KR101898198B1 - Method of increasing cytopermeability of extracellular superoxide dismutase - Google Patents
Method of increasing cytopermeability of extracellular superoxide dismutase Download PDFInfo
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- KR101898198B1 KR101898198B1 KR1020160112667A KR20160112667A KR101898198B1 KR 101898198 B1 KR101898198 B1 KR 101898198B1 KR 1020160112667 A KR1020160112667 A KR 1020160112667A KR 20160112667 A KR20160112667 A KR 20160112667A KR 101898198 B1 KR101898198 B1 KR 101898198B1
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- sod
- protein
- rheumatoid arthritis
- present
- superoxide dismutase
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Abstract
본 발명은 세포외 분비 슈퍼옥사이드 디스뮤테이즈(EC-SOD 또는 SOD3) 단백질의 세포 투과성을 증가시키는 방법에 관한 것이다. 더욱 자세하게는 아연 2가 양이온을 이용하여 EC-SOD 단백질의 세포 투과성을 증가시키는 방법 및 이를 이용한 염증성 질환 예방 또는 치료용 조성물에 관한 것이다.
본 발명의 방법에 의하면 EC-SOD의 세포 투과성이 증진되어 항염증 활성 등 EC-SOD의 생리학적 활성이 극대화되어 염증성 질환의 예방 또는 치료 효과가 매우 높게 발휘될 수 있다. The present invention relates to a method for increasing the cellular permeability of extracellular secretory superoxide dismutase (EC-SOD or SOD3) proteins. More particularly, the present invention relates to a method for increasing the cell permeability of EC-SOD protein using zinc divalent cations and a composition for preventing or treating inflammatory diseases using the same.
According to the method of the present invention, the cell permeability of EC-SOD is enhanced, and the physiological activity of EC-SOD such as anti-inflammatory activity is maximized, so that the effect of preventing or treating inflammatory diseases can be highly exerted.
Description
본 발명은 세포외 분비 슈퍼옥사이드 디스뮤테이즈(EC-SOD 또는 SOD3) 단백질의 세포 투과성을 증가시키는 방법에 관한 것이다. 더욱 자세하게는 아연 2가 양이온을 이용하여 EC-SOD 단백질의 세포 투과성을 증가시키는 방법 및 이를 이용한 염증성 질환 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a method for increasing the cellular permeability of extracellular secretory superoxide dismutase (EC-SOD or SOD3) proteins. More particularly, the present invention relates to a method for increasing the cell permeability of EC-SOD protein using zinc divalent cations and a composition for preventing or treating inflammatory diseases using the same.
활성산소를 제거할 뿐만 아니라 다른 항산화 효소가 작용할 수 있도록 하여 세포를 보호하는 기능을 가지는 SOD(superoxide dismutase)는 구리와 아연 원자를 가지고 있는 Cu/Zn SOD(SOD 1), 망간 원자를 가지고 있는 Mn SOD(SOD 2) 및 세포 표면 또는 세포 외액에 존재하는 EC-SOD(extracellular superoxide dismutase)로 구분된다.SOD (superoxide dismutase), which has the function of protecting cells by allowing other antioxidant enzymes to act as well as removing active oxygen, is composed of Cu / Zn SOD (SOD 1) having copper and zinc atoms, Mn SOD (SOD 2) and extracellular superoxide dismutase (EC-SOD) present on the cell surface or extracellular fluid.
특히, EC-SOD는 Cu/Zn SOD와 같이 구리 및 아연 원자를 가지고 있으나, C-말단 쪽에 헤파린 결합 도메인(heparin binding domain)이 존재한다는 특징이 있다. EC-SOD는 헤파린 결합 도메인을 가지고 있기 때문에 세포막의 헤파린에 결합되어 세포막을 보호하는 역할을 할 것으로 추정되고 있다. In particular, EC-SOD has copper and zinc atoms as Cu / Zn SOD, but heparin binding domain is present at the C-terminal side. Since EC-SOD has a heparin-binding domain, it is presumed that it plays a role of protecting cell membrane by binding to heparin of cell membrane.
문헌에 따르면, EC-SOD는 혈장과 세포외 기질 등에서 생체 방어작용을 담당하고 있는 것으로 알려져 있다(Marklund et al, Biochem. J. 266, 213-219, 1990; Su et al., Am J Respir Cell Mol Biol., Feb 16(2), 162-70, 1997; Luoma et al., Thromb. Vasc. Bio. 18, 157-167,1998). 아울러, EC-SOD의 헤파린 결합 도메인이 핵 위치 신호(nuclear localization signal)로 작용하여 흉선(thymus)과 고환 세포(testis cell)에서 핵 안에 위치하여 산화적 스트레스로부터 게놈 DNA를 보호하고 산화 환원 반응에 민감한 전사작용을 조절한다고 보고된 바 있다(Ookawara T et al., BBRC, 296, 54-61, 2002).According to the literature, EC-SOD is known to be responsible for bio-defense in plasma and extracellular matrix (Marklund et al, Biochem., 266, 213-219, 1990; Su et al., Am J Respir Cell Mol Biol., Feb 16 (2), 162-70, 1997; Luoma et al., Thromb., Vasc. Bio., 18, 157-167, 1998). In addition, the heparin-binding domain of EC-SOD acts as a nuclear localization signal and is located in the nucleus of thymus and testis cell to protect genomic DNA from oxidative stress, (Ookawara T et al., BBRC, 296, 54-61, 2002).
그러나 앞서 언급한 바와 같이, 항산화기능이 우수한 것으로 확인되고 있는 EC-SOD는 그 자체로는 안정성과 활성에 문제가 있는 것으로 알려지고 있으며, 뿐만 아니라 세포내 투과성이 낮아 세포내에서 항산화 기능을 적절히 수행하지 못하고 있다. 따라서, EC-SOD의 항산화 기능을 극대화시키기 위하여 세포투과성 및 세포내 축적을 증가시키는 방법에 대한 연구가 절실히 요구되어 왔다.However, as mentioned above, EC-SOD, which has been confirmed to have excellent antioxidant properties, is known to have a problem in its stability and activity. In addition, since it has low intracellular permeability, I can not. Therefore, there is a desperate need to study methods for increasing cell permeability and intracellular accumulation in order to maximize the antioxidant function of EC-SOD.
이에 본 발명자들은 EC-SOD의 세포투과성이 증가되는 방법과 세포내 EC-SOD를 다량 축적시킬 수 있는 방법에 대하여 연구하였으며, 아연 2가 양이온을 이용한 EC-SOD의 세포 투과성을 증가시키는 방법을 개발하여 본 발명을 완성하였다.The present inventors have studied how to increase the cell permeability of EC-SOD and how to accumulate a large amount of EC-SOD in the cell, and developed a method of increasing the cell permeability of EC-SOD using zinc divalent cation Thereby completing the present invention.
따라서, 본 발명의 목적은 EC-SOD(Extracellular superoxide dismutase) 단백질과 아연 2가 양이온을 세포에 접촉시키는 단계를 포함하는 EC-SOD 단백질의 세포 투과성을 증가시키는 방법을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a method for increasing the cell permeability of an EC-SOD protein comprising contacting an EC-SOD (extracellular superoxide dismutase) protein with a zinc divalent cation.
본 발명의 또 다른 목적은 EC-SOD(Extracellular superoxide dismutase) 단백질 및 아연 2가 양이온을 유효성분으로 포함하는 염증성 질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다. It is still another object of the present invention to provide a pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases comprising an EC-SOD (Extracellular superoxide dismutase) protein and a zinc divalent cation as an active ingredient.
본 발명의 또 다른 목적은 EC-SOD(Extracellular superoxide dismutase) 단백질 및 아연 2가 양이온을 유효성분으로 포함하는 염증성 질환 개선용 식품 조성물을 제공하는 것이다. It is still another object of the present invention to provide a food composition for improving inflammatory diseases comprising an EC-SOD (Extracellular superoxide dismutase) protein and a zinc divalent cation as an active ingredient.
본 발명의 또 다른 목적은 EC-SOD(Extracellular superoxide dismutase) 단백질 및 아연 2가 양이온을 유효성분으로 포함하는 염증성 피부질환 개선용 화장료 조성물을 제공하는 것이다. It is still another object of the present invention to provide a cosmetic composition for improving inflammatory skin diseases comprising an EC-SOD (Extracellular superoxide dismutase) protein and a zinc divalent cation as an active ingredient.
따라서, 본 발명의 목적을 달성하기 위하여 본 발명은 EC-SOD(Extracellular superoxide dismutase) 단백질과 아연 2가 양이온을 세포에 접촉시키는 단계를 포함하는 EC-SOD 단백질의 세포 투과성을 증가시키는 방법을 제공한다.Accordingly, in order to accomplish the object of the present invention, the present invention provides a method for increasing the cell permeability of an EC-SOD protein comprising contacting an EC-SOD (extracellular superoxide dismutase) protein with a zinc divalent cation .
본 발명의 다른 목적을 달성하기 위하여 본 발명은 EC-SOD(Extracellular superoxide dismutase) 단백질 및 아연 2가 양이온을 유효성분으로 포함하는 염증성 질환 예방 또는 치료용 약학적 조성물을 제공한다. In order to achieve the other object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising an EC-SOD (Extracellular superoxide dismutase) protein and a zinc divalent cation as an active ingredient.
본 발명의 다른 목적을 달성하기 위하여 본 발명은 EC-SOD(Extracellular superoxide dismutase) 단백질 및 아연 2가 양이온을 유효성분으로 포함하는 염증성 질환 개선용 식품 조성물을 제공한다.In order to accomplish still another object of the present invention, there is provided a food composition for improving inflammatory diseases, which comprises an EC-SOD (Extracellular superoxide dismutase) protein and a zinc divalent cation as an active ingredient.
본 발명의 다른 목적을 달성하기 위하여 본 발명은 EC-SOD(Extracellular superoxide dismutase) 단백질 및 아연 2가 양이온을 유효성분으로 포함하는 염증성 피부질환 개선용 화장료 조성물을 제공한다. In order to accomplish still another object of the present invention, there is provided a cosmetic composition for the treatment of inflammatory skin diseases comprising EC-SOD (Extracellular superoxide dismutase) protein and zinc divalent cation as an active ingredient.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
달리 규정되지 않는 한, 본 발명에 사용된 기술적 및 과학 용어들은 본 발명이 속하는 분야에서 통상의 지식을 가진 자가 이해하는 바와 동일한 의미를 갖는다.Unless defined otherwise, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
본 명세서에서 사용된‘EC-SOD’는 extracellular superoxide dismutase의 약자로 세포외 슈퍼옥사이드 디스뮤테이즈 또는 SOD3로 표기될 수 있다. 바람직하게는 인간, 마우스, 소, 말, 돼지 등 포유동물의 EC-SOD 일 수 있으며, 더욱 바람직하게는 인간 유래 EC-SOD일 수 있다. 일실시예에서는 인간 재조합 SOD3(rhSOD)로 실험을 수행하였으며, 도면에서 Full이라고 기재된 것 또한 인간 재조합 SOD3로 간주될 수 있다.As used herein, 'EC-SOD' is an abbreviation for extracellular superoxide dismutase and may be expressed as extracellular superoxide dismutase or SOD3. Preferably, it may be EC-SOD of a mammal such as a human, a mouse, a cow, a horse, a pig, and more preferably a human-derived EC-SOD. In one embodiment, experiments with human recombinant SOD3 (rhSOD) were performed, and those described as Full in the figures can also be considered human recombinant SOD3.
본 명세서에서 사용된‘형질도입’은 유전 정보를 유기체, 특히 진핵세포 내로 도입함을 의미한다. 이는 상기 유전 정보, 특히 DNA를 도입시키는데 있어서 당업자에게 공지된 임의의 가능한 방법들, 예를 들어 DNA-충전된 입자들로의 포격, 원형질체를 이용한 형질전환, DNA의 미세주입, 전기천공, 컴피턴트(competent) 세포의 접합 또는 형질전환, 화학물질 또는 아그로박테리아-매개된 형질전환을 포함한다. 유전 정보란 유전자 구역, 하나의 유전자 또는 다수의 유전자들을 의미한다. 유전 정보는, 예를들어 벡터나 유리 핵산(예를 들어, DNA, RNA)의 보조하에 그리고 임의의 다른 방식에 의해 세포내로 도입될 수 있으며, 재조합에 의해 숙주 게놈내로 혼입되거나 세포에서 유리 형태로 존재할 수 있다. As used herein, " transduction " means introducing genetic information into organisms, particularly eukaryotic cells. This may be accomplished by any of the possible methods known to those skilled in the art for introducing the genetic information, particularly DNA, such as, for example, bombardment with DNA-filled particles, transformation using protoplasts, microinjection of DNA, electroporation, binding or transformation of competent cells, chemical or Agrobacterium-mediated transformation. Genetic information refers to a gene region, a gene, or a number of genes. The genetic information can be introduced into the cell, for example, under the aid of a vector or a free nucleic acid (e. G., DNA, RNA) and by any other means, introduced into the host genome by recombination, Can exist.
본 명세서에서 사용된 아연 2가 양이온은 2개의 양 전하를 가진 아연 이온을 일컫는 것이다. 사용될 수 있는 이러한 아연의 특정한 형태는 아연 2가 양이온의 염산염, 아세트산염, 탄산염, 시트르산염 및 황산염 형태와 같은 염 형태 (예를 들어, 제약학적으로 허용가능한 염 형태)를 포함한다. 본 발명에서 사용하기 위해 바람직한 아연 2가 양이온은 염 형태인 염화아연이다.As used herein, a zinc divalent cation refers to a zinc ion having two positive charges. Certain forms of such zinc that may be used include salt forms (e. G., Pharmaceutically acceptable salt forms) such as the hydrochloride, acetate, carbonate, citrate and sulfate forms of zinc diatomic cations. Preferred zinc divalent cations for use in the present invention are zinc chloride in salt form.
본 명세서에서 사용된 ‘접촉’이라는 용어는 본 발명의 아연 2가 양이온을 포함하는 용액을 EC-SOD 단백질과 세포를 침지시키는 액체 매질과 혼합하는 것을 의미한다. 본 발명의 아연 2가 양이온을 포함하는 용액은 또한 다른 성분, 예를 들어, 버퍼 등을 포함할 수 있다. 본 발명의 아연 2가 양이온은 운반 장치(예: 피펫을 기본으로 하는 장치 또는 주사기를 기본으로 하는 장치)를 사용하여 EC-SOD 단백질과 세포를 침지시킨 매질에 가할 수 있다.As used herein, the term " contacting " means mixing a solution comprising the zinc di-cation of the present invention with a liquid medium that immerses the cells in an EC-SOD protein. The solution containing the zinc divalent cation of the present invention may also contain other components, such as buffers and the like. The zinc divalent cations of the present invention can be added to media immersed with EC-SOD protein and cells using a delivery device (e.g., a device based on a pipette or a syringe based device).
보다 구체적으로, 본 발명은 (a) EC-SOD(Extracellular superoxide dismutase) 유전자를 숙주세포에 형질도입하는 단계; (b) 상기 형질도입한 숙주세포로부터 EC-SOD 단백질을 정제하는 단계; 및 (c) 상기 정제된 EC-SOD 단백질과 아연 2가 양이온을 세포에 접촉시키는 단계를 포함하는 (d) 정제된 EC-SOD 단백질을 인체내 피하, 근육, 정맥내 또는 다른경로로 주입시 아연 2가 양이온과 동시에 주입하여 인체조직내 세포들에 침투성을 증가시키는 방법을 포함하는 EC-SOD 단백질의 세포 투과성을 증가시키는 방법도 함께 제공한다.More particularly, the present invention relates to a method for producing a recombinant vector comprising the steps of (a) transfecting an EC-SOD (Extracellular superoxide dismutase) gene into a host cell; (b) purifying the EC-SOD protein from the transfected host cell; And (c) contacting the purified EC-SOD protein with zinc divalent cations to the cells. (D) injecting the purified EC-SOD protein subcutaneously, intramuscularly, intravenously or by other routes in the body, 2 < / RTI > cations in order to increase the permeability of the EC-SOD protein.
상기 (a) 단계의 EC-SOD 유전자를 숙주세포에 형질 도입하는 것은 핵산을 숙주세포에 도입하는 어떤 방법도 포함되며, 당업자에게 공지된 형질전환기술에 의해 수행될 수 있다. 바람직하게는 미세사출법(microprojectile bombardment), 전기충격유전자전달법(electroporation), 인산 칼슘(CaPO4) 침전, 염화 칼슘(CaCl2) 침전, PEG-매개 융합법(PEG-mediated fusion), 미세주입법(microinjection) 및 리포좀 매개법(liposome-mediated method) 등이 포함되나 이로 제한되지 않는다.Transformation of the EC-SOD gene of step (a) into a host cell includes any method of introducing the nucleic acid into a host cell, and can be carried out by a transformation technique known to a person skilled in the art. Preferably, microprojectile bombardment, electroporation, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, PEG-mediated fusion, microinjection ) And liposome-mediated method, and the like.
본 발명의 상기 EC-SOD(Extracellular superoxide dismutase) 유전자는 서열번호 1로 표시되는 것을 특징으로 한다. 본 발명의 EC-SOD는 헤파린 결합 도메인을 가지는 인간 재조합 EC-SOD 유전자인 것을 특징으로 한다. 본 발명의 재조합 EC-SOD 단백질은 전체 길이는 222개의 아미노산으로 이루어져 있으며, 바람직하게는 서열번호 2의 아미노산 서열을 가질 수 있다.The EC-SOD (Extracellular superoxide dismutase) gene of the present invention is characterized by being represented by SEQ ID NO: 1. The EC-SOD of the present invention is characterized by being a human recombinant EC-SOD gene having a heparin-binding domain. The recombinant EC-SOD protein of the present invention has a total length of 222 amino acids, preferably an amino acid sequence of SEQ ID NO: 2.
본 발명의 상기 숙주세포는 효모, 고초균(CHO cell), 대장균, 곤충세포 및 인간화세포로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 한다. 숙주세포는 원핵세포 또는 진핵세포를 사용할 수 있으나, 바람직하게는 숙주세포로는 대장균(Escherichia coli), 바실러스 서브틸리스(Bacillus subtilis), 스트렙토마이세스(Streptomyces), 슈도모나스(Pseudomonas), 프로테우스 미라빌리스(Proteus mirabilis) 또는 스타필로코쿠스(Staphylococcus)와 같은 원핵 숙주 세포, 진균(예를 들어, 아스퍼질러스(Aspergillus)), 효모(예를 들어, 피키아 패스토리스(Pichiapastoris), 사카로마이세스 세르비지애(Saccharomyces cerevisiae), 쉬조사카로마세스(Schizosaccharomyces), 뉴로스포라 크라사(Neurospora crassa) 등과 같은 하등 진핵세포, 곤충 세포, 식물 세포, 포유동물 등을 포함하는 고등 진핵생물 유래의 세포를 숙주세포로 사용할 수 있으나 이에 제한되지는 않는다. 더 바람직하게는 본 발명의 숙주세포는 대장균을 사용할 수 있다.The host cell of the present invention is characterized in that it is any one selected from the group consisting of yeast, Bacillus subtilis (CHO cell), Escherichia coli, insect cells and humanized cells. The host cell may be a prokaryotic cell or an eukaryotic cell. Preferably, the host cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Prokaryotic host cells such as Proteus mirabilis or Staphylococcus, fungi such as Aspergillus, yeast such as Pichiapastoris, Derived from higher eukaryotes, including eukaryotic cells such as Saccharomyces cerevisiae, Schizosaccharomyces, Neurospora crassa, insect cells, plant cells, mammals and the like. Cells can be used as host cells, but are not limited thereto. More preferably, E. coli can be used as a host cell of the present invention.
본 발명에서 사용된 표준 재조합 DNA 및 분자 클로닝 기술은 당해 분야에 널리 공지되어 있고, 다음 문헌에 기재되어 있다(Sambrook, J., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989); Silhavy, T. J., Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984); Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience (1987)).Standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art and are described in Sambrook, J., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold (1984); Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc., Spring Harbor, NY (1989); Silhavy, TJ; Experiments with Gene Fusions; Cold Spring Harbor Laboratory; and Wiley-lnterscience (1987)).
본 발명의 상기 정제 방법은 한외여과법, 염석법, 투석법 및 크로마토그래피로 이루어진 군에서 선택되는 적어도 한 가지 방법에 의해서 실시되는 것을 특징으로 하며, 바람직하게는 겔-여과 컬럼(gel-filtration column)에 의해 분리, 정제하는 것이다.The purification method of the present invention is characterized in that it is carried out by at least one method selected from the group consisting of ultrafiltration, salting-out, dialysis and chromatography, preferably a gel-filtration column, To be separated and purified.
상기 (b) 단계의 EC-SOD의 정제는 당업계에 공지된 다양한 분리 및 정제 방법을 통해 수행할 수 있으며, 예를 들어, 염석(황산암모늄 침전 및 인산나트륨 침전), 용매 침전(아세톤, 에탄올 등을 이용한 단백질 분획 침전), 투석, 겔 여과, 이온 교환 크로마토그래피, 역상 컬럼 크로마토그래피 및 친화성 크로마토그래피 등의 기법을 단독 또는 조합으로 적용시켜 본 발명의 재조합 EC-SOD 단백질을 정제할 수 있다. 바람직하게는 겔-여과 컬럼(gel-filtration column)에 의한 크로마토그래피의 방법으로 정제하는 것이다.The purification of the EC-SOD in step (b) can be carried out through various separation and purification methods known in the art, for example, salting out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (acetone, ethanol , Etc.), dialysis, gel filtration, ion exchange chromatography, reverse phase column chromatography and affinity chromatography, alone or in combination, to purify the recombinant EC-SOD protein of the present invention . Preferably by means of chromatography on a gel-filtration column.
한편, 본 발명의 일실시예에 따르면, 본 발명의 방법에서 제조한 재조합 EC-SOD 단백질은 인간 EC-SOD와 동일하게 SOD 활성을 가지고 있으며, 이에 아연 2가 양이온을 첨가하면 세포투과성 및 세포내 축적량을 더욱 증가시킬 수 있다.Meanwhile, according to one embodiment of the present invention, the recombinant EC-SOD protein produced by the method of the present invention has the same SOD activity as human EC-SOD, and when zinc divalent cation is added thereto, The accumulation amount can be further increased.
본 발명의 일실시예에 따르면, 본 발명의 방법에서 제조한 재조합 EC-SOD 단백질과 ZnCl2를 함께 세포에 처리하면 단일 EC-SOD 단백질만 처리할 때 보다 세포내 염증 인자인 NF-kB p65의 활성을 더욱 효과적으로 억제할 수 있다.According to one embodiment of the present invention, the cells treated with the recombinant EC-SOD protein and ZnCl2 produced by the method of the present invention are more effective than the single EC-SOD protein only in the activity of the intracellular inflammatory factor NF-kB p65 Can be more effectively suppressed.
본 발명의 일실시예에 따르면, 본 발명의 방법에서 제조한 재조합 EC-SOD 단백질과 ZnCl2를 함께 세포에 처리하면 세포내 염증성 사이토카인인 IL-1β, IL-1α, TNFα, IL-8 및 IL-6의 mRNA 발현을 단일 EC-SOD 단백질만 처리할 때 보다 더욱 효과적으로 억제할 수 있다.In accordance with one embodiment of the present invention, the recombinant EC-SOD protein and ZnCl2 produced in the method of the present invention are treated together with the cells to inhibit the intracellular inflammatory cytokines IL-1β, IL-1α, TNFα, -6 mRNA expression can be more effectively inhibited than when treated with only a single EC-SOD protein.
본 발명의 방법은 또한, 정제된 EC-SOD 단백질을 인체 내 피하, 근육, 정맥내 또는 다른 경로를 통해 투여할 때, 아연 2가 양이온과 동시에 투여하여 인체조직내 세포들에 EC-SOD의 침투성을 증가시키는 방법을 포함한다. 즉, EC-SOD를 아연 2가 양이온과 함께 인체로 투여함으로써, EO-SOD의 세포 투과성을 증진시켜 생리학적 활성을 극대화 할 수 있는 것이다. EC-SOD와 아연 2가 양이온을 투여하는 순서는 특별히 제한되지 않는다. EC-SOD와 아연 2가 양이온을 동시에 투여할 수도 있으며, 둘 중에 어느 하나를 먼저 투여한 후 일정한 시간 간격을 두고 다른 하나를 투여할 수도 있다. 가장 적절한 투여순서는 당업계에서 통상의 기술자가 반복적인 실험관찰을 통해 결정할 수 있을 것이다. The method of the present invention may further comprise administering the purified EC-SOD protein subcutaneously, intramuscularly, intravenously, or via another route in the body, with simultaneous administration of a zinc divalent cation to cause permeation of EC-SOD / RTI > That is, by administering EC-SOD together with a zinc divalent cation to the human body, the cell permeability of EO-SOD can be enhanced and the physiological activity can be maximized. The order of administration of EC-SOD and zinc divalent cations is not particularly limited. EC-SOD and zinc divalent cations may be administered simultaneously. Alternatively, one of the two may be administered first and the other one may be administered at a predetermined time interval. The most appropriate dosing sequence may be determined by routine experimentation with routine experimentation in the art.
한편, 본 발명은 EC-SOD(Extracellular superoxide dismutase) 단백질 및 아연 2가 양이온을 유효성분으로 포함하는 염증성 질환 예방 또는 치료용 약학적 조성물을 제공한다. The present invention also provides a pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases comprising an EC-SOD (Extracellular superoxide dismutase) protein and a zinc divalent cation as an active ingredient.
상기 아연 2가 양이온은 이미 언급한 바와 동일하게 사용될 수 있는 이러한 아연의 특정한 형태는 아연 2가 양이온의 염산염, 아세트산염, 탄산염, 시트르산염 및 황산염 형태와 같은 염 형태 (예를 들어, 제약학적으로 허용가능한 염 형태)를 포함한다. 본 발명에서 사용하기 위해 바람직한 아연 2가 양이온은 염 형태인 염화아연이다.This particular form of zinc, which can be used in the same way as already mentioned, can be used in the form of salts such as hydrochlorides, acetates, carbonates, citrates and sulphate salts of zinc divalent cations (e. Acceptable salt forms). Preferred zinc divalent cations for use in the present invention are zinc chloride in salt form.
상기 염증성 질환은 피부염, 알레르기, 아토피, 천식, 건선, 주사, 여드름, 편평태선, 선상 태선, 색소성 피부질환, 수포성 피부질환, 결막염, 당뇨성 망막질환, 건성 안,치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 위염, 크론병, 대장염, 치질, 통풍, 강직성 척추염, 류마티스 열, 루푸스, 섬유근통 (fibromyalgia), 건선관절염, 골관절염, 류마티스관절염, 견관절주위염, 건염, 건초염, 건주위염, 근육염, 간염, 방광염, 신장염, 쇼그렌 증후군(sjogren's syndrome), 다발성 경화증, 및 급성 및 만성 염증 질환으로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하지만 이에 제한되는 것은 아니다. The inflammatory disease is selected from the group consisting of dermatitis, allergy, atopy, asthma, psoriasis, injection, acne, squamous cell, glandular gland, pigmented skin disease, watery skin disease, conjunctivitis, diabetic retinopathy, dry eye, periodontitis, Rheumatic fever, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder periitis, tendonitis, hay fever, tendonitis, gingivitis, gastroenteritis, But are not limited to, those selected from the group consisting of inflammation, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases.
본 발명에 따른 약학적 조성물은 상기 EC-SOD 및 아연 2가 양이온만을 함유하거나 약학적으로 허용되는 담체와 함께 적합한 형태로 제형화 될 수 있으며, 부형제 또는 희석제를 추가로 함유할 수 있다. 상기 담체로는 모든 종류의 용매, 분산매질, 수중유 또는 유중수 에멀젼, 수성 조성물, 리포좀, 마이크로비드 및 마이크로좀이 포함된다.The pharmaceutical composition according to the present invention may be formulated into a suitable form containing only the EC-SOD and the zinc divalent cation or a pharmaceutically acceptable carrier, and may further contain an excipient or a diluent. Such carriers include all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.
약학적으로 허용되는 담체로는 예컨대, 경구 투여용 담체 또는 비경구 투여용 담체를 추가로 포함할 수 있다. 경구 투여용 담체는 락토스, 전분, 셀룰로스 유도체, 마그네슘 스테아레이트, 스테아르산 등을 포함할 수 있다. 아울러, 경구투여용으로 사용되는 다양한 약물전달물질을 포함할 수 있다. 또한, 비경구 투여용 담체는 물, 적합한 오일, 식염수, 수성 글루코오스 및 글리콜 등을 포함할 수 있으며, 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르 브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸-또는 프로필-파라벤 및 클로로부탄올이 있다. 본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현택제 등을 추가로 포함할 수 있다. 그 밖의 약학적으로 허용되는 담체 및 제제는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다(Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery materials used for oral administration. In addition, the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and preparations can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).
본 발명의 조성물은 인간을 비롯한 포유동물에 어떠한 방법으로도 투여할 수 있다. 예를 들면, 경구 또는 비경구적으로 투여할 수 있다. 비경구적인 투여방법으로는 이에 한정되지는 않으나, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장내 투여일 수 있다.The composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI >
본 발명의 약학적 조성물은 상술한 바와 같은 투여 경로에 따라 경구 투여용 또는 비경구 투여용 제제로 제형화 할 수 있다.The pharmaceutical composition of the present invention can be formulated into oral preparations or parenteral administration preparations according to the administration route as described above.
경구 투여용 제제의 경우에 본 발명의 조성물은 분말, 과립, 정제, 환제, 당의정제, 캡슐제, 액제, 겔제, 시럽제, 슬러리제, 현탁액 등으로 당업계에 공지된 방법을 이용하여 제형화될 수 있다. 예를 들어, 경구용 제제는 활성성분을 고체 부형제와 배합한 다음 이를 분쇄하고 적합한 보조제를 첨가한 후 과립 혼합물로 가공함으로써 정제 또는 당의정제를 수득할 수 있다. 적합한 부형제의 예로는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨 및 말티톨 등을 포함하는 당류와 옥수수 전분, 밀 전분, 쌀 전분 및 감자 전분 등을 포함하는 전분류, 셀룰로즈,메틸 셀룰로즈, 나트륨 카르복시메틸셀룰로오즈 및 하이드록시프로필메틸-셀룰로즈 등을 포함하는 셀룰로즈류, 젤라틴, 폴리비닐피롤리돈 등과 같은 충전제가 포함될 수 있다. 또한, 경우에 따라 가교결합 폴리비닐피롤리돈, 한천, 알긴산 또는 나트륨 알기네이트 등을 붕해제로 첨가할 수 있다. 나아가, 본 발명의 약학적 조성물은 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include, but are not limited to, sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.
비경구 투여용 제제의 경우에는 주사제, 크림제, 로션제, 외용연고제, 오일제, 보습제, 겔제, 에어로졸 및 비강 흡입제의 형태로 당업계에 공지된 방법으로 제형화할 수 있다. 이들 제형은 모든 제약 화학에 일반적으로 공지된 처방서인 문헌(Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA,1995)에 기재되어 있다.In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa., 1995, which is a commonly known formulary for all pharmaceutical chemistries.
본 발명의 조성물의 총 유효량은 단일 투여량(single dose)으로 환자에게 투여될 수 있으며, 다중 투여량(multiple dose)으로 장기간 투여되는 분할 치료 방법(fractionated treatment protocol)에 의해 투여될 수 있다. 본 발명의 약학적 조성물은 질환의 정도에 따라 유효성분의 함량을 달리할 수 있다. 바람직하게 본 발명의 약학적 조성물의 바람직한 전체 용량은 1일당 환자 체중 1㎏ 당 약 0.0001㎍ 내지 10,000mg, 가장 바람직하게는 0.1㎍ 내지 500mg일 수 있다. 그러나 상기 약학적 조성물의 용량은 제제화 방법, 투여 경로 및 치료 횟수뿐만 아니라 환자의 연령, 체중, 건강 상태, 성별, 질환의 중증도, 식이 및 배설율등 다양한 요인들을 고려하여 환자에 대한 유효 투여량이 결정되는 것이므로, 이러한 점을 고려할 때 당 분야의 통상적인 지식을 가진 자라면 본 발명의 조성물의 적절한 유효 투여량을 결정할 수 있을 것이다. 본 발명에 따른 약학적 조성물은 본 발명의 효과를 보이는 한 그 제형, 투여 경로 및 투여 방법에 특별히 제한되지 아니한다. The total effective amount of the composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses. In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Preferably, the total preferred dose of the pharmaceutical composition of the present invention may be from about 0.0001 μg to 10,000 mg, and most preferably from 0.1 μg to 500 mg per kilogram of patient body weight per day. However, the dosage of the pharmaceutical composition may be determined depending on various factors such as the formulation method, administration route and frequency of treatment, as well as the patient's age, body weight, health condition, sex, severity of disease, diet and excretion rate, It will be possible to determine the appropriate effective dose of the composition of the present invention by those of ordinary skill in the art in view of this point. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.
본 발명은 또한 EC-SOD(Extracellular superoxide dismutase) 단백질 및 아연 2가 양이온을 유효성분으로 포함하는 염증성 질환 개선용 식품 조성물을 제공한다. The present invention also provides a food composition for improving inflammatory diseases, which comprises an EC-SOD (Extracellular superoxide dismutase) protein and a zinc divalent cation as an active ingredient.
본 발명에 따른 식품 조성물은 기능성 식품(functional food), 영양보조제(nutritional supplement), 건강식품(health food) 및 식품첨가제(food additives) 등의 모든 형태를 포함한다. 상기 유형들은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다.The food composition according to the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. These types can be prepared in various forms according to conventional methods known in the art.
예를 들면, 건강식품으로는 본 발명의 식품 조성물 자체를 차, 쥬스 및 드링크의 형태로 제조하여 음용하도록 하거나, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. 또한 본 발명의 식품 조성물은 염증성 질환에 효과가 있다고 알려진 공지의 물질 또는 활성 성분과 함께 혼합하여 조성물의 형태로 제조할 수 있다.For example, as a health food, the food composition itself of the present invention may be prepared in the form of tea, juice, and drink, and may be ingested, granulated, encapsulated, and powdered. In addition, the food composition of the present invention can be prepared in the form of a composition by mixing with known substances or active ingredients known to be effective for inflammatory diseases.
또한 기능성 식품으로는 음료(알콜성 음료 포함), 과실 및 그의 가공식품 (예를 들어 과일 통조림, 병조림, 잼, 마아말레이드 등), 어류, 육류 및 그 가공식품(예를 들어 햄, 소시지콘비이프 등), 빵류 및 면류(예를 들어 우동, 메밀국수, 라면, 스파게티, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예를 들어 버터, 치이즈 등), 식용식물유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예를 들어 된장, 간장, 소스 등) 등에 본 발명의 식품 조성물을 첨가하여 제조할 수 있다.Functional foods also include beverages (including alcoholic beverages), fruits and their processed foods (such as canned fruits, bottled, jam, maalmalade, etc.), fish, meats and processed foods such as ham, (Such as udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, , Vegetable protein, retort food, frozen food, various kinds of seasoning (for example, soybean paste, soy sauce, sauce, etc.).
본 발명에 따른 식품 조성물의 바람직한 함유량으로는 이에 한정되지 않지만 바람직하게는 최종적으로 제조된 식품 총 중량 중 0.0001 내지 50 중량% 이다. 본 발명의 식품 조성물을 식품첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다.The preferred content of the food composition according to the present invention is not limited thereto, but is preferably 0.0001 to 50% by weight based on the total weight of the final food. In order to use the food composition of the present invention in the form of a food additive, it may be used in the form of powder or concentrate.
본 발명은 또한 EC-SOD(Extracellular superoxide dismutase) 단백질 및 아연 2가 양이온을 유효성분으로 포함하는 염증성 피부질환 개선용 화장료 조성물을 제공한다. The present invention also provides a cosmetic composition for the treatment of inflammatory skin diseases comprising EC-SOD (Extracellular superoxide dismutase) protein and zinc divalent cation as an active ingredient.
본 발명에서 상기 염증성 피부질환은 건선, 아토피, 어린선, 천포창 등의 수포성 피부질환, 여드름, 주사(Rosacea), 홍반성 루푸스, 피부염, 선상태선, 편평태선, 색소성 피부질환, 피부노화 및 피부주름으로 이루어진 군에서 선택될 수 있으나 이에 제한되는 것은 아니다. In the present invention, the inflammatory skin disease is selected from the group consisting of psoriasis, atopic dermatitis, pustular skin diseases such as pemphigus and pemphigus, acne, Rosacea, lupus erythematosus, dermatitis, Skin wrinkles, < / RTI > and the like.
본 발명의 화장료 조성물은 다양한 형태로 제조될 수 있는데, 예컨대, 에멀젼, 로션, 크림(수중유적형, 유중수적형, 다중상), 용액, 현탁액(무수 및 수계), 무수 생성물(오일 및 글리콜계), 젤, 마스크, 팩, 분말 등의 제형으로 제조될 수 있다.The cosmetic composition of the present invention can be prepared in various forms such as emulsion, lotion, cream (aquatic type, water type, multiphase), solution, suspension (anhydrous and aquatic), anhydrous product ), A gel, a mask, a pack, a powder, and the like.
본 발명의 조성물은 그 유효성분을 포함하는 이외에 화장료 제제에 있어서 수용가능한 담체를 포함할 수 있다. 여기서 "화장료 제제에 있어서 수용가능한 담체"란 화장료 제제에 포함될 수 있는 이미 공지되어 사용되고 있는 화합물 또는 조성물이거나 앞으로 개발될 화합물 또는 조성물로서 피부와의 접촉시 인체가 적응 가능한 이상의 독성을 지니지 않는 것을 말한다.The composition of the present invention may contain an acceptable carrier in cosmetic formulations other than those containing the active ingredient. As used herein, the term " acceptable carrier for a cosmetic preparation "refers to a compound or composition that is already known and used in the cosmetic preparation, or a compound or composition to be developed in the future, and has no toxicity that the human body can adapt to when contacted with skin.
상기 담체는 본 발명의 조성물에 그것의 전체 중량에 대하여 약 1 중량 % 내지 약 99.99 중량 %, 바람직하게는 조성물의 중량의 약 50 중량% 내지 약 99 중량 %로 포함될 수 있다. The carrier may be included in the composition of the present invention in an amount of from about 1% by weight to about 99.99% by weight, preferably from about 50% by weight to about 99% by weight of the composition, based on the total weight thereof.
그러나 상기 비율은 화장료의 전술한 바의 제형에 따라 또 그것의 구체적인 적용 부위(얼굴이나 손)나 그것의 바람직한 적용량 등에 따라 달라지는 것이기 때문에, 상기 비율은 어떠한 측면으로든 본 발명의 범위를 제한하는 것으로 이해되어서는 안 된다. However, since the ratio depends on the above-mentioned formulation of the cosmetic composition and its specific application site (face or hands) or its preferable application amount, the ratio is limited in any aspect to the scope of the present invention It should not be.
한편 상기 담체로서는 알코올, 오일, 계면활성제, 지방산, 실리콘 오일, 습윤제, 보습제, 점성 변형제, 유제, 안정제, 자외선 차단제, 발색제, 향료 등이 예시될 수 있다. Examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosifiers, emulsifiers, stabilizers, sunscreens, coloring agents and perfumes.
상기 담체로서 사용될 수 있는 알코올, 오일, 계면활성제, 지방산, 실리콘 오일, 습윤제, 보습제, 점성 변형제, 유제, 안정제, 자외선 차단제, 발색제, 향료 로 사용될 수 있는 화합물/조성물 등은 이미 당업계에 공지되어 있기 때문에 당업자라면 적절한 해당 물질/조성물을 선택하여 사용할 수 있다.The compounds / compositions which can be used as the carrier and which can be used as alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosifiers, emulsions, stabilizers, sunscreens, A person skilled in the art can select and use appropriate substances / compositions.
본 발명의 화장료 조성물은 당업계에 알려진 피부 보습 활성 물질, 항아토피 활성 물질, 항여드름 활성 물질, 미백 활성 물질 등 기능성 물질을 추가로 포함할 수도 있다.The cosmetic composition of the present invention may further comprise a functional substance such as a skin moisturizing active substance, an anti-atopic active substance, an anti-acne active substance, a whitening active substance, etc., which are known in the art.
따라서 본 발명은 세포외 분비 슈퍼옥사이드 디스뮤테이즈(EC-SOD 또는 SOD3) 단백질의 세포 투과성을 증가시키는 방법 및 EC-SOD 단백질의 세포내 축적량을 증가시키는 방법을 제공한다. 이는 염증성 질환의 치료시 아연 2가 양이온을 EC-S0D 단백질에 처리함으로써 더욱 효과적인 염증성질환 치료를 기대 할 수 있으며, 다양한 의약 및 화장품 산업에 적용될 수 있어 효과적이다.Accordingly, the present invention provides a method for increasing the cell permeability of extracellular secretory superoxide dismutase (EC-SOD or SOD3) protein and a method for increasing the intracellular accumulation of EC-SOD protein. This is because treatment of inflammatory diseases with zinc divalent cation to EC-SOD protein is expected to be more effective in treating inflammatory diseases and applicable to various medicinal and cosmetic industries.
도 1은 CuSO4 와 ZnCl2를 각각 0, 10, 25 또는 50uM 포함하는 배지에서 세포를 배양한 후 배지와 세포내부에서 SOD3와 209E의 단백질 양을 측정한 웨스턴 블롯 사진이다(A: HaCa T cell, B: 203 T cell).
도 2는 SOD3와 209E 단백질을 세포배양에 처리한 후 추가적으로 ZnCl2를 처리한 군과 그렇지 않은 군으로부터 세포질 내, 핵 내 및 세포외의 SOD3 단백질 양을 측정한 웨스턴 블롯 사진이다.
도 3A는 SOD3와 209E 단백질을 세포배양에 처리한 후, 추가적으로 ZnCl2를 처리한 군과 그렇지 않은 군에서의 SOD3의 세포내 축적 양태를 형광 현미경을 통해 측정한 사진이며, 도 3B와 도 3C는 각각 ZnCl2와 PBS를 처리했을 때 FITC 감도를 그래프로 나타낸 것이다.
도 4는 ZnCl2를 각각 0, 1, 5, 10uM를 포함하는 배지에 rhSOD3의 유무에 따른 염증억제 인자 NF-kB의 활성을 측정한 웨스턴 블롯 사진이다.
도 5는 SOD3와 209E로 각각 형질도입된 HaCa T 세포에 ZnCl2와 TNFα+INFγ 처리 할 때, stat3와 stat1의 인산화와 SOD3 단백질의 양을 측정한 웨스턴 블롯 사진이다.
도 6은 rhSOD3와 209E를 ZnCl2와 같이 배양한 군과 그렇지 않은 군에 염증성 자극을 준 후 염증시 많이 나타나는 사이토카인의 mRNA 발현량을 분석한 그래프이다(A: IL-1β, B: IL-1α, C: TNFα, D: IL-8, E: IL-6). FIG. 1 is a Western blot photograph showing the amounts of SOD3 and 209E proteins in the medium and the cells after culturing the cells in a medium containing CuSO 4 and ZnCl 2 at 0, 10, 25, or 50 μM, respectively (A: HaCa T cell , B: 203 T cell).
Figure 2 is a Western blot photograph the cytoplasm the protein SOD3 and 209E from the group treated with ZnCl 2 and then further and it is not in the group treated cell culture and measuring the amount of protein SOD3 other chromosomal and cellular.
FIG. 3A is a photograph of SOD3 and 209E proteins treated with cell culture, followed by addition of ZnCl 2 and non-SOD3 cells, and FIG. 3B and FIG. 3C are photographs The graph shows FITC sensitivity when treated with ZnCl 2 and PBS, respectively.
FIG. 4 is a Western blot photograph showing the activity of the inflammation-suppressing factor NF-kB according to the presence or absence of rhSOD3 in a medium containing ZnCl 2 at 0, 1, 5, and 10 uM, respectively.
FIG. 5 is a Western blot photograph showing the amounts of phosphorylation of stat3 and stat1 and the amount of SOD3 protein when HaCa T cells transduced with SOD3 and 209E, respectively, were treated with ZnCl 2 and TNFα + INFγ.
6 is an analysis of the mRNA expression levels of cytokines that appear much when given after an inflammatory stimulus and the
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
<실험 방법><Experimental Method>
1) 세포배양1) Cell culture
HaCa T cell line은 CLSCell Line Services (Heidelberg, Germany)로부터 얻었다. 세포 배양 배지로는 10 % 소태아혈청(FBS), 1 % l-글루타민, 1 % 피루브산 나트륨(HyClone), 및 1 % antibiotic-antimycotic(HyClone)이 첨가된 high glucose DMEM (HyClone)이 사용되었으며, 세포는 37 ℃의 5 % CO2 가습배양기(humidified incubator) 속에서 배양되었다. 70 ~ 80 %컨플루언트(confluent) 일 때 세포를 실험에 사용하였다. 293 T 세포 또한 HaCa T 세포와 마찬가지로 10 % FBS를 포함하는 DMEM 배지에 보관하였다.The HaCa T cell line was obtained from CLSCell Line Services (Heidelberg, Germany). High glucose DMEM (HyClone) supplemented with 10% fetal bovine serum (FBS), 1% l-glutamine, 1% sodium pyruvate and 1% antibiotic-antimycotic (HyClone) Cells were cultured in a humidified incubator at 37 ° C in a 5% CO 2 incubator. Cells were used for experiments when 70-80% confluent. 293 T cells were also stored in DMEM medium containing 10% FBS as in HaCa T cells.
2) rhSOD3 단백질 발현을 위한 형질전환2) Transformation for rhSOD3 protein expression
서열번호 2로 표시되는 Met1부터 Glu227에 해당하는 인간 SOD3 단백질과 서열번호 4으로 표시되는 C말단 헤파린 결합 도메인이 결실된 209E 변이체 단백질은 C말단에 His6tag 또는 녹색 형광 단백질(EGFP)를 포함한다. 상기 단백질들은 수득하기 위하여 서열번호 1로 표시되는 hSOD3 유전자와 서열번호 3으로 표시되는 209E 변이체 유전자를 각각 HindIII와 EcoRI 또는 HindIII와 XbaI를 사용하여 pcDNA3.1(Invitrogen)에 삽입하였다. 플라스미드 제조업체의 지침에 따라, hSOD3(human SOD3)와 209E 변이체가 각각 인코팅된 플라스미드를 293T-EBNA와 HaCaT 세포에 Attractene(Qiagen)을 이용하여 형질전환 시켰다. 형질전환 24시간 후에 배양액을 무혈청 DMEM(DulbeccoModified Eagle Medium)로 배지를 교체하였다. 그 후 안정적으로 rhSOD3를 발현하는 293T-EBNA 세포를 G418(Invitrogen)을 사용하여 선별하고 FACS(fluorescence-activated cell sorting)를 이용하여 농축하였다. 293TEBNA와 HacaT는 10% FBS를 포함하는 DMEM 배지에 보관하였다.The human SOD3 protein corresponding to Met1 through Glu227 represented by SEQ ID NO: 2 and the 209E mutant protein lacking the C-terminal heparin binding domain represented by SEQ ID NO: 4 include a His6 tag or a green fluorescent protein (EGFP) at the C terminus. In order to obtain the above proteins, the hSOD3 gene of SEQ ID NO: 1 and the 209E mutant gene of SEQ ID NO: 3 were inserted into pcDNA3.1 (Invitrogen) using HindIII and EcoRI or HindIII and XbaI, respectively. Following plasmid manufacturer's instructions, plasmids coated with hSOD3 (human SOD3) and 209E variants, respectively, were transformed with 293T-EBNA and HaCaT cells using Attractene (Qiagen). After 24 hours of transformation, the medium was replaced with serum-free DMEM (Dulbecco Modified Eagle Medium). Then, 293T-EBNA cells stably expressing rhSOD3 were selected using G418 (Invitrogen) and concentrated using fluorescence-activated cell sorting (FACS). 293 TEBNA and HacaT were stored in DMEM medium containing 10% FBS.
3) rhSOD3 단백질 발현 및 정제3) Expression and purification of rhSOD3 protein
형질주입한 세포를 5일간 배양 후, rhSOD3를 포함하는 배양액을 수집하고 HiTrap 킬레이팅 HP 컬럼 (GE Healthcare)에 로드하여 여과하였다. 로딩 후, 컬럼은 컬럼부피의 50배 이상의 세척 완충액(50 mM NaPO4, 500 mM NaCl 및 30 mM 이미다졸)으로 세척하였다. 이어서 rhSOD3와 209E단백질은 500mM 이미다졸을 포함하는 용출 버퍼로 수득하였다. 그 후 PBS(Phosphate Buffered Saline) 또는 상기 세척 완충액(50 mM NaPO4, 500 mM NaCl 및 30 mM 이미다졸) 상에서 투석하였다. 정제된 rhSOD3의 농도는 BSA 표준 곡선에 기초하였고, BSA 표준 곡선 분석시 단백질 분석 염료(Bio-Rad)를 이용하였다.After transfected cells were cultured for 5 days, the culture containing rhSOD3 was collected, loaded on a HiTrap chelating HP column (GE Healthcare), and filtered. After loading, the column was washed with the washing buffer at least 50 times the column volume (50 mM NaPO 4, 500 mM NaCl and 30 mM imidazole). The rhSOD3 and 209E proteins were then obtained with an elution buffer containing 500 mM imidazole. And then dialyzed against PBS (Phosphate Buffered Saline) or the wash buffer (50 mM NaPO 4 , 500 mM NaCl and 30 mM imidazole). The concentration of purified rhSOD3 was based on the BSA standard curve, and the protein analysis dye (Bio-Rad) was used for BSA standard curve analysis.
4) rhSOD3의 세포투과성 측정4) Cell permeability measurement of rhSOD3
HaCaT 세포와 HEK293 T 세포는 1 x 106 cells/well의 세포 밀도로 6 well plate에 배양하였다. 24시간 후에 starvation 된 세포에 rhSOD3 또는 209E 단백질을 넣고 ZnCl2 또는 CuSO4 각각 0, 10, 25, 50uM를 같이 처리한 배지와 그렇지 않은 배지 두 그룹으로 나누어 1시간동안 배양 하였다. rhSOD3와 209E 단백질을 각각 배지, 세포 용해물로부터 SOD3 마우스 단일항체와 Horseradish peroxidase conjugated goat anti-mouse IgG (Invitrogen)을 이용하여 웨스턴 블롯으로 검출하였다.HaCaT cells and HEK293 T cells were cultured in 6-well plates at a cell density of 1 × 10 6 cells / well. Twenty-four hours later, rhSOD3 or 209E protein was added to the starved cells. The cells were treated with either ZnCl 2 or CuSO 4 , 0, 10, 25, or 50 μM, respectively, for 1 hour. The rhSOD3 and 209E proteins were detected by Western blotting using SOD3 mouse monoclonal antibody and Horseradish peroxidase-conjugated goat anti-mouse IgG (Invitrogen) from the cell lysate, respectively.
5) rhSOD3의 세포 핵 내 축적량 측정5) Measurement of accumulation of rhSOD3 in the cell nucleus
HaCaT 세포는 30-40 % 컨플루언트(confluent) 상태로 커버슬립(cover slip)에 분주한 뒤, 1 x 106 cells/well의 세포 밀도로 6 well plate에 보관되었다. 24시간 후에 starvation 된 HaCa T 세포에 rhSOD3 또는 209E 단백질과 ZnCl2 10uM를 같이 처리한 배지와 rhSOD3 또는 209E 단백질만을 처리한 두 그룹으로 나누어 1시간동안 배양 하였다. 세포는 PBS로 세척하였고, 상온에서 15분 동안 4 % 파라포름알데히드로 고정하였다. HaCaT cells were placed on a cover slip at 30-40% confluent and stored in 6-well plates at a cell density of 1 x 10 6 cells / well. Twenty-four hours later, starved HaCa T cells were treated with rhSOD3 or 209E protein and 10 uM of ZnCl 2 in the same medium, and cultured for one hour in two groups treated with only rhSOD3 or 209E protein. Cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature.
세포질과 세포핵 내로 rhSOD3와 209E 단백질이 얼마나 축적되었는지 확인하기 위하여, 세포는 5 % triton X-100 으로 용해하였고, 염색체 DNA를 변성시키기 위해 2N HCl/Triton 에서 배양하였다. 세포에서 HCl을 제거하기 위해 1회 세척한 뒤에, 비특이적 결합(nonspecific binding)을 차단하기 위해 1 % FBS 알부민에서 처리하였다. SOD3의 내부화(Internalization) 정도는 SOD3 마우스 단일항체와 FITC conjugated goat anti-mouse IgG (Invitrogen)을 이용하여 염색하여 공초점 형광현미경(confocal laser scanning microscope, Carl Zeiss LSM 510)으로 확인하였다.To determine the accumulation of rhSOD3 and 209E proteins into the cytoplasm and nucleus, cells were lysed with 5% triton X-100 and cultured in 2N HCl / Triton to denature chromosomal DNA. After one wash to remove HCl in the cells, they were treated with 1% FBS albumin to block nonspecific binding. The degree of internalization of SOD3 was determined by confocal laser scanning microscope (Carl Zeiss LSM 510) by staining with SOD3 mouse monoclonal antibody and FITC conjugated goat anti-mouse IgG (Invitrogen).
6) rhSOD3의 항염증 효과 6) Anti-inflammatory effect of rhSOD3
70 % confluency 상태의 인간각질형성세포(Human keratinocyte cell line)인 HaCa T 세포는 무혈청 DMEM 배지상에서 6시간동안 굶주렸다. 그리고 10 ng/ml TNF-α를 처리하였다. HaCa T 세포에 ZnCl2를 첨가하고 rhSOD3 단백질을 첨가한 그룹과 그렇지 않은 그룹으로 나누어 200 units/ml에 상응하는 양을 유지하였다. 세포에 직접적으로 단백질 분해 효소 억제제를 함유하는 SDS 샘플 버퍼를 첨가하여 24 시간동안 배양하였다. 그 후, 세포를 용해시켜 p-NF-κB p65 항체를 사용하여 인산화된 NF-κB p65의 양을 웨스턴블롯을 통해 분석하였다. HaCa T cells, a human keratinocyte cell line at 70% confluency, starved for 6 hours on serum-free DMEM medium. And treated with 10 ng / ml TNF-α. HaCa T cells were supplemented with ZnCl 2 and maintained in an amount corresponding to 200 units / ml in groups with and without rhSOD3 protein. SDS sample buffer containing proteinase inhibitor directly was added to the cells and cultured for 24 hours. The cells were then lysed and the amount of phosphorylated NF-κB p65 was analyzed using Western blot using p-NF-κB p65 antibody.
7) ZnCl7) ZnCl 22 의 염증 억제 효과 측정Of inflammation
70 % confluency 상태의 인간각질형성세포(Human keratinocyte cell line)인 HaCa T 세포는 무혈청 DMEM 배지상에서 6시간동안 굶주렸다. 그리고 10 ng/ml TNF-α와 100 U/ml IFN-γ를 처리하였다. HaCa T 세포에 rhSOD3 또는 209E 단백질을 처리하고, 그 후 ZnCl2를 첨가한 그룹과 그렇지 않은 그룹으로 나누어 100 units/ml에 상응하는 양을 유지하였다. 세포에 직접적으로 단백질 분해 효소 억제제를 함유하는 SDS 샘플 버퍼를 첨가하여 24 시간동안 배양하였다. p-STAT1, p-STAT3, GAPDH 및 hSOD3는 항-GAPDH (Santa Cruz Biotechnology), 및 항-hSOD3 (AbCam) 항체를 사용하여 웨스턴블롯을 통해 분석하였다. 또한, qRT-PCR(quantitative reverse transcription-polymerase chain reaction) analysis 를 이용하여, rhSOD3와 rhSOD3에 ZnCl2를 같이 배양한 그룹에서 IL-1β, IL-1α, IL-6, IL-8 및 TNF-α의 발현을 억제하는지, 발현을 억제한다면 ZnCl2를 처리하지 않은 군보다 더 강한 발현 억제 효과를 지니는지에 대하여 조사하였다. RNA는 모두 TRIzol reagent (Invitrogen)을 이용하여 분리하였다. 총 RNA의 1 microgram이 QuantiTech reverse Transcription kit (Qiagen)에 의해 역전사 되었다. 적어도 3개의 독립적인 RNA 샘플을 이용해 qRT-PCR analysis가 수행되었는데, 이 때 KAPATMSYBRFASTq-PCR Master Mix(2x) universal 과 함께 Rotorgene TM6000(Corbett life Science)가 사용되었다. mRNA의 상대적인 양은, 대조군으로서 GAPDH와 함께 ΔCt 방법(delta Ct = Ct (gene) - Ct (control gene, GAPDH))을 사용하여 산출되었다. 실험에 사용된 프라이머들은 Qiagen으로 부터 구입하였다. (hIL-1b;cat.QT00021385, hIL-1a; cat.QT00001127, hTNF-a;cat.QT00029162, hIL-8; cat.QT00000322, hIL-6; cat.QT00083720). HaCa T cells, a human keratinocyte cell line at 70% confluency, starved for 6 hours on serum-free DMEM medium. And treated with 10 ng / ml TNF-α and 100 U / ml IFN-γ. HaCa T cells were treated with rhSOD3 or 209E protein, then divided into groups with and without ZnCl 2 , and maintained a corresponding dose of 100 units / ml. SDS sample buffer containing proteinase inhibitor directly was added to the cells and cultured for 24 hours. p-STAT1, p-STAT3, GAPDH and hSOD3 were analyzed by Western blot using anti-GAPDH (Santa Cruz Biotechnology) and anti-hSOD3 (AbCam) antibodies. IL-1β, IL-6, IL-8, and TNF-α in rhSOD3 and rhSOD3 co-cultured with ZnCl 2 using qRT-PCR Or suppression of expression, the effect of inhibiting the expression of ZnCl 2 was stronger than that of the group not treated with ZnCl 2 . RNA was isolated using TRIzol reagent (Invitrogen). One microgram of total RNA was reverse transcribed by the QuantiTech reverse transcription kit (Qiagen). QRT-PCR analysis was performed using at least three independent RNA samples, in which Rotorgene TM6000 (Corbett life Science) was used with KAPATMSYBRFASTq-PCR Master Mix (2x) universal. The relative amount of mRNA was calculated using the delta Ct method (delta Ct = Ct (gene) - Ct (control gene, GAPDH)) with GAPDH as a control. The primers used in the experiments were purchased from Qiagen. (hIL-1b; cat.QT00021385, hIL-1a; cat.QT00001127, hTNF-a; cat.QT00029162, hIL-8; cat.QT00000322, hIL-6; cat.QT00083720).
<실험 결과><Experimental Results>
ZnClZnCl 22 에 의한 rhSOD3의 세포투과성 증가Increased cell permeability of rhSOD3 by
CuSO4 와 ZnCl2를 각각 0, 10, 25 또는 50uM 포함하는 배지에서 세포를 배양한 후 배지와 세포내부에서 SOD3와 209E의 단백질 양을 웨스턴 블롯으로 측정하였다. 도 1의 A는 HaCaT 세포를 대상으로 실험하였으며, 도 1의 B는 293T 세포를 대상으로 실험하였다. Cells were cultured in a medium containing CuSO 4 and ZnCl 2 at 0, 10, 25, or 50 μM, respectively, and the amounts of SOD3 and 209E proteins were determined by Western blotting in the medium and cells. FIG. 1A shows HaCaT cells and FIG. 1B shows 293T cells.
도 1A의 결과에 따르면, CuSO4를 투과촉매제로 첨가하였을 때, rhSOD3가 배지에서 세포 내부로 투과하기는 하나, 농도의존적으로 감소하는 경향을 보였다(도 1A 좌측상단 패널). 반면, ZnCl2를 투과촉매제로 첨가하였을 때 농도의존적으로 세포투과성이 증가되는 것을 확인할 수 있었다(도 1A 우측상단 패널). 한편, 헤파린 결합 도메인이 결실된 209E 단백질은 ZnCl2의 농도가 증가함에 따라 배지에 존재하는 209E 단백질이 감소하는 것을 보였으나, 세포용해물에서는 유의미한 농도변화를 보이지 않았다(도 1A 우측하단 패널).According to the results shown in Fig. 1A, when CuSO 4 was added as a permeation catalyst, rhSOD3 permeated into the cells from the medium but tended to decrease in a concentration-dependent manner (Fig. 1A upper left panel). On the other hand, when ZnCl 2 was added as a permeation catalyst, it was confirmed that the cell permeability was increased in a concentration-dependent manner (FIG. 1A, upper right panel). On the other hand, the 209E protein in which the heparin-binding domain was deleted showed a decrease in the 209E protein present in the medium as the concentration of ZnCl 2 increased, but showed no significant concentration change in the cell lysate (FIG. 1A, lower right panel).
293 T 세포에서도 유사하게, CuSO4를 투과촉매제로 첨가하였을 때 rhSOD3와 209E 단백질이 세포 내부로 투과하지 못하는 것으로 관찰되었으며(도 1B 좌측 패널), 오직 ZnCl2를 투과촉매제로 처리하였을 때 헤파린 도메인을 가지는 rhSOD3 단백질이 세포 내부로 투과한다는 것을 알 수 있었다(도 1B 우측상단 패널).Similarly, in 293 T cells, when CuSO 4 was added as a permeation catalyst, it was observed that rhSOD3 and 209E protein did not penetrate into the cells (left panel of FIG. 1B), and only ZnCl 2 was treated with a permeation catalyst, (Fig. 1B, upper right panel).
이는 2가 금속이온 중 아연이 헤파린 도메인과 상호작용하여 단백질을 핵 내부로 투과시킨다는 것을 암시한다.This implies that zinc among the divalent metal ions interacts with the heparin domain to transmit the protein into the nucleus.
Zn 또는 ZnClZn or ZnCl 22 에 의한 rhSOD3의 세포 핵 내 축적량 증가Increase in accumulation of rhSOD3 in the cell nucleus
세포질과 세포핵 내로 rhSOD3와 209E 단백질이 얼마나 축적되었는지 확인하기 위하여 웨스턴 블롯 실험을 실시하였다. 도 2의 A는 세포질에서의 rhSOD3 또는 209E의 축적을 나타내며, 도 2의 B는 핵과 상청액(supernatant)에서의 rhSOD3 또는 209E의 축적을 나타낸다.Western blot experiments were performed to determine the accumulation of rhSOD3 and 209E proteins in cytoplasm and nuclei. Figure 2 A shows the accumulation of rhSOD3 or 209E in the cytoplasm and Figure 2B shows the accumulation of rhSOD3 or 209E in the nucleus and supernatant.
도 2에 나타난 바에 따르면, Zn을 10uM 처리한 군 중 rhSOD3 단백질을 처리한 실험군에서 다량의 rhSOD3 단백질이 세포질로 또는 핵 내부로 투과되어 축적된 것을 확인할 수 있었다. 반면, Zn을 처리하지 않은 군과 209E 단백질을 처리한 군에서는 세포 내부로의 이동 또는 축적을 관찰할 수 없었다. 또한, 209E 단백질이 상청액에서도 관찰되지 않는 것으로 보아 Zn이 단백질의 안정성에도 영향을 미친다는 것을 유추할 수 있었다.As shown in FIG. 2, it was confirmed that a large amount of rhSOD3 protein was accumulated in the cytoplasm or nucleus in the experimental group treated with rhSOD3 protein among the groups treated with 10uM of Zn. On the other hand, migration or accumulation into the cell could not be observed in the group treated with Zn and the group treated with 209E protein. In addition, 209E protein was not observed in the supernatant, suggesting that Zn also affects protein stability.
한편, 세포질과 세포핵 내의 rhSOD3 또는 209E 단백질 분포를 시각적으로 확인하기 위하여, rhSOD3 및 209E 단백질을 FITC(Fluorescein isothiocyanate)로 표지하여 면역형광법을 실시하였다. 도 3의 A는 ZnCl2와 PBS(대조군)을 처리하였을 때, rhSOD3와 209E 단백질의 이동을 관찰한 사진이며, 도 3의 B와 C는 도 3A의 사진을 수치화하여 도식화한 그래프이다.On the other hand, in order to visually confirm the distribution of rhSOD3 or 209E protein in the cytoplasm and nucleus, rhSOD3 and 209E proteins were labeled with FITC (Fluorescein isothiocyanate) to perform immunofluorescence. FIG. 3A is a photograph of migration of rhSOD3 and 209E protein when treated with ZnCl 2 and PBS (control group), and FIGS. 3B and 3C are graphs obtained by digitizing the photograph of FIG. 3A.
도 3A의 결과에 따르면, 앞에서 언급한 결과와 유사하게도 ZnCl2를 처리한 군 중에서 rhSOD3 단백질을 처리한 실험군에서 다량의 rhSOD3 단백질이 세포질로 또는 핵 내부로 투과되어 축적된 것을 확인할 수 있었다. FITC 감도를 도식화한 그래프에서도, ZnCl2를 처리한 군에서 더욱 높은 세포 투과성 및 세포내 축적을 보였으며, 특히, rhSOD3를 처리한 실험군에서 많은 양의 rhSOD3가 세포 내부에 축적되었음을 확인할 수 있었다.According to the results of FIG. 3A, it was confirmed that the earlier-mentioned results and rhSOD3 Similarly, a large amount of protein in the test group treated with rhSOD3 protein in group treated with ZnCl 2 is transmitted to the cytoplasm or as a nuclear accumulation inside. In the graph plotted with FITC sensitivity, ZnCl 2- treated group showed higher cell permeability and intracellular accumulation. In particular, rhSOD3-treated group was found to accumulate large amounts of rhSOD3 in the cells.
rhSOD3의 항염증 효과Anti-inflammatory effect of rhSOD3
세포 내부로 투과하여 축적된 rhSOD3의 정상적으로 항염증 효과를 보이는지, 항염증 효과를 보인다면, 본 발명의 2가 금속이온 촉매제 ZnCl2를 확인하기 위한 실험을 실시하였다. 염증반응을 유도한 HaCaT 세포에 ZnCl2를 처리한 후 rhSOD3 단백질을 처리한 그룹과 그렇지 않은 그룹으로 나누어 NF-kB p65의 인산화 정도를 측정하였다. An experiment was conducted to confirm the divalent metal ion catalyst ZnCl 2 of the present invention if the rhSOD 3 permeated and accumulated inside the cell showed normal anti-inflammatory effect or anti-inflammatory effect. HaCaT cells induced by inflammation were treated with ZnCl 2 and then divided into rhSOD3-treated group and non-treated group, and the degree of phosphorylation of NF-kB p65 was measured.
그 결과, 도 4에 나타난 바와 같이, ZnCl2만을 처리한 군에서는 p-NF-kB p65 단백질의 양이 변화가 없었으나(도 4A), ZnCl2와 rhSOD3를 함께 처리한 군에서는 p-NF-kB p65 단백질이 ZnCl2의 농도 의존적으로 감소함을 알 수 있었다.As a result, as shown in Figure 4, in the group treated only with ZnCl 2 p-NF-kB p65, but the amount of protein did not change (Fig. 4A), the group treated with ZnCl 2 and rhSOD3 p-NF- kB p65 protein was decreased by ZnCl 2 concentration.
이것은 rhSOD3 단백질이 NF-kB p65 활성화를 억제한다는 것을 의미하며, ZnCl2의 농도의존적으로 NF-kB p65의 인산화가 억제되는 것을 의미한다.This means that the rhSOD3 protein inhibits NF-kB p65 activation, which means that the phosphorylation of NF-kB p65 is inhibited in a concentration-dependent manner by ZnCl 2 .
ZnClZnCl 22 의 염증 억제 효과Of inflammation
HaCaT 세포에 rhSOD3 또는 209E 단백질을 처리하고, 그 후 ZnCl2를 첨가한 그룹과 그렇지 않은 그룹으로 나누어 염증 자극을 주어 stat1과 stat3의 인산화를 관찰하고, 염증시 많이 나타나는 사이토카인인 IL-1β, IL-1α, TNFα, IL-8 및 IL-6의 mRNA 발현량을 측정하였다.Handle rhSOD3 or 209E protein in HaCaT cells, and then a given inflammatory stimuli divided into groups that are not a group the addition of ZnCl 2 cytokines appear much when observing the phosphorylation of stat1 and stat3, and inflammation, IL-1β, IL -1α, TNFα, IL-8, and IL-6 were measured.
그 결과 도 5에 보이는 바와 같이, rhSOD3 단백질을 처리한 HaCaT 세포에서는 stat3와 stat1의 인산화가 효과적으로 억제되었음을 확인할 수 있었다. 특히, rhSOD3 단백질에 촉매활성제인 ZnCl2를 처리할 때 더욱 효과적으로 염증반응을 억제하는 것을 확인할 수 있었다. 반면, 209E 단백질을 처리한 HaCaT 세포에서는 stat3와 stat1의 인산화를 유의미한 억제효과를 확인할 수 없었다. As a result, as shown in Fig. 5, it was confirmed that HaCaT cells treated with rhSOD3 protein effectively inhibited the phosphorylation of stat3 and stat1. In particular, when rhSOD3 protein was treated with ZnCl 2 , which is a catalytic activator, it was confirmed that the inflammatory reaction was inhibited more effectively. On the other hand, in HaCaT cells treated with 209E protein, significant inhibition of stat3 and stat1 phosphorylation could not be confirmed.
마찬가지로 염증성 사이토카인의 mRNA 발현량 실험결과에서도 도 6에 보이는 바와 같이, rhSOD3와 ZnCl2를 함께 처리한 HaCa T 세포에서 염증성 사이토카인의 발현이 효과적으로 감소함을 확인할 수 있었다.Similarly, as shown in FIG. 6, the expression level of inflammatory cytokine mRNA was effectively reduced in HaCa T cells treated with rhSOD3 and ZnCl 2 .
본 발명은 세포외 분비 슈퍼옥사이드 디스뮤테이즈(EC-SOD 또는 SOD3) 단백질의 세포 투과성을 증가시키는 방법 및 EC-SOD 단백질의 세포내 축적량을 증가시키는 방법을 제공한다. 이는 염증성 질환의 치료시 2가 아연이온을 EC-S0D 단백질과 함께 처리함으로써 더욱 효과적인 염증성질환 치료 효과를 기대 할 수 있으며, 다양한 의약 및 화장품 산업에 적용될 수 있어 산업상 이용가능성이 높다.The present invention provides a method for increasing the cellular permeability of extracellular secretory superoxide dismutase (EC-SOD or SOD3) protein and a method for increasing the intracellular accumulation of EC-SOD protein. It is expected that treatment of inflammatory diseases with divalent zinc ions together with EC-S0D protein can be more effective for treating inflammatory diseases and can be applied to various medicines and cosmetics industries, thus being highly industrially applicable.
<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Method of increasing cytopermeability of extracellular superoxide dismutase <130> NP14-0122 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> EC-SOD sequence <400> 1 atgctggcgc tactgtgttc ctgcctgctc ctggcagccg gtgcctcgga cgcctggacg 60 ggcgaggact cggcggagcc caactctgac tcggcggagt ggatccgaga catgtacgcc 120 aaggtcacgg agatctggca ggaggtcatg cagcggcggg acgacgacgg cacgctccac 180 gccgcctgcc aggtgcagcc gtcggccacg ctggacgccg cgcagccccg ggtgaccggc 240 gtcgtcctct tccggcagct tgcgccccgc gccaagctcg acgccttctt cgccctggag 300 ggcttcccga ccgagccgaa cagctccagc cgcgccatcc acgtgcacca gttcggggac 360 ctgagccagg gctgcgagtc caccgggccc cactacaacc cgctggccgt gccgcacccg 420 cagcacccgg gcgacttcgg caacttcgcg gtccgcgacg gcagcctctg gaggtaccgc 480 gccggcctgg ccgcctcgct cgcgggcccg cactccatcg tgggccgggc cgtggtcgtc 540 cacgctggcg aggacgacct gggccgcggc ggcaaccagg ccagcgtgga gaacgggaac 600 gcgggccggc ggctggcctg ctgcgtggtg ggcgtgtgcg ggcccgggct ctgggagcgc 660 caggcgcggg agcactcaga gcgcaagaag cggcggcgcg agagcgagtg caaggccgcc 720 tga 723 <210> 2 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> EC-SOD protein <400> 2 Met Leu Ala Leu Leu Cys Ser Cys Leu Leu Leu Ala Ala Gly Ala Ser 1 5 10 15 Asp Ala Trp Thr Gly Glu Asp Ser Ala Glu Pro Asn Ser Asp Ser Ala 20 25 30 Glu Trp Ile Arg Asp Met Tyr Ala Lys Val Thr Glu Ile Trp Gln Glu 35 40 45 Val Met Gln Arg Arg Asp Asp Asp Gly Thr Leu His Ala Ala Cys Gln 50 55 60 Val Gln Pro Ser Ala Thr Leu Asp Ala Ala Gln Pro Arg Val Thr Gly 65 70 75 80 Val Val Leu Phe Arg Gln Leu Ala Pro Arg Ala Lys Leu Asp Ala Phe 85 90 95 Phe Ala Leu Glu Gly Phe Pro Thr Glu Pro Asn Ser Ser Ser Arg Ala 100 105 110 Ile His Val His Gln Phe Gly Asp Leu Ser Gln Gly Cys Glu Ser Thr 115 120 125 Gly Pro His Tyr Asn Pro Leu Ala Val Pro His Pro Gln His Pro Gly 130 135 140 Asp Phe Gly Asn Phe Ala Val Arg Asp Gly Ser Leu Trp Arg Tyr Arg 145 150 155 160 Ala Gly Leu Ala Ala Ser Leu Ala Gly Pro His Ser Ile Val Gly Arg 165 170 175 Ala Val Val Val His Ala Gly Glu Asp Asp Leu Gly Arg Gly Gly Asn 180 185 190 Gln Ala Ser Val Glu Asn Gly Asn Ala Gly Arg Arg Leu Ala Cys Cys 195 200 205 Val Val Gly Val Cys Gly Pro Gly Leu Trp Glu Arg Gln Ala Arg Glu 210 215 220 His Ser Glu Arg Lys Lys Arg Arg Arg Glu Ser Glu Cys Lys Ala Ala 225 230 235 240 <210> 3 <211> 681 <212> RNA <213> Artificial Sequence <220> <223> 209E mutant sequence <400> 3 atgctggcgc tactgtgttc ctgcctgctc ctggcagccg gtgcctcgga cgcctggacg 60 ggcgaggact cggcggagcc caactctgac tcggcggagt ggatccgaga catgtacgcc 120 aaggtcacgg agatctggca ggaggtcatg cagcggcggg acgacgacgg cacgctccac 180 gccgcctgcc aggtgcagcc gtcggccacg ctggacgccg cgcagccccg ggtgaccggc 240 gtcgtcctct tccggcagct tgcgccccgc gccaagctcg acgccttctt cgccctggag 300 ggcttcccga ccgagccgaa cagctccagc cgcgccatcc acgtgcacca gttcggggac 360 ctgagccagg gctgcgagtc caccgggccc cactacaacc cgctggccgt gccgcacccg 420 cagcacccgg gcgacttcgg caacttcgcg gtccgcgacg gcagcctctg gaggtaccgc 480 gccggcctgg ccgcctcgct cgcgggcccg cactccatcg tgggccgggc cgtggtcgtc 540 cacgctggcg aggacgacct gggccgcggc ggcaaccagg ccagcgtgga gaacgggaac 600 gcgggccggc ggctggcctg ctgcgtggtg ggcgtgtgcg ggcccgggct ctgggagcgc 660 caggcgcggg agcactcaga g 681 <210> 4 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> 209E mutant protein <400> 4 Met Leu Ala Leu Leu Cys Ser Cys Leu Leu Leu Ala Ala Gly Ala Ser 1 5 10 15 Asp Ala Trp Thr Gly Glu Asp Ser Ala Glu Pro Asn Ser Asp Ser Ala 20 25 30 Glu Trp Ile Arg Asp Met Tyr Ala Lys Val Thr Glu Ile Trp Gln Glu 35 40 45 Val Met Gln Arg Arg Asp Asp Asp Gly Thr Leu His Ala Ala Cys Gln 50 55 60 Val Gln Pro Ser Ala Thr Leu Asp Ala Ala Gln Pro Arg Val Thr Gly 65 70 75 80 Val Val Leu Phe Arg Gln Leu Ala Pro Arg Ala Lys Leu Asp Ala Phe 85 90 95 Phe Ala Leu Glu Gly Phe Pro Thr Glu Pro Asn Ser Ser Ser Arg Ala 100 105 110 Ile His Val His Gln Phe Gly Asp Leu Ser Gln Gly Cys Glu Ser Thr 115 120 125 Gly Pro His Tyr Asn Pro Leu Ala Val Pro His Pro Gln His Pro Gly 130 135 140 Asp Phe Gly Asn Phe Ala Val Arg Asp Gly Ser Leu Trp Arg Tyr Arg 145 150 155 160 Ala Gly Leu Ala Ala Ser Leu Ala Gly Pro His Ser Ile Val Gly Arg 165 170 175 Ala Val Val Val His Ala Gly Glu Asp Asp Leu Gly Arg Gly Gly Asn 180 185 190 Gln Ala Ser Val Glu Asn Gly Asn Ala Gly Arg Arg Leu Ala Cys Cys 195 200 205 Val Val Gly Val Cys Gly Pro Gly Leu Trp Glu Arg Gln Ala Arg Glu 210 215 220 His Ser Glu 225 <110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Method of increasing cytoperability of extracellular superoxide dismutase <130> NP14-0122 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> EC-SOD sequence <400> 1 atgctggcgc tactgtgttc ctgcctgctc ctggcagccg gtgcctcgga cgcctggacg 60 ggcgaggact cggcggagcc caactctgac tcggcggagt ggatccgaga catgtacgcc 120 aaggtcacgg agatctggca ggaggtcatg cagcggcggg acgacgacgg cacgctccac 180 gccgcctgcc aggtgcagcc gtcggccacg ctggacgccg cgcagccccg ggtgaccggc 240 gtcgtcctct tccggcagct tgcgccccgc gccaagctcg acgccttctt cgccctggag 300 ggcttcccga ccgagccgaa cagctccagc cgcgccatcc acgtgcacca gttcggggac 360 ctgagccagg gctgcgagtc caccgggccc cactacaacc cgctggccgt gccgcacccg 420 cagcacccgg gcgacttcgg caacttcgcg gtccgcgacg gcagcctctg gaggtaccgc 480 gccggcctgg ccgcctcgct cgcgggcccg cactccatcg tgggccgggc cgtggtcgtc 540 cacgctggcg aggacgacct gggccgcggc ggcaaccagg ccagcgtgga gaacgggaac 600 gcgggccggc ggctggcctg ctgcgtggtg ggcgtgtgcg ggcccgggct ctgggagcgc 660 caggcgcggg agcactcaga gcgcaagaag cggcggcgcg agagcgagtg caaggccgcc 720 tga 723 <210> 2 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> EC-SOD protein <400> 2 Met Leu Ala Leu Leu Cys Ser Cys Leu Leu Leu Ala Ala Gly Ala Ser 1 5 10 15 Asp Ala Trp Thr Gly Glu Asp Ser Ala Glu Pro Asn Ser Asp Ser Ala 20 25 30 Glu Trp Ile Arg Asp Met Tyr Ala Lys Val Thr Glu Ile Trp Gln Glu 35 40 45 Val Met Gln Arg Arg Asp Asp Asp Gly Thr Leu His Ala Ala Cys Gln 50 55 60 Val Gln Pro Ser Ala Thr Leu Asp Ala Gln Pro Arg Val Thr Gly 65 70 75 80 Val Val Leu Phe Arg Gln Leu Ala Pro Arg Ala Lys Leu Asp Ala Phe 85 90 95 Phe Ala Leu Glu Gly Phe Pro Thr Glu Pro Asn Ser Ser Ser Ala 100 105 110 Ile His Val His Gln Phe Gly Asp Leu Ser Gln Gly Cys Glu Ser Thr 115 120 125 Gly Pro His Tyr Asn Pro Leu Ala Val Pro His Pro Gln His Pro Gly 130 135 140 Asp Phe Gly Asn Phe Ala Val Arg Asp Gly Ser Leu Trp Arg Tyr Arg 145 150 155 160 Ala Gly Leu Ala Ala Ser Leu Ala Gly Pro His Ser Ile Val Gly Arg 165 170 175 Ala Val Val Val His Ala Gly Glu Asp Asp Leu Gly Arg Gly Gly Asn 180 185 190 Gln Ala Ser Val Glu Asn Gly Asn Ala Gly Arg Arg Leu Ala Cys Cys 195 200 205 Val Val Gly Val Cys Gly Pro Gly Leu Trp Glu Arg Gln Ala Arg Glu 210 215 220 His Ser Glu Arg Lys Lys Arg Arg Arg Glu Ser Glu Cys Lys Ala Ala 225 230 235 240 <210> 3 <211> 681 <212> RNA <213> Artificial Sequence <220> <223> 209E mutant sequence <400> 3 atgctggcgc tactgtgttc ctgcctgctc ctggcagccg gtgcctcgga cgcctggacg 60 ggcgaggact cggcggagcc caactctgac tcggcggagt ggatccgaga catgtacgcc 120 aaggtcacgg agatctggca ggaggtcatg cagcggcggg acgacgacgg cacgctccac 180 gccgcctgcc aggtgcagcc gtcggccacg ctggacgccg cgcagccccg ggtgaccggc 240 gtcgtcctct tccggcagct tgcgccccgc gccaagctcg acgccttctt cgccctggag 300 ggcttcccga ccgagccgaa cagctccagc cgcgccatcc acgtgcacca gttcggggac 360 ctgagccagg gctgcgagtc caccgggccc cactacaacc cgctggccgt gccgcacccg 420 cagcacccgg gcgacttcgg caacttcgcg gtccgcgacg gcagcctctg gaggtaccgc 480 gccggcctgg ccgcctcgct cgcgggcccg cactccatcg tgggccgggc cgtggtcgtc 540 cacgctggcg aggacgacct gggccgcggc ggcaaccagg ccagcgtgga gaacgggaac 600 gcgggccggc ggctggcctg ctgcgtggtg ggcgtgtgcg ggcccgggct ctgggagcgc 660 caggcgcggg agcactcaga g 681 <210> 4 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> 209E mutant protein <400> 4 Met Leu Ala Leu Leu Cys Ser Cys Leu Leu Leu Ala Ala Gly Ala Ser 1 5 10 15 Asp Ala Trp Thr Gly Glu Asp Ser Ala Glu Pro Asn Ser Asp Ser Ala 20 25 30 Glu Trp Ile Arg Asp Met Tyr Ala Lys Val Thr Glu Ile Trp Gln Glu 35 40 45 Val Met Gln Arg Arg Asp Asp Asp Gly Thr Leu His Ala Ala Cys Gln 50 55 60 Val Gln Pro Ser Ala Thr Leu Asp Ala Gln Pro Arg Val Thr Gly 65 70 75 80 Val Val Leu Phe Arg Gln Leu Ala Pro Arg Ala Lys Leu Asp Ala Phe 85 90 95 Phe Ala Leu Glu Gly Phe Pro Thr Glu Pro Asn Ser Ser Ser Ala 100 105 110 Ile His Val His Gln Phe Gly Asp Leu Ser Gln Gly Cys Glu Ser Thr 115 120 125 Gly Pro His Tyr Asn Pro Leu Ala Val Pro His Pro Gln His Pro Gly 130 135 140 Asp Phe Gly Asn Phe Ala Val Arg Asp Gly Ser Leu Trp Arg Tyr Arg 145 150 155 160 Ala Gly Leu Ala Ala Ser Leu Ala Gly Pro His Ser Ile Val Gly Arg 165 170 175 Ala Val Val Val His Ala Gly Glu Asp Asp Leu Gly Arg Gly Gly Asn 180 185 190 Gln Ala Ser Val Glu Asn Gly Asn Ala Gly Arg Arg Leu Ala Cys Cys 195 200 205 Val Val Gly Val Cys Gly Pro Gly Leu Trp Glu Arg Gln Ala Arg Glu 210 215 220 His Ser Glu 225
Claims (7)
A method of increasing the cellular permeability of an EC-SOD protein comprising contacting an EC-SOD (Extracellular superoxide dismutase) protein comprising a heparin binding domain with a zinc divalent cation.
2. The method according to claim 1, wherein the EC-SOD protein is represented by SEQ ID NO: 2.
A pharmaceutical composition for the prophylaxis or treatment of an inflammatory disease comprising an EC-SOD (Extracellular superoxide dismutase) protein comprising a heparin binding domain and a zinc divalent cation as an active ingredient.
The method according to claim 3, wherein the inflammatory disease is selected from the group consisting of dermatitis, allergy, atopic dermatitis, acne, injection, squamous cell carcinoma, scleroderma, pigmented skin disease, watery skin disease, asthma, conjunctivitis, dry eye, diabetic retinopathy, Rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, Wherein the composition is any one selected from the group consisting of cachexia, nephritis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, chronic kidney disease, sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases.
An extracellular superoxide dismutase (EC-SOD) protein comprising a heparin-binding domain and a zinc divalent cation as an active ingredient.
A cosmetic composition for the treatment of inflammatory skin diseases, comprising an EC-SOD (Extracellular superoxide dismutase) protein comprising a heparin-binding domain and a zinc divalent cation as an active ingredient.
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| BBRC, Vol. 348, pp. 450-458 (2006) |
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| WO2024096540A1 (en) * | 2022-10-31 | 2024-05-10 | 재단법인 오송첨단의료산업진흥재단 | Method for producing recombinant sod3 protein |
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