KR20210110266A - Composition for the prevention or treatment of bone disease, including soluble CCR2 stem cells as an active ingredient - Google Patents

Composition for the prevention or treatment of bone disease, including soluble CCR2 stem cells as an active ingredient Download PDF

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KR20210110266A
KR20210110266A KR1020210111650A KR20210111650A KR20210110266A KR 20210110266 A KR20210110266 A KR 20210110266A KR 1020210111650 A KR1020210111650 A KR 1020210111650A KR 20210111650 A KR20210111650 A KR 20210111650A KR 20210110266 A KR20210110266 A KR 20210110266A
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osteoarthritis
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KR102446150B1 (en
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김석중
조미라
나현식
이선영
최시영
권지예
조근형
김구영
김선애
고은정
최정원
백진아
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가톨릭대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons

Abstract

The present invention relates to a composition for prevention or treatment of bone disease, comprising stem cells induced to express soluble CCR2 as an active ingredient. It has been confirmed that mesenchymal stem cells, into which soluble CCR2 or an E3 domain of soluble CCR2 according to the present invention is introduced, neutralize MCP-1, a bone disease factor, and thus reduce collagen epitope (CTX-II) and collagen metabolism factors (MMP1 and MMP3) related to collagen absorption in vitro or vi vivo. In addition, it has been confirmed that an expression of SOX9 gene and anti-inflammatory cytokines (TGF-β and IL-10) related to cartilage differentiation is significantly increased. Thus, the composition has an excellent regenerative ability for osteoarthritis and excellent pain suppression and alleviation effects, and thus can be effectively used for the prevention or treatment of bone diseases such as osteoarthritis.

Description

Soluble CCR2 발현 유도된 줄기세포를 유효성분으로 포함하는 골질환의 예방 또는 치료용 조성물{Composition for the prevention or treatment of bone disease, including soluble CCR2 stem cells as an active ingredient}Composition for the prevention or treatment of bone disease, including soluble CCR2 expression-induced stem cells as an active ingredient {Composition for the prevention or treatment of bone disease, including soluble CCR2 stem cells as an active ingredient}

본 발명은 Soluble CCR2(sCCR2) 발현 유도된 줄기세포를 유효성분으로 포함하는 골질환의 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating bone disease, comprising stem cells induced in Soluble CCR2 (sCCR2) expression as an active ingredient.

골관절염(Osteoarthritis, OA)은 연골에서 흔히 발생하는 퇴행성 관절질환이며, 인구 노령화 추세에 따라 임상에서 흔히 볼 수 있는 노인성 질환으로 관절연골의 퇴행성 변화를 기반으로 병변이 시작되어 운동 시 통증과 부종, 관절 강직감 및 점진적인 운동장애를 일으킨다. Osteoarthritis (OA) is a degenerative joint disease that occurs frequently in cartilage, and is a geriatric disease commonly seen in clinical practice according to the aging of the population. It causes stiffness and gradual movement disorders.

골관절염의 치료에는 체중감량, 근육강화운동, 단순진통제, 비스테로이드 소염제, 관절내 스테로이드 주사 및 수술 등의 운동요법, 약물요법, 수술요법이 실시되고 있다. 하지만, 스테로이드 소염제는 위장관 장애 등의 합병증을 일으킬 수 있고, 그 외에 스테로이드제의 관절 내 주사요법은 전신부작용 및 스테로이드의 반복주사에 따른 연골의 파괴, 감염의 위험성이 따르게 되므로 적극적인 치료법이 될 수 없다. 또한, 본질적으로 퇴행성 질병의 진행을 억제시키거나 차단시켜 줄 수 있는 골관절염의 치료법이 미비한 실정이다.For the treatment of osteoarthritis, exercise therapy such as weight loss, muscle strengthening exercises, simple analgesics, non-steroidal anti-inflammatory drugs, intra-articular steroid injections and surgery, drug therapy, and surgery are implemented. However, steroid anti-inflammatory drugs can cause complications such as gastrointestinal disorders, and other intra-articular injection therapy of steroids cannot be an active treatment because there is a risk of systemic side effects and cartilage destruction and infection due to repeated injections of steroids. . In addition, there is a lack of treatment for osteoarthritis that can essentially inhibit or block the progression of degenerative diseases.

KRUS 10-181982710-1819827 BB

본 발명의 목적은 CCR2(C-C chemokine receptor type 2)를 포함하는 재조합 벡터가 형질도입된 줄기세포 또는 이의 배양액을 유효성분으로 포함하는 골질환의 예방 또는 치료용 조성물을 제공하는 것이다. It is an object of the present invention to provide a composition for preventing or treating bone disease, comprising as an active ingredient a stem cell transduced with a recombinant vector containing CCR2 (C-C chemokine receptor type 2) or a culture solution thereof.

본 발명의 또 다른 목적은 CCR2(C-C chemokine receptor type 2)의 E3 도메인을 포함하는 재조합 벡터가 형질도입된 줄기세포 또는 이의 배양액을 유효성분으로 포함하는 골질환의 예방 또는 치료용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for the prevention or treatment of bone diseases comprising, as an active ingredient, stem cells transduced with a recombinant vector comprising the E3 domain of CCR2 (CC chemokine receptor type 2) or a culture solution thereof. .

본 발명의 또 다른 목적은 상기의 조성물을 포함하는, 골질환 치료용 키트를 제공하는 것이다.Another object of the present invention is to provide a kit for treating bone disease, comprising the above composition.

상기 목적을 달성하기 위하여, CCR2(C-C chemokine receptor type 2)를 포함하는 재조합 벡터가 형질도입된 줄기세포 또는 이의 배양액을 유효성분으로 포함하는 골질환의 예방 또는 치료용 조성물을 제공한다. In order to achieve the above object, there is provided a composition for the prevention or treatment of bone diseases comprising, as an active ingredient, stem cells transduced with a recombinant vector containing CCR2 (C-C chemokine receptor type 2) or a culture solution thereof.

본 발명의 일실시예에 있어서, 상기 CCR2를 암호화하는 폴리뉴클레오타이드는 서열번호 2의 염기서열로 이루어진 것일 수 있다. In one embodiment of the present invention, the polynucleotide encoding CCR2 may consist of the nucleotide sequence of SEQ ID NO: 2.

본 발명의 일실시예에 있어서, 상기 CCR2는 서열번호 1의 아미노산 서열로 이루어진 것일 수 있다.In one embodiment of the present invention, the CCR2 may consist of the amino acid sequence of SEQ ID NO: 1.

본 발명의 일실시예에 있어서, 상기 줄기세포는 성체줄기세포, 배아줄기세포, 중간엽 줄기세포, 종양 줄기세포 및 유도만능줄기세포로 이루어진 군에서 선택된 1종 이상의 세포인 것일 수 있다. In one embodiment of the present invention, the stem cells may be one or more cells selected from the group consisting of adult stem cells, embryonic stem cells, mesenchymal stem cells, tumor stem cells, and induced pluripotent stem cells.

본 발명의 일실시예에 있어서, 상기 중간엽줄기세포는 골관절염 환자의 말초혈액 또는 지방조직으로부터 분리된 것일 수 있다. In one embodiment of the present invention, the mesenchymal stem cells may be isolated from peripheral blood or adipose tissue of a patient with osteoarthritis.

본 발명의 일실시예에 있어서, 상기 상기 골질환은 골관절염(Osteoarthritis)인 것일 수 있다. In one embodiment of the present invention, the bone disease may be osteoarthritis (Osteoarthritis).

또한, 본 발명은 CCR2(C-C chemokine receptor type 2)의 E3 도메인을 포함하는 재조합 벡터가 형질도입된 줄기세포 또는 이의 배양액을 유효성분으로 포함하는 골질환의 예방 또는 치료용 조성물을 제공한다. In addition, the present invention provides a composition for preventing or treating bone disease comprising, as an active ingredient, a stem cell transduced with a recombinant vector comprising the E3 domain of CCR2 (C-C chemokine receptor type 2) or a culture solution thereof.

본 발명의 일실시예에 있어서, 상기 E3 도메인은 Fc절편(fragment)을 포함하는 것일 수 있다. In an embodiment of the present invention, the E3 domain may include an Fc fragment.

본 발명의 일실시예에 있어서, CCR2를 암호화하는 폴리뉴클레오타이드는 서열번호 4의 염기서열로 이루어진 것일 수 있다. In one embodiment of the present invention, the polynucleotide encoding CCR2 may consist of the nucleotide sequence of SEQ ID NO: 4.

본 발명의 일실시예에 있어서, CCR2는 서열번호 3의 아미노산 서열로 이루어진 것일 수 있다. In one embodiment of the present invention, CCR2 may consist of the amino acid sequence of SEQ ID NO: 3.

본 발명의 일실시예에 있어서, 상기 줄기세포는 성체줄기세포, 배아줄기세포, 중간엽 줄기세포, 종양 줄기세포 및 유도만능줄기세포로 이루어진 군에서 선택된 1종 이상의 세포인 것일 수 있다. In one embodiment of the present invention, the stem cells may be one or more cells selected from the group consisting of adult stem cells, embryonic stem cells, mesenchymal stem cells, tumor stem cells, and induced pluripotent stem cells.

본 발명의 일실시예에 있어서, 상기 중간엽줄기세포는 골관절염 환자의 말초혈액 또는 지방조직으로부터 분리된 것일 수 있다. In one embodiment of the present invention, the mesenchymal stem cells may be isolated from peripheral blood or adipose tissue of a patient with osteoarthritis.

본 발명의 일실시예에 있어서, 상기 골질환은 골관절염(Osteoarthritis)인 것일 수 있다. In one embodiment of the present invention, the bone disease may be osteoarthritis (Osteoarthritis).

또한, 본 발명은 상기의 조성물을 포함하는, 골질환 치료용 키트을 제공한다.In addition, the present invention provides a kit for treating bone disease, comprising the composition.

본 발명에 따른 CCR2 또는 CCR2의 E3 도메인이 도입된 중간엽 줄기세포는 in vitro 또는 vi vivo 상에서, 골 질환 인자인 MCP-1을 중화하여, 콜라겐 체내 흡수와 관련된 콜라겐 에피토프(CTX-Ⅱ) 및 콜라겐 대사인자(MMP1 및 MMP3)을 감소시킴을 확인하였다. 또한, 연골 분화와 관련된 SOX9 유전자, 항염증 사이토카인(TGF-β 및 IL-10)의 발현을 유의적으로 증가시킴을 확인하였다. 따라서, 골관절염에 대한 재생능이 우수하고 통증억제 및 완화 효과가 우수한 바, 골관절염과 같은 골질환의 예방 또는 치료에 유용하게 사용될 수 있다. The mesenchymal stem cells into which CCR2 or the E3 domain of CCR2 according to the present invention are introduced neutralize MCP-1, a bone disease factor, in vitro or in vivo, and thus, a collagen epitope (CTX-II) and collagen related to collagen absorption into the body. It was confirmed that the metabolic factors (MMP1 and MMP3) were reduced. In addition, it was confirmed that the expression of SOX9 gene, anti-inflammatory cytokines (TGF-β and IL-10) related to cartilage differentiation was significantly increased. Therefore, it has excellent regenerative ability for osteoarthritis and excellent pain suppression and alleviation effects, and thus can be usefully used for the prevention or treatment of bone diseases such as osteoarthritis.

도 1a는 건강한 사람(healthy) 또는 골관절염(Osteoarthritis, OA) 환자의 활막액에서의 human IL-1β, human IL-17, human VEGF, human MCP1 등을 포함하는 염증성 사이토카인의 분비량을 측정한 결과(왼쪽부터)를 나타낸 것이고, 그 중 MCP1에 대해서는 류마티스 관절염 환자 또는 골관절염 환자에서의 분비량을 비교한 결과(가장 오른쪽)를 나타낸 것이다.
도 1b는 환자 유래의 연골세포에 MCP1를 처리한 후, 연골 세포의 분화와 관련되는 Collagen type 2a 및 SOX9의 mRNA 발현량, catalytic enzyme인 MMP1 및 MMP3의 발현량, 통증인자인 TRPV2의 발현량을 비교한 결과를 나타낸 것이다.
도 2는 골관절염 동물 모델의 연골 조직 내에서 MCP1의 발현 여부를 면역화학염색법을 통해 확인한 결과이다.
도 3은 본 발명의 일실시예에 따른 sCCR2 E3-Fc 과발현 재조합 벡터 지도를 나타낸 것이다.
도 4는 본 발명의 일실시예에 따른 sCCR2 과발현 재조합 벡터 지도를 나타낸 것이다.
도 5는 HEK293 세포에 sCCR2 재조합 벡터를 도입한 후 과발현되는지 여부를 확인한 결과이다.
도 6은 골관절염 동물 모델에 sCCR2(E3-Fc) 과발현 재조합 벡터를 주입한 후, 회복된 대퇴골 관절을 해부현미경으로 관찰한 것이다.
도 7은 골관절염 동물 모델에 sCCR2(E3-Fc) 과발현 재조합 벡터를 주입한 후, 손상된 연골 및 회복된 연골을 Safranin O 염색으로 확인한 결과이다.
도 8은 골관절염 동물 모델에서 sCCR2 재조합 벡터 주입 후, 발을 떼는데 걸리는 시간(Paw Withdrawal Latency, s, 초), 얼마만큼의 무게를 주었을 때 발을 떼는지(Paw Withdrawal Threshold, g) 및 우측 뒷다리 무게를 측정한 결과를 나타낸 것이다.
도 9는 골관절염 동물 모델의 관절 부위를 Safranin O 염색을 한 후 연골 손상 정도를 비교하였고, OARSI score 및 Mankin score 결과를 나타낸 것이다.
도 10은 골관절염 동물 모델에서 sCCR2 과발현시킨 후 micro CT 분석 결과를 나타낸 것이다.
도 11은 골관절염 동물 모델의 연골 조직 내에서 IL-1β, IL-6 및 MMP13의 발현여부를 면역화학염색법을 통해 확인한 결과를 나타낸 것이다.
도 12는 sCCR2가 발현하는 중간엽줄기세포의 특성을 분석한 결과로서, 도 12a는 positive marker를 분석한 결과이고, 도 12b는 negative marker를 분석한 결과를 나타낸 것이다.
도 13은 골관절염 동물 모델에 sCCR2가 발현하는 중간엽줄기세포를 주입한 후, 발을 떼는데 걸리는 시간(Paw Withdrawal Latency, s, 초), 얼마만큼의 무게를 주었을 때 발을 떼는지(Paw Withdrawal Threshold, g) 및 우측 뒷다리 무게를 측정한 결과를 나타낸 것이다.
도 14는 pSecTag2-rat CCR2의 아미노산 서열(서열번호 7)을 나타낸 것이다.
도 15는 pSecTag2-rat CCR2E3-FC의 아미노산 서열(서열번호 8)을 나타낸 것이다.
도 16은 pSecTag2-rat CCR2의 염기 서열(서열번호 9)을 나타낸 것이다.
도 17은 pSecTag2-rat CCR2E3-FC의 염기 서열(서열번호 10)을 나타낸 것이다.
도 18은 골관절염 동물 모델에 sCCR2(E3-Fc)가 발현하는 중간엽 줄기세포를 주입한 후, MCP-1 중화를 확인한 것이다.
도 19는 골관절염 동물 모델에 sCCR2(E3-Fc)가 발현하는 중간엽 줄기세포를 주입한 후, SOX9의 mRNA 발현의 증가를 확인한 것이다.
도 20은 골관절염 동물 모델에 sCCR2(E3-Fc)가 발현하는 중간엽 줄기세포를 주입한 후, MMP1 및 MMP3의 발현 억제를 확인한 것이다.
도 21은 골관절염 동물 모델에 sCCR2(E3-Fc)가 발현하는 중간엽 줄기세포를 주입한 후, 항염증 사이토카인의 분비가 증가한 것을 확인한 것이다.
도 22는 골관절염 동물 모델에 sCCR2(E3-Fc)가 발현하는 중간엽 줄기세포를 주입한 후, CTX-Ⅱ발현량의 감소를 확인한 것이다.Figure 1a is a result of measuring the secretion amount of inflammatory cytokines including human IL-1β, human IL-17, human VEGF, human MCP1, etc. in the synovial fluid of a healthy person or osteoarthritis (OA) patient ( from the left), and among them, for MCP1, the result of comparing the secretion amount in patients with rheumatoid arthritis or osteoarthritis (far right) is shown.
Figure 1b shows the mRNA expression levels of collagen type 2a and SOX9 related to the differentiation of chondrocytes, the expression levels of catalytic enzymes MMP1 and MMP3, and the expression level of TRPV2, a pain factor, after treating patient-derived chondrocytes with MCP1. The comparison results are shown.
2 is a result of confirming the expression of MCP1 in the cartilage tissue of an animal model of osteoarthritis through immunochemical staining.
3 shows a map of the sCCR2 E3-Fc overexpressing recombinant vector according to an embodiment of the present invention.
4 shows a map of the sCCR2 overexpressing recombinant vector according to an embodiment of the present invention.
5 is a result of confirming whether or not overexpression of the sCCR2 recombinant vector is introduced into HEK293 cells.
6 is a dissection microscope of the restored femoral joint after injection of a recombinant vector overexpressing sCCR2(E3-Fc) into an animal model of osteoarthritis.
7 is a result of confirming the damaged cartilage and the restored cartilage by Safranin O staining after injecting the sCCR2(E3-Fc) overexpressing recombinant vector into the osteoarthritis animal model.
8 shows the time it takes to release the paw (Paw Withdrawal Latency, s, seconds) after injection of the sCCR2 recombinant vector in an animal model of osteoarthritis, how much weight it takes to release the foot (Paw Withdrawal Threshold, g), and the right hind limb The weight measurement results are shown.
9 is a comparison of the degree of cartilage damage after Safranin O staining of the joint site of an animal model of osteoarthritis, and shows the results of OARSI score and Mankin score.
10 shows the results of micro CT analysis after overexpression of sCCR2 in an animal model of osteoarthritis.
11 shows the results of confirming the expression of IL-1β, IL-6 and MMP13 in the cartilage tissue of an animal model of osteoarthritis through immunochemical staining.
12 is a result of analyzing the characteristics of mesenchymal stem cells expressing sCCR2, FIG. 12a is a result of analyzing a positive marker, and FIG. 12b is a result of analyzing a negative marker.
Figure 13 is after injection of mesenchymal stem cells expressing sCCR2 in the osteoarthritis animal model, the time it takes to take off the foot (Paw Withdrawal Latency, s, seconds), how much weight it takes to release the foot (Paw Withdrawal) Threshold, g) and the results of measuring the weight of the right hind leg are shown.
14 shows the amino acid sequence of pSecTag2-rat CCR2 (SEQ ID NO: 7).
15 shows the amino acid sequence of pSecTag2-rat CCR2E3-FC (SEQ ID NO: 8).
16 shows the nucleotide sequence (SEQ ID NO: 9) of pSecTag2-rat CCR2.
17 shows the nucleotide sequence (SEQ ID NO: 10) of pSecTag2-rat CCR2E3-FC.
18 is a view illustrating the neutralization of MCP-1 after injection of mesenchymal stem cells expressing sCCR2 (E3-Fc) into an animal model of osteoarthritis.
19 shows an increase in SOX9 mRNA expression after injection of mesenchymal stem cells expressing sCCR2 (E3-Fc) into an animal model of osteoarthritis.
20 is a view illustrating inhibition of MMP1 and MMP3 expression after injection of mesenchymal stem cells expressing sCCR2 (E3-Fc) into an animal model of osteoarthritis.
FIG. 21 shows that after injection of mesenchymal stem cells expressing sCCR2 (E3-Fc) into an animal model of osteoarthritis, the secretion of anti-inflammatory cytokines was increased.
22 is a view showing a decrease in CTX-II expression level after injection of mesenchymal stem cells expressing sCCR2 (E3-Fc) into an animal model of osteoarthritis.

본 발명의 용어, "CCR2(C-C chemokine receptor type 2)"는 chemokine receptor로서, CCR2 유전자에 의해 인코딩되는 단백질로서, 단핵구를 유인하는 MCP1(monocyte chemoattractant protein-1 or CCL2)의 receptor이다. As used herein, the term "CCR2 (CC chemokine receptor type 2)" is a chemokine receptor, a protein encoded by the CCR2 gene, and a receptor of monocyte chemoattractant protein-1 or CCL2 (MCP1) that attracts monocytes.

본 발명은 CCR2(C-C chemokine receptor type 2)를 포함하는 재조합 벡터가 형질도입된 줄기세포 또는 이의 배양액을 유효성분으로 포함하는 골질환의 예방 또는 치료용 조성물을 제공할 수 있다. The present invention can provide a composition for preventing or treating bone disease, comprising, as an active ingredient, a stem cell transduced with a recombinant vector containing CCR2 (C-C chemokine receptor type 2) or a culture solution thereof.

또한, 본 발명은 CCR2(C-C chemokine receptor type 2)의 E3 도메인을 포함하는 재조합 벡터가 형질도입된 줄기세포 또는 이의 배양액을 유효성분으로 포함하는 골질환의 예방 또는 치료용 조성물을 제공할 수 있다. In addition, the present invention can provide a composition for preventing or treating bone disease, comprising as an active ingredient a stem cell transduced with a recombinant vector comprising the E3 domain of CCR2 (C-C chemokine receptor type 2) or a culture solution thereof.

바람직하게, 상기 CCR2 단백질은 서열번호 1의 아미노산 서열로 이루어진 것이거나, CCR2 단백질의 E3 도메인으로서 서열번호 3의 아미노산 서열로 이루어진 것일 수 있다. 상기 CCR2 단백질을 암호화하는 폴리뉴클레오타이드는 서열번호 2의 염기서열로 이루어진 것이거나, CCR2 단백질의 E3 도메인을 암호화하는 폴리뉴클레오타이드로서 서열번호 4의 염기서열로 이루어진 것일 수 있다. Preferably, the CCR2 protein may consist of the amino acid sequence of SEQ ID NO: 1, or may consist of the amino acid sequence of SEQ ID NO: 3 as the E3 domain of the CCR2 protein. The polynucleotide encoding the CCR2 protein may consist of the nucleotide sequence of SEQ ID NO: 2, or may consist of the nucleotide sequence of SEQ ID NO: 4 as a polynucleotide encoding the E3 domain of the CCR2 protein.

또한, 본 발명에 따른 상기 CCR2 단백질은 바람직하게 서열번호 1의 아미노산 서열로 이루어진 폴리펩타이드에 대해 기능적 동등물일 수 있다. 상기 "기능적 동등물"이란, 아미노산의 부가, 치환 또는 결실의 결과, 서열번호 1의 아미노산 서열과 적어도 60%, 바람직하게는 70%, 보다 바람직하게는 80% 이상의 서열 상동성을 갖는 것으로서, 본 발명의 CCR2와 실질적으로 동질의 활성을 나타내는 폴리펩타이드를 말한다. 상기 "실질적으로 동질의 활성"이란 상기 CCR2의 활성을 의미한다. 상기 기능적 동등물에는, 예를 들어, 본 발명에 따른 CCR2의 아미노산 서열의 아미노산 중 일부가 치환되거나, 결실 또는 부가된 아미노산 서열 변형체가 포함될 수 있다. 아미노산의 치환은 바람직하게는 보존적 치환일 수 있으며, 천연에 존재하는 아미노산의 보존적 치환의 예는 다음과 같다; 지방족 아미노산(Gly, Ala, Pro), 소수성 아미노산(Ile, Leu, Val), 방향족 아미노산(Phe, Tyr, Trp), 산성 아미노산(Asp, Glu), 염기성 아미노산(His, Lys, Arg, Gln, Asn) 및 황함유 아미노산(Cys, Met). 아미노산의 결실은 바람직하게는 본 발명의 CCR2의 활성에 직접 관여하지 않는 부분에 위치할 수 있다. 또한, 상기 기능적 동등물의 범위에는 CCR2의 기본 골격 및 이의 생리 활성을 유지하면서 폴리펩타이드의 일부 화학 구조가 변형된 폴리펩타이드 유도체도 포함될 수 있다. 예를 들어, 본 발명의 폴리펩타이드의 안정성, 저장성, 휘발성 또는 용해도 등을 변경시키기 위한 구조변경 및 생리활성을 유지하면서 다른 단백질과 융합으로 만들어진 융합 단백질 등이 이에 포함될 수 있다.In addition, the CCR2 protein according to the present invention may preferably be a functional equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. The "functional equivalent" means having at least 60%, preferably 70%, more preferably 80% or more sequence homology with the amino acid sequence of SEQ ID NO: 1 as a result of addition, substitution or deletion of amino acids, It refers to a polypeptide that exhibits substantially the same activity as CCR2 of the present invention. The "substantially homogeneous activity" means the activity of the CCR2. The functional equivalent may include, for example, an amino acid sequence variant in which some of the amino acids of the amino acid sequence of CCR2 according to the present invention are substituted, deleted, or added. The substitution of amino acids may preferably be conservative substitutions, and examples of conservative substitutions of naturally occurring amino acids are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn) ) and sulfur-containing amino acids (Cys, Met). Deletion of amino acids may preferably be located in a region not directly involved in the activity of CCR2 of the present invention. In addition, the scope of the functional equivalent may include a polypeptide derivative in which some chemical structures of the polypeptide are modified while maintaining the basic backbone of CCR2 and its physiological activity. For example, fusion proteins made by fusion with other proteins while maintaining structural changes and physiological activity to change the stability, storage, volatility or solubility of the polypeptide of the present invention may be included therein.

또한, 상기 CCR2 단백질을 암호화하는 폴리뉴클레오타이드는 플라스미드 또는 바이러스 벡터와 같은 발현 벡터 내로 공지의 방법에 의해 도입시킨 후 상기 발현 벡터를 당업계에 공지된 다양한 방법으로 형질도입(transduction) 또는 형질주입(transfection)에 의해 발현형으로 표적 세포 내에 도입시킬 수 있다.In addition, the polynucleotide encoding the CCR2 protein is introduced by a known method into an expression vector such as a plasmid or a viral vector, and then the expression vector is transduced or transfected by various methods known in the art. ) can be introduced into target cells by expression type.

플라스미드 발현 벡터는 사람에게 사용할 수 있는 FDA의 승인된 유전자 전달방법으로 사람 세포에 직접적으로 플라스미드 DNA를 전달하는 방법으로서(Nabel, E G et al, Science, 249:1285-1288, 1990), 플라스미드 DNA는 바이러스 벡터와는 달리 균질하게 정제될 수 있는 장점이 있다. 본 발명에서 사용할 수 있는 플라스미드 발현 벡터로는 당업계에 공지된 포유동물 발현 플라스미드를 사용할 수 있으며, 본 발명의 일실시예에서는 pSecTag2A 벡터를 사용하여 CCR2 유전자가 삽입된 재조합 발현벡터인 pSecTag2A-CCR2, pSecTag2A-CCR2E3 벡터를 제조하였다.The plasmid expression vector is an FDA-approved gene delivery method that can be used in humans, and is a method of directly delivering plasmid DNA into human cells (Nabel, EG et al, Science, 249:1285-1288, 1990). Unlike a viral vector, it has the advantage that it can be purified homogeneously. As a plasmid expression vector that can be used in the present invention, mammalian expression plasmids known in the art can be used. In one embodiment of the present invention, pSecTag2A-CCR2, a recombinant expression vector into which the CCR2 gene is inserted using pSecTag2A vector, The pSecTag2A-CCR2E3 vector was constructed.

본 발명에 따른 핵산을 포함하는 플라스미드 발현 벡터(plasmid expression vector)는 당업계에 공지된 방법, 예를 들어, 일시적 형질주입(transient transfection), 미세주사(microinjection), 형질도입(transduction), 세포융합, 칼슘 포스페이트 침전법, 리포좀 매개된 형질주입(liposome-mediated transfection), DEAE 덱스트란-매개된 형질주입(DEAE Dextran-mediated transfection), 폴리브렌-매개된 형질주입(polybrene-mediated trans fection), 전기침공법(electropora tion), 유전자 건(gene gun) 및 세포 내로 DNA를 유입시키기 위한 다른 공지의 방법에 의해 목적세포 내로 도입할 수 있고, 이에 한정되는 것은 아니다(Wu et al, J Bio Chem, 267:963-967, 1992; Wu et al, Bio Chem, 263:14621-14624, 1988).The plasmid expression vector containing the nucleic acid according to the present invention can be prepared by methods known in the art, for example, transient transfection, microinjection, transduction, cell fusion. , calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, electrophoresis It can be introduced into the target cell by an invasion method (electroporation), a gene gun (gene gun), and other known methods for introducing DNA into the cell, but is not limited thereto (Wu et al, J Bio Chem, 267) :963-967, 1992; Wu et al, Bio Chem, 263:14621-14624, 1988).

또한, 상기 CCR2를 발현시킬 수 있는 벡터는 공지의 방법에 의해 세포, 조직 또는 체내로 투여될 수 있는데, 예를 들면, 국소적으로, 비경구, 경구, 비강, 정맥, 근육 내, 피하 내 또는 다른 적절한 수단에 의해 투여될 수 있다. 특히, 상기 벡터는 표적 조직 또는 표적 세포를 치료하기 위한 유효량으로 직접 주사할 수 있다.In addition, the vector capable of expressing CCR2 may be administered to cells, tissues or the body by a known method, for example, topically, parenterally, orally, nasally, intravenously, intramuscularly, subcutaneously or It may be administered by other suitable means. In particular, the vector can be directly injected in an amount effective to treat a target tissue or target cell.

본 발명에 따른 조성물은 골질환, 바람직하게는 골관절염을 예방 및 치료할 수 있는 약학적 조성물로 사용될 수 있으며, 상기 약학적 조성물은 약학적으로 허용되는 담체를 추가로 포함할 수 있다. 상기에서 "약학적으로 허용되는"이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증 등과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 약학적으로 허용되는 담체로는, 예를 들면, 락토스, 전분, 셀룰로스 유도체, 마그네슘 스테아레이트, 스테아르산 등과 같은 경구투여용 담체 및 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등과 같은 비경구 투여용 담체 등이 있으며, 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 그 밖의 약학적으로 허용되는 담체로는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다(Remington's Pharmaceutical Sciences, 19th ed, Mack Publishing Company, Easton, PA, 1995). 본 발명에 따른 약학적 조성물은 상술한 바와 같은 약학적으로 허용되는 담체와 함께 당업계에 공지된 방법에 따라 적합한 형태로 제형화 될 수 있다. 즉, 본 발명의 약학적 조성물은 공지의 방법에 따라 다양한 비경구 또는 경구 투여용 형태로 제조될 수 있으며, 비경구 투여용 제형의 대표적인 것으로는 주사용 제형으로 등장성 수용액 또는 현탁액이 바람직하다. 주사용 제형은 적합한 분산제 또는 습윤제 및 현탁화제를 사용하여 당업계에 공지된 기술에 따라 제조할 수 있다. 예를 들면, 각 성분을 식염수 또는 완충액에 용해시켜 주사용으로 제형화될 수 있다. 또한, 경구 투여용 제형으로는, 분말, 과립, 정제, 환약 및 캡슐 등이 있고, 이에 한정되는 것은 아니다.The composition according to the present invention may be used as a pharmaceutical composition capable of preventing and treating bone diseases, preferably osteoarthritis, and the pharmaceutical composition may further include a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like and water, suitable oils, saline, aqueous glucose and glycols for parenteral administration. and a carrier, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed, Mack Publishing Company, Easton, PA, 1995). The pharmaceutical composition according to the present invention may be formulated in a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms according to known methods, and an isotonic aqueous solution or suspension is preferred as an injectable dosage form as a representative dosage form for parenteral administration. Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving it in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules.

상기와 같은 방법으로 제형화된 약학적 조성물은 유효량으로 경구, 경피, 피하, 정맥 또는 근육을 포함한 여러 경로를 통해 투여될 수 있다. 상기에서 "유효량" 이란 환자에게 투여하였을 때, 예방 또는 치료 효과를 나타내는 양을 말한다. 본 발명에 따른 약학적 조성물의 투여량은 투여 경로, 투여 대상, 연령, 성별 체중, 개인차 및 질병 상태에 따라 적절히 선택할 수 있다. 바람직하게는, 본 발명의 약학적 조성물은 질환의 정도에 따라 유효성분의 함량을 달리할 수 있으나, 바람직하게는 0.1 ~ 1000 ㎎/체중kg/day의 유효용량으로 하루에 수회 반복 투여될 수 있다. 또한, 본 발명의 조성물은 골질환을 예방, 개선 또는 치료하는 효과를 가지는 공지의 화합물과 병행하여 투여할 수도 있다.The pharmaceutical composition formulated in the above manner may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular. As used herein, the term "effective amount" refers to an amount exhibiting a preventive or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may be appropriately selected according to the route of administration, administration, age, sex and weight, individual differences and disease state. Preferably, the pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease, but may be administered repeatedly several times a day, preferably at an effective dose of 0.1 to 1000 mg/weight kg/day . In addition, the composition of the present invention may be administered in combination with a known compound having an effect of preventing, improving or treating bone disease.

이하, 본 발명을 실시예를 통하여 더욱 상세히 설명하기로 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

실시예 1. 골관절염 환자에서 MCP1의 발현 증가 및 병인 반응 상관성 규명Example 1. Identification of the correlation between increased expression of MCP1 and etiologic response in osteoarthritis patients

본 발명자들은 골관절염의 병인성을 규명하기 위하여, 건강한 사람(healthy) 또는 골관절염(Osteoarthritis, OA) 환자의 활막액에서 여러 종류의 염증성 사이토카인의 분비량을 비교하는 실험을 수행하였다. 상기 분비량의 측정은 공지된 방법인 ELIZA를 통해 진행하였다. The present inventors performed an experiment comparing the secretion amount of various types of inflammatory cytokines in the synovial fluid of a healthy person or a patient with osteoarthritis (OA) in order to identify the etiology of osteoarthritis. The measurement of the secretion amount was carried out through a known method, ELIZA.

그 결과, human IL-1β, human IL-17, human VEGF(vascular endothelial growth factor), human MCP-1(monocyte chemoattractant protein 1) 등은 건강한 사람에 비하여 골관절염 환자의 활막액에서 분비량이 현저히 증가됨을 확인하였다(도 1a). 특히, 본 발명자들은 류마티스 관절염 환자와 골관절염 환자에서의 MCP-1 분비량을 확인하였고, 그 결과, 류마티스 관절염 환자에 비해 골관절염 환자의 활막액에서는 MCP-1 분비량이 현저히 증가되어 있음을 확인하였다(도 1a 오른쪽 그래프). As a result, it was confirmed that the secretion of human IL-1β, human IL-17, human vascular endothelial growth factor (VEGF), and human MCP-1 (monocyte chemoattractant protein 1) from the synovial fluid of osteoarthritis patients was significantly increased compared to healthy people. (Fig. 1a). In particular, the present inventors confirmed the amount of MCP-1 secretion in rheumatoid arthritis patients and osteoarthritis patients, and as a result, it was confirmed that the MCP-1 secretion amount was significantly increased in the synovial fluid of osteoarthritis patients compared to rheumatoid arthritis patients (Fig. 1a). right graph).

이후, 본 발명자들은 환자 유래의 연골세포에 MCP-1을 처리한 후 유전자의 발현량에 변화가 있는지 확인하는 실험을 수행하였다. 상기 실험을 공지된 방법의 RT-PCR로 수행되었고, 이에 대한 프라이머 서열은 하기와 같다. Then, the present inventors performed an experiment to determine whether there is a change in the expression level of the gene after treating the patient-derived chondrocytes with MCP-1. The above experiment was performed by RT-PCR of a known method, and the primer sequences for this were as follows.

유전자gene 방향direction 서열order collagen 2acollagen 2a forwardforward 5'-TCT ACC CCA ATC CAG CAA AC-3'5'-TCT ACC CCA ATC CAG CAA AC-3' reversereverse 5'-GTT GGG AGC CAG ATT GTC AT-3'5'-GTT GGG AGC CAG ATT GTC AT-3' SOX9SOX9 forwardforward 5'-ACT TGC ACA ACG CCG AG-3'5'-ACT TGC ACA ACG CCG AG-3' reversereverse 5'-CTG GTA CTT GTA ATC CGG GTG-3'5'-CTG GTA CTT GTA ATC CGG GTG-3' MMP1MMP1 forwardforward 5'-AGT CCA AGA GAA TGG CCG AG-3'5'-AGT CCA AGA GAA TGG CCG AG-3' reversereverse 5'-GCA GCG TCA AGT TTA ACT GGA A-3'5'-GCA GCG TCA AGT TTA ACT GGA A-3' MMP3MMP3 forwardforward 5'-CTC ACA GAC CTG ACT CGG TT-3'5'-CTC ACA GAC CTG ACT CGG TT-3' reversereverse 5'-CAC GCC TGA AGG AAG AGA TG-3'5'-CAC GCC TGA AGG AAG AGA TG-3' TRPV2TRPV2 forwardforward 5'-AGT CAA CCT CAA CTA CCG AAA GG-3'5'-AGT CAA CCT CAA CTA CCG AAA GG-3' reversereverse 5'-CCG CAT TGA AGA GCC GAT CT-3'5'-CCG CAT TGA AGA GCC GAT CT-3'

그 결과, 연골세포의 분화와 관련되는 Collagen type 2a 와 SOX9 유전자의 mRNA 발현량이 감소되었고, collagenase로 알려진 MMP1(matrix metalloproteinase 1) 및 MMP3(matrix metalloproteinase 3)의 mRNA 유전자의 발현량이 현저하게 증가됨을 확인하였다(도 1b). 또한, 통증활성인자로 알려진 TRPV2(transient receptor potential cation channel subfamily V member 2)의 mRNA 발현량 역시 증가되었음을 확인하였다(도 1b). As a result, it was confirmed that the mRNA expression levels of collagen type 2a and SOX9 genes related to the differentiation of chondrocytes were decreased, and the mRNA expression levels of MMP1 (matrix metalloproteinase 1) and MMP3 (matrix metalloproteinase 3), also known as collagenases, were significantly increased. (Fig. 1b). In addition, it was confirmed that the mRNA expression level of TRPV2 (transient receptor potential cation channel subfamily V member 2) known as a pain activator was also increased (FIG. 1b).

또한, 본 발명자들은 골관절염 동물 모델의 연골 조직을 분리한 후 MCP1이 발현되는지 여부를 확인하는 실험을 수행하였다. 상기 실험은 공지된 면역화학염색법을 통해 수행되었다. 그 결과, 정상 대조군에 비하여 골관절염 동물 모델에서는 MCP1의 발현이 현저하게 증가되었음을 확인하였다(도 2). In addition, the present inventors performed an experiment to determine whether MCP1 is expressed after isolating the cartilage tissue of the animal model of osteoarthritis. The experiment was performed through a known immunochemical staining method. As a result, it was confirmed that the expression of MCP1 was significantly increased in the osteoarthritis animal model compared to the normal control group ( FIG. 2 ).

따라서, 본 발명자들은 MCP1의 발현량 또는 분비량이 증가되는 경우 골관절염을 야기시킬 수 있는 주요한 인자임을 확인하였다. Therefore, the present inventors confirmed that when the expression or secretion of MCP1 is increased, it is a major factor that can cause osteoarthritis.

실시예 2. Rat sCCR2(soluble C-C chemokine receptor type 2) 과발현의 재조합 벡터 제작 및 sCCR2 과발현 확인Example 2. Construction of a recombinant vector of Rat sCCR2 (soluble C-C chemokine receptor type 2) overexpression and confirmation of sCCR2 overexpression

본 발명자들은 MCP1에 대한 receptor로 알려진 CCR2(C-C chemokine receptor type 2) 또는 CCR2의 서열 중 MCP1의 결합부위(binding site)로 알려진 E3 도메인(domain)과 Fc 절편(fragment)를 연결시킨 complex 서열을 PCR을 이용하여 합성하였다. 이후, 제한효소(restriction enzyme)를 이용하여 pSecTag2A 벡터에 상기 CCR2 또는 E3-Fc를 삽입하여 재조합 벡터를 제작하였다. CCR2 또는 E3-Fc 를 발현할 수 있는 재조합 벡터는 도 3 또는 도 4의 개열 지도를 갖고 있다. CCR2가 삽입된 재조합 벡터의 구체적인 서열 정보는 도 14 및 도 16에 나타내었고, E3-Fc가 삽입된 재조합 벡터의 구체적인 서열 정보는 도 15 및 도 17에 나타내었다. The present inventors PCR the complex sequence linking the E3 domain known as the binding site of MCP1 and the Fc fragment among the sequences of CCR2 (CC chemokine receptor type 2) or CCR2, which is known as a receptor for MCP1. was synthesized using Thereafter, the CCR2 or E3-Fc was inserted into the pSecTag2A vector using a restriction enzyme to prepare a recombinant vector. A recombinant vector capable of expressing CCR2 or E3-Fc has the cleavage map of FIG. 3 or FIG. 4 . Specific sequence information of the recombinant vector into which CCR2 is inserted is shown in FIGS. 14 and 16, and specific sequence information of the recombinant vector into which E3-Fc is inserted is shown in FIGS. 15 and 17.

이후, 본 발명자들은 제작된 재조합 벡터를 HEK293 세포에 도입하여 sCCR2 단백질이 발현되는지 여부를 확인하는 실험을 수행하였다. 상기 실험은 공지된 웨스턴 블럿팅을 통해 수행되었다. 그 결과, R-E3#1(pSecTag2A-rat CCR2E3 #1 construct) 에서 sCCR2의 발현이 확인되었다(도 5).Thereafter, the present inventors introduced the prepared recombinant vector into HEK293 cells and performed an experiment to determine whether the sCCR2 protein was expressed. The experiment was performed through known Western blotting. As a result, the expression of sCCR2 was confirmed in R-E3#1 (pSecTag2A-rat CCR2E3 #1 construct) (FIG. 5).

실시예 3. sCCR2(E3-Fc) 과발현의 재조합 벡터에 의한 연골 손상 회복Example 3. Repair of cartilage damage by recombinant vector overexpressing sCCR2 (E3-Fc)

본 발명자들은 골관절염 동물 모델의 연골 내에서 연골의 회복을 확인하기 위하여, 랫(rat)의 대퇴골(Femur)을 채취하여 해부현미경 및 Safronin O 염색법으로 연골의 손상 정도를 분석하였다. In order to confirm the recovery of cartilage within the cartilage of an animal model of osteoarthritis, the present inventors collected rat femurs and analyzed the degree of cartilage damage using a dissecting microscope and Safronin O staining.

그 결과, sCCR2(E3-Fc) 처리 군에서는 대조군(Mock)에 비해 연골의 손상이 회복된 것을 확인하였다.(도 6 및 도 7).As a result, it was confirmed that cartilage damage was recovered in the sCCR2(E3-Fc) treated group compared to the control group (Mock) ( FIGS. 6 and 7 ).

실시예 4. sCCR2 과발현에 의한 골관절염의 치료 효과Example 4. Therapeutic effect of osteoarthritis by sCCR2 overexpression

4.1. 골관절염 동물 모델4.1. Osteoarthritis Animal Model

본 발명자들은 sCCR2를 과발현시킨 경우 골관절염의 치료에 효과가 있는지 확인하기 위하여 골관절염 동물 모델을 대상으로 골관절염 통증완화 효과를 실험하였다. The present inventors tested the effect of alleviating osteoarthritis pain in an animal model of osteoarthritis in order to confirm whether overexpression of sCCR2 is effective in the treatment of osteoarthritis.

구체적으로, 골관절염 동물모델을 제작하기 위해 200~250g인 5주령 수컷 Wistar 랫(rat)를 21~22℃의 온도에서 명암주기(light-dark cycle)를 12시간 간격으로 사육하였고, 살균한 물과 사료를 공급하여 키웠다. 이후, 골관절염 유도를 위해 랫(rat)의 오른쪽 무릎에 3mg/50μl의 용량으로 생리식염수에 용해시킨 MIA(Monosodium iodoacetate, Sigma, ST. Louis, MO)를 투여하여 골관절염을 유도하였다.Specifically, to produce an osteoarthritis animal model, 5-week-old male Wistar rats weighing 200 to 250 g were bred at a temperature of 21 to 22 ° C in a light-dark cycle at 12 hour intervals, and sterilized water and Feed was supplied and raised. Then, for osteoarthritis induction, MIA (Monosodium iodoacetate, Sigma, ST. Louis, MO) dissolved in physiological saline at a dose of 3 mg/50 μl was administered to the right knee of a rat to induce osteoarthritis.

4.2. 통증 분석4.2. pain analysis

MIA 투여 후, 상기 실시예 2에서 제작된 재조합 벡터를 골관절염 동물 모델의 관절강내 주사를 통해 주입 후 세포막 투과성을 증가시켜 주기위해 electropotator를 사용하여 80mV로 관절강을 5회 자극하였다. MIA 투여 후 4일, 7일, 11일 및 13일이 경과한 후, 각각 골관절염 유발 동물의 행동력에 미치는 영향을 평가하는 지표인 통증정도를 측정하였다. After MIA administration, the recombinant vector prepared in Example 2 was injected through an intra-articular injection of an animal model of osteoarthritis, and then the joint cavity was stimulated 5 times with 80 mV using an electropotator to increase cell membrane permeability. After 4 days, 7 days, 11 days, and 13 days after MIA administration, the degree of pain, which is an index for evaluating the effect on the behavior of the osteoarthritis-induced animals, was measured.

구체적으로, 통증은 Dynamic Plantat Aesthsiometer(Ugo Basile, Comerio, Italy)를 이용하여 측정하였고, 기계의 통증 측정 방법은 측정 기계 위에 그물로 된 판을 얹고 그 위에 아크릴로 된 동물고정틀 안에 랫(rat)을 넣은 후, 측정기계로 약물이 주입된 오른발을 찔러 주었다. 찌른 후에 발을 떼는데 걸리는 시간(Paw Withdrawal Latency, s, 초)과 얼마만큼의 무게를 주었을 때 발을 떼는지(Paw Withdrawal Threshold, g)를 측정하여 그때 나오는 시간과 무게를 기재하여 통증 측정 그래프를 그렸고, 우측 뒷다리 무게도 측정하였다. Specifically, pain was measured using a Dynamic Plantat Aesthsiometer (Ugo Basile, Comerio, Italy), and as for the pain measurement method of the machine, a net plate is placed on the measuring machine and a rat is placed in an acrylic animal frame on it. After putting it in, the right foot, where the drug was injected, was stabbed with a measuring machine. Pain measurement graph by measuring the time it takes to release the foot after stabbing (Paw Withdrawal Latency, s, seconds) and how much weight it takes to release the foot (Paw Withdrawal Threshold, g) was drawn, and the weight of the right hind leg was also measured.

그 결과, 골관절염 랫(rat)에 sCCR2 또는 E3-Fc 재조합 벡터를 주입한 경우 대조군(Mock)에 비해 통증반응정도가 정상 랫(rat)에 거의 근접한 것으로 확인되었다(도 8). 또한, 우측 뒷다리의 무게에서도 대조군(Mock)에서는 뒷다리의 무제가 현저히 감소되었으나, sCCR2 또는 E3-Fc 재조합 벡터를 처리한 군에서는 정상과 유사한 무게를 유지하고 있음을 확인하였다(도 8).As a result, when sCCR2 or E3-Fc recombinant vector was injected into osteoarthritis rats, it was confirmed that the degree of pain response was almost close to that of normal rats compared to the control group (Mock) (FIG. 8). In addition, the weight of the right hind limb was significantly reduced in the control group (Mock), but it was confirmed that the group treated with the sCCR2 or E3-Fc recombinant vector maintained a weight similar to that of normal ( FIG. 8 ).

4.3. 연골 손상 정도 분석4.3. Analysis of the degree of cartilage damage

본 발명자들은 랫(rat)의 대퇴골(Femur) 및 경골(Tibia)을 채취하여 Safronin O 염색법으로 연골의 손상 정도를 분석하였다. 또한, Safronin O 염색을 통한 연골의 손상 정도를 OARSI score 및 Mankin score로 비교하였다. The present inventors analyzed the degree of cartilage damage by collecting the femur and tibia of a rat and using the Safronin O staining method. In addition, the degree of cartilage damage through Safronin O staining was compared with OARSI score and Mankin score.

그 결과, sCCR2 또는 E3-Fc 재조합 벡터를 처리한 군에서는 대조군(Mock)에 비해 연골이 손상되지 않았고, OARSI score(연골 특화 분석) 및 Mankin score(연골 및 염증 침윤정도 종합 분석) 에서 낮은 점수로 확인되었다(도 9). As a result, in the group treated with the sCCR2 or E3-Fc recombinant vector, the cartilage was not damaged compared to the control group (Mock), and the OARSI score (cartilage-specific analysis) and Mankin score (comprehensive analysis of cartilage and inflammatory infiltration) showed a low score. was confirmed (FIG. 9).

4.4. micro CT 분석4.4. micro CT analysis

본 발명자들은 랫(rat)의 대퇴골(Femur)을 채취하여 micro CT를 통해 연골의 손상 정도를 분석하였다. 그 결과, 연골 손상 정도에 있어서 sCCR2 또는 E3-Fc 재조합 벡터를 처리한 군에서는 대조군(Mock)에 비해 정상 랫(rat)과 유사한 수준임을 확인하였다(도 10).The present inventors analyzed the degree of cartilage damage through micro CT by collecting the femur of a rat. As a result, it was confirmed that the level of cartilage damage in the group treated with the sCCR2 or E3-Fc recombinant vector was similar to that of normal rats compared to the control group (Mock) ( FIG. 10 ).

따라서, 상기 결과로부터 sCCR2가 과발현되는 경우 골관절염의 치료 효과가 있음을 확인할 수 있었다. Therefore, from the above results, it was confirmed that the overexpression of sCCR2 has a therapeutic effect on osteoarthritis.

실시예 5. sCCR2 조절에 의한 연골 내 염증성 사이토카인 발현 억제 효과Example 5. Inhibitory effect of inflammatory cytokine expression in cartilage by sCCR2 regulation

본 발명자들은 sCCR2가 과발현되는 경우 염증성 사이토카인의 발현에 영향을 주는지 확인하기 위하여 골관절염 동물 모델의 연골 내에서 IL-1β, IL-6 및 MMP13의 발현여부를 면역화학염색법을 통해 확인하는 실험을 수행하였다. The present inventors performed an experiment to confirm the expression of IL-1β, IL-6 and MMP13 in the cartilage of an animal model of osteoarthritis through immunochemical staining to determine whether sCCR2 overexpression affects the expression of inflammatory cytokines. did.

그 결과, sCCR2 또는 E3-Fc 재조합 벡터를 처리한 군에서는 대조군(Mock)에 비해 IL-1β+ 세포, IL-6+ 세포 및 MMP13+ 세포가 현저히 감소되었음을 확인하였다(도 11).As a result, it was confirmed that IL-1β+ cells, IL-6+ cells and MMP13+ cells were significantly reduced in the group treated with the sCCR2 or E3-Fc recombinant vector compared to the control group (Mock) ( FIG. 11 ).

따라서, sCCR2는 연골 내에서 염증성 사이토카인의 발현을 억제하여 골관절염을 치료할 수 있음을 확인하였다. Therefore, it was confirmed that sCCR2 can treat osteoarthritis by inhibiting the expression of inflammatory cytokines in cartilage.

실시예 6. sCCR2 발현의 중간엽줄기세포Example 6. Mesenchymal stem cells of sCCR2 expression

6.1. 중간엽줄기세포(mesenchymal stem cell, MSC)의 획득 및 배양6.1. Acquisition and culture of mesenchymal stem cells (MSCs)

본 발명자들은 하기의 과정을 통하여 중간엽줄기세포를 획득하였다. 우선, 골관절염 환자에서 외과적 수술 후에 얻어진 지방 조직을 10％ 페니실린-스트렙토마이신을 포함한 PBS로 10회 이상 세척하여 혈액과 이물질을 제거한 후, 조직을 0.2~0.3g이 되도록 잘게 절단하였다. 0.1％ 콜라게나아제(Roche, Sandhofer Strasse, Mannheim, Germany) 용액에 넣어서 37℃, 100rpm에서 1시간 동안 반응시켰다. 100㎛ 메쉬를 이용하여 콜라게나아제에 의해서 분해된 용액층과 분해되지 않은 조각을 분리한 후, 분리된 콜라게나아제 용액에 동량의 PBS를 첨가하였다. 이어, 4℃, 1200rpm에서 5분간 원심분리하여 상층액인 지질과 지방층을 제거하고, 콜라게나아제 상층액을 제거하였다. 얻어진 중간엽줄기세포는 배양접시에 접종하여 중간우태아혈청을 포함하는 MEM(Dulbecco's modified Eagle's medium)으로 37℃, 5％ CO2 인큐베이터에서 3일에 한번 씩 배양액을 교체하면서 배양되었다. The present inventors obtained mesenchymal stem cells through the following process. First, the adipose tissue obtained after surgery in osteoarthritis patients was washed with PBS containing 10% penicillin-streptomycin 10 times or more to remove blood and foreign substances, and then the tissue was cut into 0.2-0.3 g. It was put into a 0.1% collagenase (Roche, Sandhofer Strasse, Mannheim, Germany) solution and reacted at 37° C., 100 rpm for 1 hour. After separating the solution layer decomposed by collagenase and the fragments not decomposed by using a 100 μm mesh, the same amount of PBS was added to the separated collagenase solution. Then, centrifugation was performed at 4° C. and 1200 rpm for 5 minutes to remove the supernatant lipid and fat layer, and the collagenase supernatant was removed. The obtained mesenchymal stem cells were inoculated in a culture dish and cultured with MEM (Dulbecco's modified Eagle's medium) containing fetal intermediate bovine serum at 37° C., 5% CO 2 in an incubator, changing the culture medium once every 3 days.

6.2. sCCR2 발현의 중간엽줄기세포 제조 및 특성 분석6.2. Preparation and characterization of mesenchymal stem cells of sCCR2 expression

본 발명자들은 sCCR2(E3-Fc)가 발현되는 중간엽줄기세포를 제조하기 위하여 X-tremeGENE™ HP DNA Transfection Reagent(Roche)를 이용하였고, sCCR2 또는 E3-Fc 재조합 벡터를 리포좀으로 둘러 싼 후 생체막으로 들어가게 하는 원리로 중간엽줄기세포에 도입하였다. The present inventors used X-tremeGENE™ HP DNA Transfection Reagent (Roche) to prepare mesenchymal stem cells expressing sCCR2 (E3-Fc), and surround the sCCR2 or E3-Fc recombinant vector with liposomes with a biofilm. It was introduced into mesenchymal stem cells by the principle of entering.

sCCR2이 발현되는 중간엽줄기세포는 마커(CD29, CD44, CD105, CD31, CD34 및 HLA-DR(Human Leukocyte Antigen - antigen D Related))를 이용하여 세포의 특성을 분석하는 실험을 수행하였다. For mesenchymal stem cells expressing sCCR2, an experiment was performed to characterize the cells using markers (CD29, CD44, CD105, CD31, CD34, and Human Leukocyte Antigen - Antigen D Related (HLA-DR)).

그 결과, sCCR2(E3-Fc)이 발현되는 중간엽줄기세포의 경우 CD29, CD44 및 CD105는 양성이며, CD31, CD34 및 HLA-DR(Human Leukocyte Antigen - antigen D Related)은 음성의 면역학적 특징을 나타내었다(도 12a, 12b). As a result, CD29, CD44 and CD105 were positive for sCCR2 (E3-Fc)-expressing mesenchymal stem cells, and CD31, CD34, and Human Leukocyte Antigen - Antigen D Related (HLA-DR) were negative immunologically characteristic. shown (Fig. 12a, 12b).

실시예 7. sCCR2 발현의 중간엽줄기세포에 의한 골관절염의 치료 효과Example 7. Therapeutic effect of sCCR2 expression on osteoarthritis by mesenchymal stem cells

7.1. sCCR2 발현의 중간엽줄기세포에 의한 통증분석7.1. Pain analysis by mesenchymal stem cells of sCCR2 expression

본 발명자들은 sCCR2(E3-Fc)가 발현되는 중간엽줄기세포가 골관절염의 치료에 효과가 있는지 확인하기 위하여 골관절염 동물 모델을 대상으로 골관절염 통증완화 효과를 실험하였다. 골관절염 동물 모델은 상기 실시예 4.1.과 같다.The present inventors tested the pain relief effect of osteoarthritis in an animal model of osteoarthritis in order to confirm whether mesenchymal stem cells expressing sCCR2 (E3-Fc) are effective in the treatment of osteoarthritis. The osteoarthritis animal model is the same as in Example 4.1.

sCCR2(E3-Fc)가 도입된 중간엽 줄기세포(3x105 cells)는 골관절염 동물 모델에 혈관내 주사(intravenous injection)로 주입되었고, 대조군으로서 sCCR2(E3-Fc)가 도입되지 않은 중간엽줄기세포가 주입된 군, Celecoxib(80 mg/kg)이 주입된 군 및 아무것도 주입되지 않은 군으로 나누어 상기 실시예 4.2. 에서와 같은 통증 분석을 수행하였다. Mesenchymal stem cells (3x10 5 cells) into which sCCR2 (E3-Fc) was introduced were injected into an animal model of osteoarthritis by intravenous injection, and as a control, mesenchymal stem cells into which sCCR2 (E3-Fc) was not introduced. is divided into a group injected, a group injected with Celecoxib (80 mg/kg), and a group not injected with anything in Example 4.2. Pain analysis was performed as in

그 결과, sCCR2(E3-Fc)가 도입되어 발현되는 중간엽 줄기세포를 주입한 군에서는 골관절염 유도 후 8일 이후부터 대조군에 비하여 통증반응정도가 골관절염을 유도하지 않은 상태와 거의 동일한 정도로 확인되었다(도 13). 즉, 골관절염 유도 후 8일 이후부터 정상과 거의 동일한 통증반응정도로 회복되었음을 확인하였다. As a result, in the group injected with mesenchymal stem cells in which sCCR2 (E3-Fc) was introduced and expressed, the degree of pain response was found to be approximately the same as that of the non-osteoarthritis-inducing state from 8 days after osteoarthritis induction compared to the control group ( 13). That is, it was confirmed that after 8 days after osteoarthritis induction, the pain response level was almost the same as normal.

따라서, sCCR2(E3-Fc)가 발현되는 중간엽줄기세포를 주입할 경우 골관절염의 치료 효과가 있음을 확인할 수 있었다. Therefore, it was confirmed that the injection of mesenchymal stem cells expressing sCCR2 (E3-Fc) has a therapeutic effect on osteoarthritis.

7.2. sCCR2 발현의 중간엽줄기세포의 MCP-1 억제 확인7.2. Confirmation of MCP-1 inhibition of sCCR2 expression in mesenchymal stem cells

sCCR2(E3-Fc) 발현 중간엽줄기세포에서 MOCK 대비 MCP-1이 발현이 감소하는지 여부를 확인하는 실험을 수행하였다. 상기 실험은 공지된 ELISA를 통해 수행되었으며, 동일 조건의 두 환자의 세포에서 (P1 및 P2)에서 수행하였다. 그 결과, 대조군에 비하여 sCCR2(E3-Fc)가 발현되는 중간엽줄기세포에서 MCP-1의 발현이 유의적으로 감소되었음을 확인하였다(도 18).In sCCR2(E3-Fc)-expressing mesenchymal stem cells, an experiment was performed to determine whether the expression of MCP-1 compared to MOCK was decreased. The above experiment was performed through a known ELISA, and was performed in cells of two patients (P1 and P2) under the same conditions. As a result, it was confirmed that the expression of MCP-1 was significantly reduced in the mesenchymal stem cells expressing sCCR2 (E3-Fc) compared to the control group (FIG. 18).

7.3. sCCR2 발현의 중간엽줄기세포의 연골분화 및 대사인자 제어 확인7.3. Confirmation of chondrogenic differentiation and metabolic factor control of mesenchymal stem cells of sCCR2 expression

sCCR2(E3-Fc) 발현 중간엽줄기세포의 유전자의 발현량에 변화가 있는지 확인하는 실험을 수행하였다. 상기 실험을 공지된 방법의 realtime-PCR로 수행되었고, 이에 대한 프라이머 서열 표 1과 같다.An experiment was performed to determine whether there was a change in the expression level of the sCCR2(E3-Fc)-expressing mesenchymal stem cells. The above experiment was performed by realtime-PCR of a known method, and the primer sequences are shown in Table 1.

그 결과, 연골세포의 분화와 관련되는 SOX9 유전자의 mRNA 발현량이, sCCR2(E3-Fc) 발현 중갑엽줄기세포에서 대조군(Mock)보다 유의적으로 증가하였다(도 19). collagenase로 알려진 MMP1(matrix metalloproteinase 1) 및 MMP3(matrix metalloproteinase 3)의 mRNA 유전자의 발현량이 유의적으로 감소됨을 확인하였다(도 20).As a result, the mRNA expression level of the SOX9 gene related to the differentiation of chondrocytes was significantly increased in the sCCR2(E3-Fc)-expressing mesenchymal stem cells than in the control group (Mock) ( FIG. 19 ). It was confirmed that the expression levels of mRNA genes of matrix metalloproteinase 1 (MMP1) and matrix metalloproteinase 3 (MMP3), known as collagenase, were significantly reduced ( FIG. 20 ).

7.4. sCCR2 발현의 중간엽줄기세포의 항염증 사이토카인 발현 확인7.4. Confirmation of anti-inflammatory cytokine expression in mesenchymal stem cells of sCCR2 expression

sCCR2(E3-Fc) 발현 중간엽줄기세포에서 항염증성 사이토카인 TGF-β 및 IL-10의 발현이 증가하는지 여부를 확인하는 실험을 수행하였다. 상기 실험은 공지된 ELISA를 통해 수행되었다. An experiment was performed to determine whether the expression of anti-inflammatory cytokines TGF-β and IL-10 increased in sCCR2(E3-Fc) expressing mesenchymal stem cells. The experiment was performed through a known ELISA.

그 결과, 대조군(Mock)에 비하여 sCCR2(E3-Fc) 발현 중간엽줄기세포에서 TGF-β 및 IL-10 의 발현이 유의적으로 증가하였으며, 특히 IL-10의 발현이 유의적으로 증가되었음을 확인하였다(도 21).As a result, the expression of TGF-β and IL-10 was significantly increased in sCCR2(E3-Fc) expressing mesenchymal stem cells compared to the control (Mock), and in particular, it was confirmed that the expression of IL-10 was significantly increased. (FIG. 21).

7.5. sCCR2 발현의 중간엽줄기세포의 콜라겐 에피토프 발현 확인7.5. Confirmation of collagen epitope expression in mesenchymal stem cells of sCCR2 expression

콜라겐 에피토프인 CTX-Ⅱ는 연골의 손상이 발생하면 collagen type II 의 분해가 일어나게 되고 그 때 증가되는 요소로서, 본 발명에서는 sCCR2(E3-Fc) 발현 중간엽줄기세포를 골관절염 동물 모델의 연골세포 처리하였을 시, 혈청에서 콜라겐 에피토프인 CTX-Ⅱ의 발현 변화를 확인하고자 하였다.Collagen epitope CTX-II is a factor that increases collagen type II degradation when cartilage is damaged. In this case, it was attempted to confirm the change in the expression of CTX-II, a collagen epitope, in serum.

그 결과, 골관절염 동물 모델(MIA) 및 대조군(Mock MSC)에서 보다 sCCR2(E3-Fc) 발현 중간엽줄기세포를 처리하였을 때, CTX-Ⅱ의 발현이 유의적으로 감소하는 것을 확인하였다(도 22). As a result, it was confirmed that the expression of CTX-II was significantly reduced when treated with sCCR2(E3-Fc)-expressing mesenchymal stem cells than in the osteoarthritis animal model (MIA) and the control group (Mock MSC) (FIG. 22). ).

따라서, 본 발명의 sCCR2(E3-Fc) 발현의 중간엽줄기세포는, 골관절염 동물모델에서 확인한 바와 같이 대조군(Mock)과 비교하여, 통증 반응을 개선시켰으며(도 13), 골질환 인자인 MCP-1을 중화하여 유의적으로 감소시켰으며(도 18), 콜라겐 대사인자(MMP1 및 MMP3)들 또한 유의적으로 감소시켰다(도 20). 또한, sCCR2(E3-Fc) 발현의 중간엽줄기세포는 대조군(Mock)과 비교하여, 연골 분화와 관련된 SOX9유전자의 발현(도 19), 항염증 사이토카인(TGF-β 및 IL-10)의 발현(도 21)을 유의적으로 증가시켰으며, 콜라겐 체내 흡수와 관련된 콜라겐 에피토프(CTX-Ⅱ) 또한 sCCR2(E3-Fc) 발현의 중간엽줄기세포의 처리로 발현이 감소한 것을 확인하였다(도 22). 상기와 같은 결과로, 본 발명의 sCCR2(E3-Fc) 발현의 중간엽줄기세포는 골관절염에 대한 재생능이 우수하고 통증억제 및 완화 효과가 우수하여, 골관절염 치료제로서 효과적인 것을 확인하였다.Therefore, the sCCR2(E3-Fc)-expressing mesenchymal stem cells of the present invention improved the pain response compared to the control group (Mock) as confirmed in the osteoarthritis animal model (FIG. 13), and MCP, a bone disease factor It was significantly reduced by neutralizing -1 (FIG. 18), and collagen metabolic factors (MMP1 and MMP3) were also significantly decreased (FIG. 20). In addition, the sCCR2 (E3-Fc) expression of the mesenchymal stem cells was compared with the control (Mock), the expression of the SOX9 gene associated with cartilage differentiation (Fig. 19), the anti-inflammatory cytokines (TGF-β and IL-10) Expression (FIG. 21) was significantly increased, and it was confirmed that the expression of the collagen epitope (CTX-II) related to collagen absorption into the body was also reduced by treatment with mesenchymal stem cells of sCCR2 (E3-Fc) expression (FIG. 22). ). As a result of the above, it was confirmed that the sCCR2(E3-Fc)-expressing mesenchymal stem cells of the present invention have excellent regenerative ability for osteoarthritis and excellent pain suppression and alleviation effects, and are effective as a therapeutic agent for osteoarthritis.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Composition for the prevention or treatment of bone disease, including soluble CCR2 stem cells as an active ingredient <130> PN2107-369 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 375 <212> PRT <213> Artificial Sequence <220> <223> rat CCR2 <400> 1 Met Glu Asp Ser Asn Met Leu Pro Gln Phe Ile His Gly Ile Leu Ser 1 5 10 15 Thr Ser His Ser Leu Phe Pro Arg Ser Ile Gln Glu Leu Asp Glu Gly 20 25 30 Ala Thr Thr Pro Tyr Asp Tyr Asp Asp Gly Glu Pro Cys His Lys Thr 35 40 45 Ser Val Lys Gln Ile Gly Ala Trp Ile Leu Pro Pro Leu Tyr Ser Leu 50 55 60 Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val Ile Ile Ile Leu 65 70 75 80 Ile Ser Cys Lys Lys Leu Lys Ser Met Thr Asp Ile Tyr Leu Phe Asn 85 90 95 Leu Ala Ile Ser Asp Leu Leu Phe Leu Leu Thr Leu Pro Phe Trp Ala 100 105 110 His Tyr Ala Ala Asn Glu Trp Val Phe Gly Asn Ile Met Cys Lys Leu 115 120 125 Phe Thr Gly Leu Tyr His Ile Gly Tyr Phe Gly Gly Ile Phe Phe Ile 130 135 140 Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val Phe 145 150 155 160 Ala Leu Lys Ala Arg Thr Val Thr Phe Gly Val Ile Thr Ser Val Val 165 170 175 Thr Trp Val Val Ala Val Phe Ala Ser Leu Pro Gly Ile Ile Phe Thr 180 185 190 Lys Ser Glu Gln Glu Asp Asp Gln His Thr Cys Gly Pro Tyr Phe Pro 195 200 205 Thr Ile Trp Lys Asn Phe Gln Thr Ile Met Arg Asn Ile Leu Ser Leu 210 215 220 Ile Leu Pro Leu Leu Val Met Val Ile Cys Tyr Ser Gly Ile Leu His 225 230 235 240 Thr Leu Phe Arg Cys Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg 245 250 255 Leu Ile Phe Ala Ile Met Ile Val Tyr Phe Leu Phe Trp Thr Pro Tyr 260 265 270 Asn Ile Val Leu Phe Leu Thr Thr Phe Gln Glu Phe Leu Gly Met Ser 275 280 285 Asn Cys Val Val Asp Met His Leu Asp Gln Ala Met Gln Val Thr Glu 290 295 300 Thr Leu Gly Met Thr His Cys Cys Val Asn Pro Ile Ile Tyr Ala Phe 305 310 315 320 Val Gly Glu Lys Phe Arg Arg Tyr Leu Ser Ile Phe Phe Arg Lys His 325 330 335 Ile Ala Lys Asn Leu Cys Lys Gln Cys Pro Val Phe Tyr Arg Glu Thr 340 345 350 Ala Asp Arg Val Ser Ser Thr Phe Thr Pro Ser Thr Gly Glu Gln Glu 355 360 365 Val Ser Val Gly Leu Leu Gln 370 375 <210> 2 <211> 1125 <212> DNA <213> Artificial Sequence <220> <223> rat CCR2 <400> 2 atggaagaca gtaatatgtt acctcagttc atccatggca tactatcaac atctcattct 60 ctatttccaa gaagtatcca agagcttgat gagggggcca ccacaccgta tgactatgat 120 gatggtgaac cttgtcataa aaccagtgtg aagcaaattg gagcttggat cctgccccca 180 ctctactccc tggtattcat ctttggtttt gtgggcaaca tgttggtcat tataattctg 240 ataagctgta aaaagctgaa gagcatgact gatatctacc tgttcaacct ggccatctct 300 gacctgctct tcctgctcac actcccattc tgggctcact atgctgcaaa tgagtgggtc 360 tttgggaata taatgtgcaa attattcaca gggctttatc acattgggta ttttggtgga 420 atcttcttca ttatcctcct gacaattgat agatatttgg ctattgtcca tgctgtcttt 480 gctttaaaag ccaggacagt tacctttggg gtaataacaa gtgtagtcac ttgggtggtg 540 gctgtgtttg cctctctacc aggaatcata tttactaaat ctgaacaaga agatgatcag 600 catacttgtg gcccttattt tccaacaatc tggaagaatt tccaaacaat aatgaggaat 660 atcttgagtt tgatcctgcc cctacttgtc atggtcatct gctactcagg aatcctccac 720 accctgtttc gctgtaggaa tgagaaaaag aggcataggg ctgtgaggct catctttgcc 780 atcatgattg tctactttct cttctggact ccatacaata ttgttctctt cctgaccacc 840 ttccaggaat tcttgggaat gagtaactgt gtggttgaca tgcacttaga ccaggccatg 900 caggtgacag agactcttgg aatgacacac tgctgcgtta atcctatcat ttatgccttt 960 gttggtgaga agttccgaag gtatctctcc atatttttca gaaagcacat tgccaaaaat 1020 ctctgcaaac aatgcccagt tttctatagg gagacagcag accgagtgag ctcaacattt 1080 accccttcta ctggggagca agaagtctca gttgggttgc tgcag 1125 <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> rat CCR2 E3 amino acid <400> 3 Thr Thr Phe Gln Glu Phe Leu Gly Met Ser Asn Cys Val Val Asp Met 1 5 10 15 His Leu Asp Gln Ala 20 <210> 4 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> rat CCR2 E3 nucleic acid <400> 4 accaccttcc aggaattctt gggaatgagt aactgtgtgg ttgacatgca cttagaccag 60 gcc 63 <210> 5 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> rat CCR2 E3-Fc amino acid <400> 5 Thr Thr Phe Gln Glu Phe Leu Gly Met Ser Asn Cys Val Val Asp Met 1 5 10 15 His Leu Asp Gln Ala Leu Gln Glu Pro Lys Ser Cys Asp Lys Thr His 20 25 30 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 35 40 45 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 50 55 60 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 65 70 75 80 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 85 90 95 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 100 105 110 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 115 120 125 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 130 135 140 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 145 150 155 160 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 165 170 175 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 180 185 190 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 195 200 205 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 210 215 220 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 225 230 235 240 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 245 250 255 <210> 6 <211> 765 <212> DNA <213> Artificial Sequence <220> <223> rat CCR2 E3-Fc nucleic acid <400> 6 accaccttcc aggaattctt gggaatgagt aactgtgtgg ttgacatgca cttagaccag 60 gccctgcagg aacccaagag ttgcgataaa acccatacat gccccccttg tcctgcccca 120 gaactcctgg ggggtccaag cgtgttcctc tttccaccca agcccaaaga cactctgatg 180 atctcacgga ctcccgaggt gacctgcgtg gtcgtggacg tgagccacga ggatcctgaa 240 gtgaagttca actggtacgt ggatggagtc gaggtgcata atgccaagac caaaccaaga 300 gaggaacagt acaacagtac ttatcgggtc gtgtcagtgc tcaccgtcct gcaccaggac 360 tggctgaacg gaaaggagta taagtgcaaa gtgtccaata aggcactgcc cgcccctatc 420 gagaaaacca tttctaaggc aaaagggcag cctagagaac cacaggtgta cacactccct 480 ccaagccggg acgagctgac caagaaccag gtgtccctca catgtctggt caaaggcttc 540 tatccttccg atatcgctgt ggagtgggaa tctaatggac agccagagaa caattacaag 600 actacccccc ctgtgctgga ctcagatggg agcttctttc tctattctaa gctgaccgtg 660 gacaaaagta gatggcagca gggtaacgtg ttcagctgct cagtcatgca tgaggccctg 720 cataatcact acacccagaa atcactgtca ctcagcccag gaaaa 765 <210> 7 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> pSecTag2-rat CCR2 <400> 7 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Trp Val Pro Gly 1 5 10 15 Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Arg Arg Thr Lys 20 25 30 Leu Met Glu Asp Ser Asn Met Leu Pro Gln Phe Ile His Gly Ile Leu 35 40 45 Ser Thr Ser His Ser Leu Phe Pro Arg Ser Ile Gln Glu Leu Asp Glu 50 55 60 Gly Ala Thr Thr Pro Tyr Asp Tyr Asp Asp Gly Glu Pro Cys His Lys 65 70 75 80 Thr Ser Val Lys Gln Ile Gly Ala Trp Ile Leu Pro Pro Leu Tyr Ser 85 90 95 Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val Ile Ile Ile 100 105 110 Leu Ile Ser Cys Lys Lys Leu Lys Ser Met Thr Asp Ile Tyr Leu Phe 115 120 125 Asn Leu Ala Ile Ser Asp Leu Leu Phe Leu Leu Thr Leu Pro Phe Trp 130 135 140 Ala His Tyr Ala Ala Asn Glu Trp Val Phe Gly Asn Ile Met Cys Lys 145 150 155 160 Leu Phe Thr Gly Leu Tyr His Ile Gly Tyr Phe Gly Gly Ile Phe Phe 165 170 175 Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val 180 185 190 Phe Ala Leu Lys Ala Arg Thr Val Thr Phe Gly Val Ile Thr Ser Val 195 200 205 Val Thr Trp Val Val Ala Val Phe Ala Ser Leu Pro Gly Ile Ile Phe 210 215 220 Thr Lys Ser Glu Gln Glu Asp Asp Gln His Thr Cys Gly Pro Tyr Phe 225 230 235 240 Pro Thr Ile Trp Lys Asn Phe Gln Thr Ile Met Arg Asn Ile Leu Ser 245 250 255 Leu Ile Leu Pro Leu Leu Val Met Val Ile Cys Tyr Ser Gly Ile Leu 260 265 270 His Thr Leu Phe Arg Cys Arg Asn Glu Lys Lys Arg His Arg Ala Val 275 280 285 Arg Leu Ile Phe Ala Ile Met Ile Val Tyr Phe Leu Phe Trp Thr Pro 290 295 300 Tyr Asn Ile Val Leu Phe Leu Thr Thr Phe Gln Glu Phe Leu Gly Met 305 310 315 320 Ser Asn Cys Val Val Asp Met His Leu Asp Gln Ala Met Gln Val Thr 325 330 335 Glu Thr Leu Gly Met Thr His Cys Cys Val Asn Pro Ile Ile Tyr Ala 340 345 350 Phe Val Gly Glu Lys Phe Arg Arg Tyr Leu Ser Ile Phe Phe Arg Lys 355 360 365 His Ile Ala Lys Asn Leu Cys Lys Gln Cys Pro Val Phe Tyr Arg Glu 370 375 380 Thr Ala Asp Arg Val Ser Ser Thr Phe Thr Pro Ser Thr Gly Glu Gln 385 390 395 400 Glu Val Ser Val Gly Leu Leu Gln Ile Ser Ser Thr Val Ala Ala Ala 405 410 415 Arg Gly Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser 420 425 430 Ala Val Asp His His His His His His 435 440 <210> 8 <211> 314 <212> PRT <213> Artificial Sequence <220> <223> pSecTag2-rat CCR2E3-FC <400> 8 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Val Arg Ser 20 25 30 Leu Thr Thr Phe Gln Glu Phe Leu Gly Met Ser Asn Cys Val Val Asp 35 40 45 Met His Leu Asp Gln Ala Leu Gln Glu Pro Lys Ser Cys Asp Lys Thr 50 55 60 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 65 70 75 80 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 85 90 95 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 100 105 110 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 115 120 125 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 130 135 140 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 145 150 155 160 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 165 170 175 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 180 185 190 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 195 200 205 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 210 215 220 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 225 230 235 240 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 245 250 255 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 260 265 270 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 275 280 285 Pro Arg Gly Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 290 295 300 Ser Ala Val Asp His His His His His His 305 310 <210> 9 <211> 1326 <212> DNA <213> Artificial Sequence <220> <223> pSecTag2-rat CCR2 nucleic acid <400> 9 atggagacag acacactcct gctatgggta ctgctctggg ttccaggttc cactggtgac 60 gcggcccagc cggccaggcg cgcgcgccgt acgaagctta tggaagacag taatatgtta 120 cctcagttca tccatggcat actatcaaca tctcattctc tatttccaag aagtatccaa 180 gagcttgatg agggggccac cacaccgtat gactatgatg atggtgaacc ttgtcataaa 240 accagtgtga agcaaattgg agcttggatc ctgcccccac tctactccct ggtattcatc 300 tttggttttg tgggcaacat gttggtcatt ataattctga taagctgtaa aaagctgaag 360 agcatgactg atatctacct gttcaacctg gccatctctg acctgctctt cctgctcaca 420 ctcccattct gggctcacta tgctgcaaat gagtgggtct ttgggaatat aatgtgcaaa 480 ttattcacag ggctttatca cattgggtat tttggtggaa tcttcttcat tatcctcctg 540 acaattgata gatatttggc tattgtccat gctgtctttg ctttaaaagc caggacagtt 600 acctttgggg taataacaag tgtagtcact tgggtggtgg ctgtgtttgc ctctctacca 660 ggaatcatat ttactaaatc tgaacaagaa gatgatcagc atacttgtgg cccttatttt 720 ccaacaatct ggaagaattt ccaaacaata atgaggaata tcttgagttt gatcctgccc 780 ctacttgtca tggtcatctg ctactcagga atcctccaca ccctgtttcg ctgtaggaat 840 gagaaaaaga ggcatagggc tgtgaggctc atctttgcca tcatgattgt ctactttctc 900 ttctggactc catacaatat tgttctcttc ctgaccacct tccaggaatt cttgggaatg 960 agtaactgtg tggttgacat gcacttagac caggccatgc aggtgacaga gactcttgga 1020 atgacacact gctgcgttaa tcctatcatt tatgcctttg ttggtgagaa gttccgaagg 1080 tatctctcca tatttttcag aaagcacatt gccaaaaatc tctgcaaaca atgcccagtt 1140 ttctataggg agacagcaga ccgagtgagc tcaacattta ccccttctac tggggagcaa 1200 gaagtctcag ttgggttgct gcagatatcc agcacagtgg cggccgctcg aggagggccc 1260 gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat 1320 cattga 1326 <210> 10 <211> 945 <212> DNA <213> Artificial Sequence <220> <223> pSecTag2-rat CCR2E3-FC nucleic acid <400> 10 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gacgcggccc agccggccag gcgcgccgta cgaagcttga ccaccttcca ggaattcttg 120 ggaatgagta actgtgtggt tgacatgcac ttagaccagg ccctgcagga acccaagagt 180 tgcgataaaa cccatacatg ccccccttgt cctgccccag aactcctggg gggtccaagc 240 gtgttcctct ttccacccaa gcccaaagac actctgatga tctcacggac tcccgaggtg 300 acctgcgtgg tcgtggacgt gagccacgag gatcctgaag tgaagttcaa ctggtacgtg 360 gatggagtcg aggtgcataa tgccaagacc aaaccaagag aggaacagta caacagtact 420 tatcgggtcg tgtcagtgct caccgtcctg caccaggact ggctgaacgg aaaggagtat 480 aagtgcaaag tgtccaataa ggcactgccc gcccctatcg agaaaaccat ttctaaggca 540 aaagggcagc ctagagaacc acaggtgtac acactccctc caagccggga cgagctgacc 600 aagaaccagg tgtccctcac atgtctggtc aaaggcttct atccttccga tatcgctgtg 660 gagtgggaat ctaatggaca gccagagaac aattacaaga ctaccccccc tgtgctggac 720 tcagatggga gcttctttct ctattctaag ctgaccgtgg acaaaagtag atggcagcag 780 ggtaacgtgt tcagctgctc agtcatgcat gaggccctgc ataatcacta cacccagaaa 840 tcactgtcac tcagcccagg aaaacctcga ggagggcccg aacaaaaact catctcagaa 900 gaggatctga atagcgccgt cgaccatcat catcatcatc attga 945 <110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Composition for the prevention or treatment of bone disease, including soluble CCR2 stem cells as an active ingredient <130> PN2107-369 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 375 <212> PRT <213> Artificial Sequence <220> <223> rat CCR2 <400> 1 Met Glu Asp Ser Asn Met Leu Pro Gln Phe Ile His Gly Ile Leu Ser 1 5 10 15 Thr Ser His Ser Leu Phe Pro Arg Ser Ile Gln Glu Leu Asp Glu Gly 20 25 30 Ala Thr Thr Pro Tyr Asp Tyr Asp Asp Gly Glu Pro Cys His Lys Thr 35 40 45 Ser Val Lys Gln Ile Gly Ala Trp Ile Leu Pro Pro Leu Tyr Ser Leu 50 55 60 Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val Ile Ile Ile Leu 65 70 75 80 Ile Ser Cys Lys Lys Leu Lys Ser Met Thr Asp Ile Tyr Leu Phe Asn 85 90 95 Leu Ala Ile Ser Asp Leu Leu Phe Leu Leu Thr Leu Pro Phe Trp Ala 100 105 110 His Tyr Ala Ala Asn Glu Trp Val Phe Gly Asn Ile Met Cys Lys Leu 115 120 125 Phe Thr Gly Leu Tyr His Ile Gly Tyr Phe Gly Gly Ile Phe Phe Ile 130 135 140 Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val Phe 145 150 155 160 Ala Leu Lys Ala Arg Thr Val Thr Phe Gly Val Ile Thr Ser Val Val 165 170 175 Thr Trp Val Val Ala Val Phe Ala Ser Leu Pro Gly Ile Ile Phe Thr 180 185 190 Lys Ser Glu Gln Glu Asp Asp Gln His Thr Cys Gly Pro Tyr Phe Pro 195 200 205 Thr Ile Trp Lys Asn Phe Gln Thr Ile Met Arg Asn Ile Leu Ser Leu 210 215 220 Ile Leu Pro Leu Leu Val Met Val Ile Cys Tyr Ser Gly Ile Leu His 225 230 235 240 Thr Leu Phe Arg Cys Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg 245 250 255 Leu Ile Phe Ala Ile Met Ile Val Tyr Phe Leu Phe Trp Thr Pro Tyr 260 265 270 Asn Ile Val Leu Phe Leu Thr Thr Phe Gln Glu Phe Leu Gly Met Ser 275 280 285 Asn Cys Val Val Asp Met His Leu Asp Gln Ala Met Gln Val Thr Glu 290 295 300 Thr Leu Gly Met Thr His Cys Cys Val Asn Pro Ile Ile Tyr Ala Phe 305 310 315 320 Val Gly Glu Lys Phe Arg Arg Tyr Leu Ser Ile Phe Phe Arg Lys His 325 330 335 Ile Ala Lys Asn Leu Cys Lys Gln Cys Pro Val Phe Tyr Arg Glu Thr 340 345 350 Ala Asp Arg Val Ser Ser Thr Phe Thr Pro Ser Thr Gly Glu Gln Glu 355 360 365 Val Ser Val Gly Leu Leu Gln 370 375 <210> 2 <211> 1125 <212> DNA <213> Artificial Sequence <220> <223> rat CCR2 <400> 2 atggaagaca gtaatatgtt acctcagttc atccatggca tactatcaac atctcattct 60 ctatttccaa gaagtatcca agagcttgat gagggggcca ccacaccgta tgactatgat 120 gatggtgaac cttgtcataa aaccagtgtg aagcaaattg gagcttggat cctgccccca 180 ctctactccc tggtattcat ctttggtttt gtgggcaaca tgttggtcat tataattctg 240 ataagctgta aaaagctgaa gagcatgact gatatctacc tgttcaacct ggccatctct 300 gacctgctct tcctgctcac actcccattc tgggctcact atgctgcaaa tgagtgggtc 360 tttgggaata taatgtgcaa attattcaca gggctttatc acattgggta ttttggtgga 420 atcttcttca ttatcctcct gacaattgat agatatttgg ctattgtcca tgctgtcttt 480 gctttaaaag ccaggacagt tacctttggg gtaataacaa gtgtagtcac ttgggtggtg 540 gctgtgtttg cctctctacc aggaatcata tttactaaat ctgaacaaga agatgatcag 600 catacttgtg gcccttattt tccaacaatc tggaagaatt tccaaacaat aatgaggaat 660 atcttgagtt tgatcctgcc cctacttgtc atggtcatct gctactcagg aatcctccac 720 accctgtttc gctgtaggaa tgagaaaaag aggcataggg ctgtgaggct catctttgcc 780 atcatgattg tctactttct cttctggact ccatacaata ttgttctctt cctgaccacc 840 ttccaggaat tcttgggaat gagtaactgt gtggttgaca tgcacttaga ccaggccatg 900 caggtgacag agactcttgg aatgacacac tgctgcgtta atcctatcat ttatgccttt 960 gttggtgaga agttccgaag gtatctctcc atatttttca gaaagcacat tgccaaaaat 1020 ctctgcaaac aatgcccagt tttctatagg gagacagcag accgagtgag ctcaacattt 1080 accccttcta ctggggagca agaagtctca gttgggttgc tgcag 1125 <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> rat CCR2 E3 amino acid <400> 3 Thr Thr Phe Gln Glu Phe Leu Gly Met Ser Asn Cys Val Val Asp Met 1 5 10 15 His Leu Asp Gln Ala 20 <210> 4 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> rat CCR2 E3 nucleic acid <400> 4 accaccttcc aggaattctt gggaatgagt aactgtgtgg ttgacatgca cttagaccag 60 gcc 63 <210> 5 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> rat CCR2 E3-Fc amino acid <400> 5 Thr Thr Phe Gln Glu Phe Leu Gly Met Ser Asn Cys Val Val Asp Met 1 5 10 15 His Leu Asp Gln Ala Leu Gln Glu Pro Lys Ser Cys Asp Lys Thr His 20 25 30 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 35 40 45 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 50 55 60 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 65 70 75 80 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 85 90 95 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 100 105 110 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 115 120 125 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 130 135 140 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 145 150 155 160 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 165 170 175 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 180 185 190 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 195 200 205 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 210 215 220 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 225 230 235 240 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 245 250 255 <210> 6 <211> 765 <212> DNA <213> Artificial Sequence <220> <223> rat CCR2 E3-Fc nucleic acid <400> 6 accaccttcc aggaattctt gggaatgagt aactgtgtgg ttgacatgca cttagaccag 60 gccctgcagg aacccaagag ttgcgataaa acccatacat gccccccttg tcctgcccca 120 gaactcctgg ggggtccaag cgtgttcctc tttccaccca agcccaaaga cactctgatg 180 atctcacgga ctcccgaggt gacctgcgtg gtcgtggacg tgagccacga ggatcctgaa 240 gtgaagttca actggtacgt ggatggagtc gaggtgcata atgccaagac caaaccaaga 300 gaggaacagt acaacagtac ttatcgggtc gtgtcagtgc tcaccgtcct gcaccaggac 360 tggctgaacg gaaaggagta taagtgcaaa gtgtccaata aggcactgcc cgcccctatc 420 gagaaaacca tttctaaggc aaaagggcag cctagagaac cacaggtgta cacactccct 480 ccaagccggg acgagctgac caagaaccag gtgtccctca catgtctggt caaaggcttc 540 tatccttccg atatcgctgt ggagtgggaa tctaatggac agccagagaa caattacaag 600 actacccccc ctgtgctgga ctcagatggg agcttctttc tctattctaa gctgaccgtg 660 gacaaaagta gatggcagca gggtaacgtg ttcagctgct cagtcatgca tgaggccctg 720 cataatcact accaccagaa atcactgtca ctcagcccag gaaaa 765 <210> 7 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> pSecTag2-rat CCR2 <400> 7 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Trp Val Pro Gly 1 5 10 15 Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Arg Arg Thr Lys 20 25 30 Leu Met Glu Asp Ser Asn Met Leu Pro Gln Phe Ile His Gly Ile Leu 35 40 45 Ser Thr Ser His Ser Leu Phe Pro Arg Ser Ile Gln Glu Leu Asp Glu 50 55 60 Gly Ala Thr Thr Pro Tyr Asp Tyr Asp Asp Gly Glu Pro Cys His Lys 65 70 75 80 Thr Ser Val Lys Gln Ile Gly Ala Trp Ile Leu Pro Pro Leu Tyr Ser 85 90 95 Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val Ile Ile Ile 100 105 110 Leu Ile Ser Cys Lys Lys Lys Leu Lys Ser Met Thr Asp Ile Tyr Leu Phe 115 120 125 Asn Leu Ala Ile Ser Asp Leu Leu Phe Leu Leu Thr Leu Pro Phe Trp 130 135 140 Ala His Tyr Ala Ala Asn Glu Trp Val Phe Gly Asn Ile Met Cys Lys 145 150 155 160 Leu Phe Thr Gly Leu Tyr His Ile Gly Tyr Phe Gly Gly Ile Phe Phe 165 170 175 Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val 180 185 190 Phe Ala Leu Lys Ala Arg Thr Val Thr Phe Gly Val Ile Thr Ser Val 195 200 205 Val Thr Trp Val Val Ala Val Phe Ala Ser Leu Pro Gly Ile Ile Phe 210 215 220 Thr Lys Ser Glu Gln Glu Asp Asp Gln His Thr Cys Gly Pro Tyr Phe 225 230 235 240 Pro Thr Ile Trp Lys Asn Phe Gln Thr Ile Met Arg Asn Ile Leu Ser 245 250 255 Leu Ile Leu Pro Leu Leu Val Met Val Ile Cys Tyr Ser Gly Ile Leu 260 265 270 His Thr Leu Phe Arg Cys Arg Asn Glu Lys Lys Arg His Arg Ala Val 275 280 285 Arg Leu Ile Phe Ala Ile Met Ile Val Tyr Phe Leu Phe Trp Thr Pro 290 295 300 Tyr Asn Ile Val Leu Phe Leu Thr Thr Phe Gln Glu Phe Leu Gly Met 305 310 315 320 Ser Asn Cys Val Val Asp Met His Leu Asp Gln Ala Met Gln Val Thr 325 330 335 Glu Thr Leu Gly Met Thr His Cys Cys Val Asn Pro Ile Ile Tyr Ala 340 345 350 Phe Val Gly Glu Lys Phe Arg Arg Tyr Leu Ser Ile Phe Phe Arg Lys 355 360 365 His Ile Ala Lys Asn Leu Cys Lys Gln Cys Pro Val Phe Tyr Arg Glu 370 375 380 Thr Ala Asp Arg Val Ser Ser Thr Phe Thr Pro Ser Thr Gly Glu Gln 385 390 395 400 Glu Val Ser Val Gly Leu Leu Gln Ile Ser Ser Thr Val Ala Ala Ala 405 410 415 Arg Gly Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser 420 425 430 Ala Val Asp His His His His His His 435 440 <210> 8 <211> 314 <212> PRT <213> Artificial Sequence <220> <223> pSecTag2-rat CCR2E3-FC <400> 8 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Val Arg Ser 20 25 30 Leu Thr Thr Phe Gln Glu Phe Leu Gly Met Ser Asn Cys Val Val Asp 35 40 45 Met His Leu Asp Gln Ala Leu Gln Glu Pro Lys Ser Cys Asp Lys Thr 50 55 60 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 65 70 75 80 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 85 90 95 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 100 105 110 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 115 120 125 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 130 135 140 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 145 150 155 160 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 165 170 175 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 180 185 190 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 195 200 205 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 210 215 220 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 225 230 235 240 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 245 250 255 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 260 265 270 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 275 280 285 Pro Arg Gly Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 290 295 300 Ser Ala Val Asp His His His His His His 305 310 <210> 9 <211> 1326 <212> DNA <213> Artificial Sequence <220> <223> pSecTag2-rat CCR2 nucleic acid <400> 9 atggagacag acacactcct gctatgggta ctgctctggg ttccaggttc cactggtgac 60 gcggcccagc cggccaggcg cgcgcgccgt acgaagctta tggaagacag taatatgtta 120 cctcagttca tccatggcat actatcaaca tctcattctc tatttccaag aagtatccaa 180 gagcttgatg agggggccac cacaccgtat gactatgatg atggtgaacc ttgtcataaa 240 accagtgtga agcaaattgg agcttggatc ctgcccccac tctactccct ggtattcatc 300 tttggttttg tgggcaacat gttggtcatt ataattctga taagctgtaa aaagctgaag 360 agcatgactg atatctacct gttcaacctg gccatctctg acctgctctt cctgctcaca 420 ctcccattct gggctcacta tgctgcaaat gagtgggtct ttgggaatat aatgtgcaaa 480 ttattcacag ggctttatca cattgggtat tttggtggaa tcttcttcat tatcctcctg 540 acaattgata gatatttggc tattgtccat gctgtctttg ctttaaaagc caggacagtt 600 acctttgggg taataacaag tgtagtcact tgggtggtgg ctgtgtttgc ctctctacca 660 ggaatcatat tactaaatc tgaacaagaa gatgatcagc atacttgtgg cccttatttt 720 ccaacaatct ggaagaattt ccaaacaata atgaggaata tcttgagttt gatcctgccc 780 ctacttgtca tggtcatctg ctactcagga atcctccaca ccctgtttcg ctgtaggaat 840 gagaaaaaga ggcatagggc tgtgaggctc atctttgcca tcatgattgt ctactttctc 900 ttctggactc catacaatat tgttctcttc ctgaccacct tccaggaatt cttgggaatg 960 agtaactgtg tggttgacat gcacttagac caggccatgc aggtgacaga gactcttgga 1020 atgacacact gctgcgttaa tcctatcatt tatgcctttg ttggtgagaa gttccgaagg 1080 tatctctcca tatttttcag aaagcacatt gccaaaaatc tctgcaaaca atgcccagtt 1140 ttctataggg agacagcaga ccgagtgagc tcaacattta ccccttctac tggggagcaa 1200 gaagtctcag ttgggttgct gcagatatcc agcacagtgg cggccgctcg aggagggccc 1260 gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat 1320 cattga 1326 <210> 10 <211> 945 <212> DNA <213> Artificial Sequence <220> <223> pSecTag2-rat CCR2E3-FC nucleic acid <400> 10 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gacgcggccc agccggccag gcgcgccgta cgaagcttga ccaccttcca ggaattcttg 120 ggaatgagta actgtgtggt tgacatgcac ttagaccagg ccctgcagga acccaagagt 180 tgcgataaaa cccatacatg ccccccttgt cctgccccag aactcctggg gggtccaagc 240 gtgttcctct ttccacccaa gcccaaagac actctgatga tctcacggac tcccgaggtg 300 acctgcgtgg tcgtggacgt gagccacgag gatcctgaag tgaagttcaa ctggtacgtg 360 gatggagtcg aggtgcataa tgccaagacc aaaccaagag aggaacagta caacagtact 420 tatcgggtcg tgtcagtgct caccgtcctg caccaggact ggctgaacgg aaaggagtat 480 aagtgcaaag tgtccaataa ggcactgccc gcccctatcg agaaaaccat ttctaaggca 540 aaagggcagc ctagagaacc acaggtgtac acactccctc caagccggga cgagctgacc 600 aagaaccagg tgtccctcac atgtctggtc aaaggcttct atccttccga tatcgctgtg 660 gagtgggaat ctaatggaca gccagagaac aattacaaga ctaccccccc tgtgctggac 720 tcagatggga gcttctttct ctattctaag ctgaccgtgg acaaaagtag atggcagcag 780 ggtaacgtgt tcagctgctc agtcatgcat gaggccctgc ataatcacta cacccagaaa 840 tcactgtcac tcagcccagg aaaacctcga ggagggcccg aacaaaaact catctcagaa 900 gaggatctga atagcgccgt cgaccatcat catcatcatc attga 945

Claims (10)

CCR2(C-C chemokine receptor type 2)를 포함하는 재조합 벡터를 유효성분으로 포함하는 골질환의 예방 또는 치료용 조성물. A composition for preventing or treating bone diseases, comprising a recombinant vector containing CCR2 (C-C chemokine receptor type 2) as an active ingredient. 제 1 항에 있어서,
상기 CCR2를 암호화하는 폴리뉴클레오타이드는 서열번호 2의 염기서열로 이루어진 것을 특징으로 하는 조성물. The method of claim 1,
The composition, characterized in that the polynucleotide encoding CCR2 consists of the nucleotide sequence of SEQ ID NO: 2. 제 1 항에 있어서,
상기 CCR2는 서열번호 1의 아미노산 서열로 이루어진 것을 특징으로 하는 조성물.The method of claim 1,
The CCR2 composition, characterized in that consisting of the amino acid sequence of SEQ ID NO: 1. 제 1 항에 있어서,
상기 골질환은 골관절염(Osteoarthritis)인 것을 특징으로 하는 조성물. The method of claim 1,
The bone disease is a composition, characterized in that osteoarthritis (Osteoarthritis). CCR2(C-C chemokine receptor type 2)의 E3 도메인을 포함하는 재조합 벡터를 유효성분으로 포함하는 골질환의 예방 또는 치료용 조성물. A composition for preventing or treating bone disease, comprising a recombinant vector comprising the E3 domain of CCR2 (C-C chemokine receptor type 2) as an active ingredient. 제5항에 있어서, 상기 E3 도메인은 Fc절편(fragment)을 포함하는 것을 특징으로 하는, 조성물.The composition of claim 5, wherein the E3 domain comprises an Fc fragment. 제 5 항에 있어서,
상기 CCR2를 암호화하는 폴리뉴클레오타이드는 서열번호 4의 염기서열로 이루어진 것을 특징으로 하는, 조성물. 6. The method of claim 5,
The polynucleotide encoding the CCR2 is characterized in that it consists of the nucleotide sequence of SEQ ID NO: 4, the composition. 제 5 항에 있어서,
상기 CCR2는 서열번호 3의 아미노산 서열로 이루어진 것을 특징으로 하는 조성물. 6. The method of claim 5,
The CCR2 composition, characterized in that consisting of the amino acid sequence of SEQ ID NO: 3. 제 5 항에 있어서,
상기 골질환은 골관절염(Osteoarthritis)인 것을 특징으로 하는 조성물.6. The method of claim 5,
The bone disease is a composition, characterized in that osteoarthritis (Osteoarthritis). 제 1 항 또는 제 5항의 조성물을 포함하는, 골질환의 예방 또는 치료용 키트.A kit for preventing or treating bone disease, comprising the composition of claim 1 or 5.
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