then the in vitro cultured cartilage cells are transplanted into the cartilage defect to obtain a regenerated cartilage tissue close to the natural cartilage. The actual cases applied to clinical practice are steadily increasing mainly in the United States and Northern Europe (Non-Patent Document 6). The autologous chondrocyte transplantation regeneration of cartilage tissue has recently been known to be a clinically very effective procedure, but it has limitations due to its inability to be used for a wide range of cartilage damage, location of articular cartilage damage, and age of the patient . In order to solve such problems, tissue engineering regeneration method of cartilage appeared. Studies on the production of artificial cartilage have already been actively carried out in foreign countries. In the production of articular cartilage of chondrocytes differentiated from stem cells, chondrocyte-derived stem cells and biodegradable scaffolds, which are central factors, Is a cell that maintains collagen type II, which is a specific phenotype of cartilage cells, and has good proliferative capacity.
본 발명의 약제학적 조성물은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때, 제형은 오일 또는 수성 매질 중의 용액, 현탁액, 시럽제 또는 유화액 형태이거나 엑스제, 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. Here, the formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
본 발명의 조성물은 비경구 투여 제형으로 제형화될 수 있다. 비경구 투여 제형은 주사제, 또는 피부외용제일 수 있다. 피부 외용제는 크림, 겔, 연고, 피부 유화제, 피부 현탁액, 경피전달성 패치, 약물 함유 붕대, 로션, 또는 그 조합일 수 있다. 상기 피부 외용제는 통상 화장품이나 의약품 등의 피부 외용제에 사용되는 성분, 예를 들어 수성성분, 유성성분, 분말성분, 알코올류, 보습제, 중점제, 자외선흡수제, 미백제, 방부제, 산화방지제, 계면활성제, 향료, 색제, 각종 피부 영양제, 또는 이들의 조합과 필요에 따라서 적절하게 배합될 수 잇다. 상기 피부외용제는, 에데트산이나트륨, 에데트산삼나트륨, 시트르산나트륨, 폴리인산나트륨, 메타인산나트륨, 글루콘산 등의 금속봉쇄제, 카페인, 탄닌, 벨라파밀, 감초추출물, 글라블리딘, 칼린의 과실의 열수추출물, 각종 생약, 아세트산토코페롤, 글리틸리틴산, 트라넥삼산 및 그 유도체 또는 그 염등의 약제, 비타민 C, 아스코르브산 인산마그네슘, 아스코르브산글루코시드, 알부틴, 코지산, 글루코스, 프룩토스, 트레할로스 등의 당류 등도 적절하게 배합할 수 있다.The composition of the present invention can be formulated into a parenteral dosage form. The parenteral dosage form may be an injection or an external preparation for skin. The external skin preparation may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof. The external preparation for skin may be a cosmetic product or a medicament for use in a composition for external use for skin such as an aqueous component, an oily component, a powder component, an alcohol, a moisturizer, a thickening agent, an ultraviolet absorber, a whitening agent, an antiseptic, Flavorings, coloring agents, various skin nutrients, or combinations thereof, and may be appropriately blended as required. The external preparation for skin may be a metal blocker such as sodium edetate, sodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate or gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridine, Vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, fructose, fructose and other herbal medicines, various herbal medicines, tocopherol acetate, glycyrrhizic acid, Sugars such as trehalose and the like can also be appropriately compounded.
본 발명의 또 다른 양태에 따르면, 본 발명은 탈단백 소혈액 추출물 또는 이의 약제학적으로 허용 가능한 염을 유효성분으로 포함하는 골 질환 개선용 기능성 식품 조성물 또는 식품 조성물을 제공한다.According to still another aspect of the present invention, there is provided a functional food composition or a food composition for improving bone diseases, comprising an extract of deproteinized blood or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명의 탈단백 소혈액 추출물을 포함하는 조성물이 기능성 식품 조성물 또는 식품 조성물로 제조되는 경우, 유효성분으로서 탈단백 소혈액 추출물 또는 이의 약제학적으로 허용 가능한 염뿐만 아니라, 기능성 식품 또는 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제를 포함한다. 상술한 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스, 올리고당 등; 및 폴리사카라이드, 예를 들어 덱스트린, 사이클로덱스트 린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 향미제로서 천연 향미제[타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등]) 및 합성 향미제(사카린, 아스 파르탐 등)를 사용할 수 있다.When the composition comprising the deproteinized bovine blood extract of the present invention is prepared from a functional food composition or a food composition, it is preferable to use a deproteinized bovine blood extract or a pharmaceutically acceptable salt thereof as an active ingredient, Include commonly added ingredients and include, for example, proteins, carbohydrates, fats, nutrients, flavoring agents and flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings.
본 발명에 따른 기능성 식품 또는 식품 조성물이 드링크제로 제조되는 경우에는 본 발명의 유효성분인 탈단백 소혈액 추출물 또는 이의 약제학적으로 허용 가능한 염 이외에 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙, 두충 추출액, 대추 추출액 또는 감초 추출액 등을 추가로 포함시킬 수 있다.When the functional food or the food composition according to the present invention is prepared as a drink, it may be added to the deproteinized bovine blood extract or the pharmaceutically acceptable salt thereof as an active ingredient of the present invention in addition to citric acid, liquid fructose, sugar, glucose, acetic acid, , Jitian extract, jujube extract or licorice extract, and the like.
본 발명의 소혈액 유래 탈단백 추출물은 골단백질 분해효소의 억제, 연골세포의 재생 및 분화 촉진에 의한 골 질환 또는 연골질환의 예방, 치료 및 개선 효과 및 통증 완화 효과가 우수하다. 따라서, 본 발명의 소혈액 유래 탈단백 추출물을 사용하면 다양한 골 질환 또는 연골질환을 효과적으로 예방 또는 치료할 수 있고, 골 질환 또는 연골질환 개선용 조성물 및 통증 완화용 조성물을 제조할 수 있다.The bovine blood-derived deproteinized extract of the present invention is excellent in prevention of bone disease or cartilage disease by inhibition of bone protease, regeneration and differentiation of cartilage cells, and treatment, improvement and pain relief. Therefore, by using the bovine blood-derived deproteinized extract of the present invention, various bone diseases or cartilage diseases can be effectively prevented or treated, and compositions for improving bone diseases or cartilage diseases and compositions for pain relief can be produced.
도 1은 본 발명에 따른 탈단백 소 혈액 추출물의 활막세포의 염증에 대한 억제 효과를 나타낸 것이다.
도 2는 본 발명에 따른 탈단백 소 혈액 추출물의 골단백질 분해효소에 대한 발현 저해 효과를 나타낸 것이다.
도 3은 본 발명에 따른 탈단백 소 혈액 추출물의 연골세포 재생 효과를 나타낸 것이다.
도 4는 본 발명에 따른 탈단백 소 혈액 추출물의 줄기세포에 대한 연골세포로의 분화 유도 효과를 연골세포의 분화 마커 발현을 통해 나타낸 것이다.
도 5는 본 발명에 따른 탈단백 소 혈액 추출물의 줄기세포에 대한 보충 효과를 나타낸 것이다.
도 6은 본 발명에 따른 탈단백 소 혈액 추출물의 골아세포 증식 효과를 나타낸 것이다.
도 7은 본 발명에 따른 탈단백 소 혈액 추출물의 골밀도 증가 효과를 나타낸 것이다.
도 8은 본 발명에 따른 탈단백 소 혈액 추출물의 연골조직 재생 효과를 나타낸 것이다.FIG. 1 shows the inhibitory effect of the deproteinized bovine blood extract according to the present invention on inflammation of synovial cells.
FIG. 2 shows the inhibitory effect of the deproteinized bovine blood extract according to the present invention on bone proteolytic enzyme.
FIG. 3 shows the cartilage cell regeneration effect of the deproteinized blood extract according to the present invention.
FIG. 4 is a graph showing the differentiation-induced effect of the deproteinized bovine blood extract according to the present invention on the differentiation of chondrocytes into stem cells.
FIG. 5 shows supplementary effects of the deproteinized bovine blood extract according to the present invention on stem cells.
FIG. 6 shows the effect of osteoblast proliferation of the deproteinized blood extract according to the present invention.
FIG. 7 shows the effect of increasing the bone mineral density of the deproteinized blood extract according to the present invention.
FIG. 8 shows the regenerating effect of cartilage tissue of the deproteinized blood extract according to the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당 업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
소 혈액 Bovine blood 탈단백Deproteinization 추출물 제조 Extract preparation
3개월 이상 자란 소에서 주사 바늘과 진공팩을 이용하여 혈액을 수득하였다. 수득한 혈액은 4℃ 조건에서 16시간 이상 보관하였다. 수득한 소혈액은 실온에서 1시간 이상 보존하여 혈액을 응고시키고 압착기를 이용하여 압착하였다. 압착시킨 추출물은 45㎛ 필터로 여과하고 4000rpm에서 30분간 원심분리한 후 상층액만을 회수하였다. 회수한 상층액은 Amicon Ultra 3kDa 한외여과막을 이용하여 여과하였고 회수한 상층액은 0.22 ㎛ 필터로 여과하고 감압증류후 동결건조를 하여 -20℃ 냉동 조건에서 보관하였다. 효능 평가시 DMEM(Dulbeco’s Modified Eagle’s Media)에 20 ㎎/㎖이 되도록 녹여서 사용하였다.
Blood was obtained using a needle and a vacuum pack in cattle growing over 3 months. The obtained blood was stored at 4 캜 for 16 hours or more. The obtained bovine blood was stored at room temperature for 1 hour or longer to coagulate blood, and the blood was squeezed using a press. The squeezed extract was filtered with a 45 ㎛ filter, centrifuged at 4000 rpm for 30 minutes, and only supernatant was recovered. The recovered supernatant was filtered using an
염증 억제Inflammation inhibition
인간 활막세포(HFLS)는 Cell applications, INC.(Cat No. 408-05a, 미국)로부터 구입하여 사용하였다. 인간 활막세포(HFLS)은 DMEM(Dulbeco's Modified Eagle's Media)을 사용하여 배양하였다. LPS(1㎍/㎖)를 6시간 동안 처리하여 염증을 유발한 후, 실시예 1의 소혈액 탈단백 추출물을 0.2, 2 및 20㎎/㎖로 처리하였다. 염증 억제 효과에 대한 유발대조군으로 추출물 용매와 같은 DMEM 배양액을 동량 사용하였고, 양성대조군으로서 Celecoxib 10 μM을 사용하였다. 48시간이 경과한 후, 실시간 중합연쇄 반응법(Real Time-PCR)에 의해 염증 유발 인자 TNF-alpha, IL-6 및 COX-2의 발현에 대한 저해 효과를 측정하였다(표 1 참조). 소혈액 탈단백 추출물의 사용량에 따른 염증 유발 인자의 발현 저해 효과를 확인하였다(도 1).Human synovial cells (HFLS) were purchased from Cell applications, INC. (Cat No. 408-05a, USA). Human synovial cells (HFLS) were cultured using DMEM (Dulbeco's Modified Eagle's Media). After treatment with LPS (1 / / ml) for 6 hours to induce inflammation, the bovine blood deproteinized extract of Example 1 was treated with 0.2, 2 and 20 mg / ml. The same amount of DMEM culture medium as the extract solvent was used as the control group for the inflammation inhibitory effect, and 10 μM of Celecoxib was used as the positive control group. After 48 hours, the inhibitory effects on the expression of inflammatory factors TNF-alpha, IL-6 and COX-2 were measured by real-time PCR (see Table 1). And inhibited the expression of inflammatory factors depending on the amount of bovine blood deproteinized extract (FIG. 1).
NM_000594
NM_000600
NM_000963
골단백질Bone protein 분해효소 억제 Protease inhibition
마우스 대식세포주 Raw 264.7에 대하여 LPS(1㎍/㎖)를 6시간 동안 처리하여 염증을 유발한 후, 실시예 1의 소혈액 탈단백 추출물을 0.02, 0.2 및 2㎎/㎖로 처리하였다. 유발대조군으로 추출물 용매와 같은 DMEM 배양액을 동량 사용하고 양성대조군으로서 Celecoxib 10 μM을 사용하였다. 48시간이 경과한 후, 실시간 중합 연쇄 반응법에 의해 골단백질 분해효소 인자 MMP-1a, MMP-3, MMP-9, 및 MMP-13의 발현 저해 효과를 측정하였다(표 2 참조). 소혈액 탈단백 추출물의 사용량에 따른 골단백질 분해효소의 발현 저해 효과를 확인하였다(도 2). The mouse macrophage cell line Raw 264.7 was treated with LPS (1 μg / ml) for 6 hours to induce inflammation, and the bovine blood deproteinized extract of Example 1 was treated with 0.02, 0.2 and 2 mg / ml. The same amount of DMEM culture medium as the extract solvent was used as the control group and 10 μM of Celecoxib was used as the positive control group. After 48 hours, the inhibitory effects of bone protease factors MMP-1a, MMP-3, MMP-9 and MMP-13 were measured by real-time polymerization chain reaction (see Table 2). And inhibited the expression of bone proteolytic enzyme according to the amount of bovine blood deproteinized extract (FIG. 2).
NM_032006
NM_010809
NM_013599
NM_008607
연골세포의 재생Regeneration of cartilage cells
토끼의 관절에서 분리한 연골세포를 Well 당 DMEM 100㎕가 포함된 96 well plate에서 8×103 Cell/Well씩 배양하였다. 실시예 1의 소혈액 탈단백 추출물을 0.02, 0.2 및 2㎎/㎖로 처리하였다. 연골세포 재생의 음성대조군은 추출물과 동일 용량으로 PBS를 사용하였고 양성대조군으로서 소태아혈청을 10% 농도로 처리하여 사용하였다. 48시간이 경과한 후, MTS 분석법을 통해 실시예 1의 추출물의 연골 세포에 대한 재생 효과를 측정하여 농도에 따른 연골세포 재생 효과를 확인하였다(도 3 좌측). The chondrocytes isolated from the joints of rabbits were cultured at 8 × 10 3 cells / well in a 96-well plate containing 100 μl of DMEM per well. The bovine blood deproteinized extract of Example 1 was treated with 0.02, 0.2 and 2 mg / ml. As a negative control for chondrocyte regeneration, PBS was used at the same dose as the extract and 10% concentration of fetal bovine serum was used as a positive control. After 48 hours, the regeneration effect of the extract of Example 1 on chondrocytes was measured through MTS analysis, and the chondrocyte regeneration effect according to the concentration was confirmed (left side in FIG. 3).
인간 연골세포주 SW1353에 실시예 1의 소혈액 탈단백 추출물을 0.02, 0.2 및 2㎎/㎖로 처리하였다. 연골세포 재생의 양성대조군으로서 소태아혈청을 사용하였다. 48시간 후, 연골세포 성장 촉진 표지인자인 KI67 염색에 의해 연골세포의 재생효과를 확인하였다(도 3 우측).
The human chondrocyte cell line SW1353 was treated with 0.02, 0.2 and 2 mg / ml of bovine blood deproteinized extract of Example 1. Fetal serum was used as a positive control for chondrocyte regeneration. After 48 hours, the regeneration effect of chondrocytes was confirmed by KI67 staining for promoting cartilage cell growth (Fig. 3 right).
연골세포의 분화 유도Induction of chondrocyte differentiation
실시예 1의 소혈액 탈단백 추출물 0.02, 0.2 및 2㎎/㎖을 토끼의 대퇴부와 지방조직에서 분리한 골수유래줄기세포(BMSC, Bone marrow stem cell)와 지방유래줄기세포(ADSC, adipose derived stem cell)에 처리하여 연골세포로의 분화 유도 효과를 시험하였다. 연골세포 분화에 대한 음성대조군은 PBS을 추출물과 동일 용량으로 사용하였고 양성대조군은 StemPro® 연골 분화유도 키트(Thermo Fisher, A1007101)를 사용하였다. 48시간이 경과한 후, 실시간 중합 연쇄 반응법을 통해 연골 세포의 분화 표지 인자인 SOX9 발현 증가를 측정하였고(표 3), 연골분화 유도 촉진 효과를 확인하였다(도 4).(BMSC) and adipose derived stem (BMSC) stem cells isolated from the femoral and adipose tissues of rabbits by 0.02, 0.2 and 2 mg / ml of bovine blood deproteinized extract of Example 1 cell) to examine the effect of inducing differentiation into chondrocytes. The negative control for chondrocyte differentiation was used at the same dose as the PBS extract and the positive control group was StemPro® cartilage differentiation induction kit (Thermo Fisher, A1007101). After 48 hours, the expression of SOX9, which is a differentiation marker of chondrocytes, was measured through real-time polymerization chain reaction (Table 3), and the promoting effect of cartilage differentiation induction was confirmed (FIG. 4).
AY598935.1
줄기세포의 동원(Mobilization of stem cells recruitingrecruiting ) 활성) activation
손상된 조직이나 부위에 줄기세포의 생체이동은 줄기세포에 의한 치료효과를 기대할 수 있다. 소혈액 탈단백 추출물에 의한 줄기세포 동원(recruiting) 활성을 확인하기 위해 0.8㎛ 트랜스웰(transwell, Corning 3422)에 인간 골수유래 줄기세포를 5×104 cell/well씩 분주하고, 실시예 1의 소혈액 탈단백 추출물을 2㎎/㎖을 동결건조하여 0.8 ㎖ 배양액이 존재하는 트랜스웰 하단 부분에 위치시켰다 음성대조군은 소혈액 탈단백 추출물이 포함되지 않은 배양액을 사용하였다. 48시간이 경과한 후, 트랜스웰(transwell)을 통과하여 소혈액추출물이 존재하는 쪽으로 이동한 인간 골수유래 줄기세포를 크리스탈 바이올렛(Crystal Violet) 염색액으로 염색하여 세포의 동원 효과를 측정하였다. 본 발명에 따른 소혈액 탈단백 추출물은 줄기세포의 동원 효과가 우수함을 확인하였다(도 5).
The migration of stem cells to injured tissues or areas can be expected to be a therapeutic effect of stem cells. Human bone marrow-derived stem cells were dispensed at a density of 5 × 10 4 cells / well in a 0.8 μm transwell (Transwell, Corning 3422) in order to confirm stem cell recruiting activity by the bovine blood deproteinized extract. 2 mg / ml of bovine blood deproteinized extract was lyophilized and placed in the lower part of the transwell in which 0.8 ml of the culture solution was present. As the negative control, a culture solution without bovine blood deproteinized extract was used. After 48 hours, human bone marrow-derived stem cells that had passed through the transwell and moved to the presence of bovine blood extract were stained with Crystal Violet staining solution to measure the mobilization effect of the cells. It was confirmed that the bovine blood deproteinization extract according to the present invention had an excellent mobilization effect of stem cells (FIG. 5).
통증 완화Pain relief
본 발명에 따른 실시예 1의 소혈액 탈단백 추출물의 통증 완화 효과를 측정하기 위해 몸부림 테스트(Writhing test)를 실시하였다. ICR계 마우스에 실시예 1의 소혈액 탈단백 추출물을 마우스 체중 1㎏당 50, 200, 500 및 1000㎎씩 경구 및 주사 투여하고, 1시간 후에 1%(v/v) 아세트산을 0.1㎖/마우스 체중 10g 용량으로 복강 내에 주사하였다. 통증 완화 효과에 대한 양성대조군으로 아스피린(aspirin)을 70㎎/마우스 체중 1㎏ 용량으로 사용하였다. 5분이 경과한 후, 10분간 발생하는 몸부림(Writhing)과 스트레칭(stretching)의 횟수를 측정하였다(표 4). Writhing test was performed to measure the pain relieving effect of bovine blood deproteinized extract of Example 1 according to the present invention. ICR mice were orally and orally injected with the bovine blood deproteinized extract of Example 1 at a dose of 50, 200, 500 and 1000 mg per 1 kg body weight of the mouse. After 1 hour, 1% (v / v) The mice were injected intraperitoneally with a dose of 10g. As a positive control for the pain relief effect, aspirin was used at a dose of 70 mg / mouse body weight of 1 kg. After 5 minutes, the number of writhing and stretching that occurred for 10 minutes was measured (Table 4).
Extract 1000 mg / kg
상기 표 4에 나타난 바와 같이, 본 발명에 따른 소혈액 탈단백 추출물은 몸부림(writhing) 횟수를 감소시켰으므로 통증 완화 효과가 있음을 확인하였다.
As shown in Table 4, it was confirmed that the bovine blood deproteinization extract according to the present invention had a pain relieving effect because it reduced the number of writhing.
골질환Bone disease 치료 cure
본 발명에 사용한 MG-63 human osteoblastic cell(골아세포)은 한국 세포주 은행으로부터 분양 받아서 계대 배양하여 사용하였다. MG-63 human osteoblastic cell은 폴리스티렌(polystyrene) 75T 세포 배양접시에 부착시키고 페니실린(penicillin)과 스트렙토마이신(streptomycin)이 함유되고, 10% FBS(Gibco, USA)가 첨가된 DMEM(Welgene)을 사용하여 배양하였다. 배양시 습도는 95%, 온도는 37℃를 유지하면서 5% CO2를 공급하였다. 골아세포를 Well 당 DMEM 100ul가 포함된 96 well plate에서 1×104 Cell/Well씩 배양하였다. 실시예 1의 소혈액 탈단백 추출물을 0.02, 0.2, 2 ㎎/㎖로 처리하였다. 골아세포 재생의 음성대조군은 PBS를 추출물과 동일 용량으로 사용하였고 양성대조군으로서 소태아혈청을 10% 농도로 처리하여 사용하였다. 30시간이 경과한 후, PBS로 세척을 2번 실시한 후에 MTS(100㎕/well) 시약을 첨가하고 1시간 동안 37℃ 배양기에 배양한 후, MTS가 formazan으로 분해되는 양을 Microplate reader를 이용하여 490nm에서의 흡광도를 측정하여 값을 산출하였다. 분석법으로서 단기간에 대량 검색을 할 수 있는 MTS colorimetric assay를 통해 실시예 1의 소혈액 탈단백 추출물의 농도에 따른 연골 세포에 재생 효과를 측정하여 골아세포의 재생 효과를 확인하였다(도 6).
The MG-63 human osteoblastic cell (osteoblast) used in the present invention was purchased from Korean Cell Line Bank and used for subculture. MG-63 human osteoblastic cells were stained with polystyrene 75T cell culture dishes using DMEM (Welgene) supplemented with penicillin and streptomycin and supplemented with 10% FBS (Gibco, USA) Lt; / RTI > During the incubation, 5% CO 2 was supplied while maintaining the humidity at 95% and the temperature at 37 ° C. The osteoblasts were cultured in 96 well plates containing 100 μl of DMEM per well at 1 × 10 4 cells / well. The bovine blood deproteinized extract of Example 1 was treated with 0.02, 0.2 and 2 mg / ml. Negative controls for osteoclast regeneration were used at the same dose as PBS extract and 10% fetal bovine serum was used as a positive control. After 30 hours, the cells were washed twice with PBS, and the MTS (100 μl / well) reagent was added. After incubation for 1 hour at 37 ° C., the amount of MTS degradation to formazan was measured using a microplate reader Absorbance at 490 nm was measured to calculate the value. The regeneration effect on chondrocytes was measured according to the concentration of bovine blood deproteinization extract of Example 1 through an MTS colorimetric assay capable of mass-search in a short time as a method (Fig. 6).
골밀도 증가Increase in bone density
본 발명에 따른 실시예 1의 소혈액 탈단백 추출물의 골밀도 증가 효과를 측정하기 위해 난소절재 골다공증 동물 모델을 이용하였다. SD rat(오리엔트바이오, 한국)을 12주령에 난소 절재를 시행하고 1주일 뒤부터 실시예 1의 소혈액 탈단백 추출물을 SD rat 체중 1㎏당 50, 200, 500 및 1000㎎씩 경구 및 주사를 1일 간격으로 8주간 투여하였다. 난소 절재를 하지 않은 SD rat을 음성대조군으로 사용하고 유발대조군은 난소 절재를 시행하고 1주일 뒤부터 식염수 1㎖씩 경구 및 주사를 추출물과 동일한 간격으로 투여하였다. 실험동물을 마취후 대퇴골을 적출하고 골격외 다른 조직을 제거 후 건조하여 골밀도 및 골미네랄 함량을 X선 골밀도측정기(pDEXA Sabre X-ray Bone Densitometer, Norland Medical Systems, USA)를 사용하여 측정하였다. 본 발명에 따른 조성물은 골밀도(BMD, Bone Mineral Density)를 증가시키는 효과가 있음을 확인하였다(도 7).
In order to measure the bone mineral density of the bovine blood deproteinized extract of Example 1 according to the present invention, an animal model of ovariectomized osteoporosis was used. SD rats (Orient Bio, Korea) were ovariectomized at 12 weeks of age and after 1 week, the bovine deproteinized extract of Example 1 was orally and orally injected at 50, 200, 500 and 1000 mg per 1 kg of SD rats Were administered at intervals of 1 day for 8 weeks. SD rats without ovariectomy were used as a negative control group and ovariectomized rats were administered ovariectomized rats. After 1 week, 1 ml of saline solution was administered orally and injected at the same intervals as the extract. The bone mineral density and bone mineral content were measured by using an X-ray bone densitometer (pDEXA Saber X-ray Bone Densitometer, Norland Medical Systems, USA) after the femur was removed and the bone tissues were removed after drying. The composition according to the present invention has an effect of increasing BMD (Bone Mineral Density) (FIG. 7).
연골조직의 재생Regeneration of cartilage tissue
본 발명에 따른 실시예 1의 소혈액 탈단백 추출물의 연골조직 재생 효과를 측정하기 위해 MIA (monosodium iodoacetate)에 의한 골관절 손상 유발 동물모델을 이용하였다. 7주령 SD rat을 Zoletil 50(VIRBAC, France, 5 ㎎/㎏) 및 xylazine (Rompun®, Bayer AG, Germany, 2.5 ㎎/㎏)을 이용하여 마취를 실시하였다. Povidone 및 70 % alcohol을 이용하여 Monosodium iodoacetate (MIA)를 투여할 부위를 넓게 소독한 다음, MIA 50 μL (60 ㎎/㎖ solution)를 우측 슬관절에 투여하였다(유발 대조군). 1주일 뒤부터 실시예 1의 소혈액 탈단백 추출물을 마우스 체중 1㎏당 0.2 ㎎, 2 ㎎씩 무릎 주위에 국소주사하였다. 주사는 7일 간격으로 4주간 투여하였다. 실험 종료후 동물을 안락사시킨 뒤 유발부위를 적출하여 10 % 중성완충포르말린용액에 고정하였다. 고정된 조직은 탈회, 삭정, 탈수, 파라핀 포매, 박절 등 일반적인 조직처리 과정을 거쳐 조직병리학적 검사를 위한 검체를 제작한 뒤, Safranin-O 염색을 통해 연골이 생성된 부위를 관찰하였다. 본 발명에 따른 조성물은 연골조직을 증가시키는 효과가 있음을 확인하였다(도 8).In order to measure the cartilage regeneration effect of the bovine deproteinized extract of Example 1 according to the present invention, an animal model causing osteoarthritis caused by monosodium iodoacetate (MIA) was used. Seven - week old SD rats were anesthetized with Zoletil 50 (VIRBAC, France, 5 mg / kg) and xylazine (Rompun ®, Bayer AG, Germany, 2.5 mg / kg) The area to be treated with Monosodium iodoacetate (MIA) was sterilized using povidone and 70% alcohol, and 50 μL of MIA (60 ㎎ / ㎖ solution) was administered to the right knee (induced control group). After one week, the bovine blood deproteinized extract of Example 1 was injected topically to the knees about 0.2 mg and 2 mg / kg body weight. The injections were administered at intervals of 7 days for 4 weeks. After the end of the experiment, animals were euthanized and the induced area was excised and fixed in 10% neutral buffered formalin solution. The fixed tissue was subjected to general tissue treatment such as demineralization, digestion, dehydration, paraffin embedding, and dissection, and specimens were prepared for histopathological examination. Then, the area where cartilage was formed was observed through Safranin-O staining. It was confirmed that the composition according to the present invention has an effect of increasing cartilage tissue (FIG. 8).
본 발명을 바람직한 방법에 관련하여 상세히 설명하였으나, 본 발명의 범주 내에서 다른 실시형태, 개조 및 변형이 가능하다. Although the invention has been described in detail with reference to a preferred method, other embodiments, adaptations and variations are possible within the scope of the invention.
본 명세서에서 인용한 모든 참조문헌, 기사, 공보 및 특허 및 특허 출원이 온전히 본 명세서에 참조로 병합되어 있다. 따라서, 하기 청구의 범위의 진의 및 범주는 상기한 바람직한 실시형태의 설명에 제한되어서는 안 된다.All references, articles, publications and patents and patent applications cited herein are hereby incorporated by reference in their entirety. Accordingly, the spirit and scope of the following claims should not be limited to the description of the preferred embodiments described above.
Claims (32)
상기 조성물은 줄기세포의 골 조직으로의 이동을 유도하는, 줄기세포로부터 연골세포 분화 유도 또는 촉진용 조성물.13. The method of claim 12,
Wherein said composition induces migration of stem cells to bone tissue, wherein said composition induces or promotes the differentiation of chondrocytes from stem cells.
상기 추출물은 연골세포의 SOX9 발현을 증가시키는, 줄기세포로부터 연골세포 분화 유도 또는 촉진용 조성물.13. The method of claim 12,
Wherein said extract enhances SOX9 expression of chondrocytes, and induces or promotes chondrocyte differentiation from stem cells.
상기 줄기세포는 골수 유래 또는 지방조직 유래인, 줄기세포로부터 연골세포 분화 유도 또는 촉진용 조성물.13. The method of claim 12,
Wherein said stem cell is derived from bone marrow or fat tissue, and induces or promotes differentiation of chondrocytes from stem cells.
상기 줄기세포는 골수 유래 또는 지방조직 유래인, 줄기세포로부터 연골세포 분화 방법.19. The method of claim 18,
Wherein said stem cells are derived from bone marrow or adipose tissue.
상기 분화는 SOX9의 발현 증가에 의해 매개되는, 줄기세포로부터 연골세포 분화 방법.19. The method of claim 18,
Wherein said differentiation is mediated by increased expression of SOX9.
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| US11466254B2 (en) | 2017-10-18 | 2022-10-11 | Seoul National University R & Db Foundation | Composition containing, as active ingredient, culture of chicken bone marrow-derived osteochondral progenitor cells for promoting osteogenesis or inducing chondrogenic differentiation |
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