KR101627920B1 - Development of a novel emphysema-related animal model using insulin-like growth factor 2 (IGF2) - Google Patents
Development of a novel emphysema-related animal model using insulin-like growth factor 2 (IGF2) Download PDFInfo
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- KR101627920B1 KR101627920B1 KR1020140088396A KR20140088396A KR101627920B1 KR 101627920 B1 KR101627920 B1 KR 101627920B1 KR 1020140088396 A KR1020140088396 A KR 1020140088396A KR 20140088396 A KR20140088396 A KR 20140088396A KR 101627920 B1 KR101627920 B1 KR 101627920B1
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- igf2
- emphysema
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Abstract
본 발명은 폐기종 유발 동물 모델의 제조 방법에 관한 것으로, 보다 구체적으로, 인슐린 유사 인자 2(Insuline-like growth factor 2; IGF2) 단백질을 과발현시키는 단계를 포함하는, 폐기종 유발 동물 모델의 제조 방법에 관한 것이다. 본 발명의 방법에 의해 제조된 형질전환 동물은 기존 모델과 달리 폐 조직 중에서 폐 상피세포에서만 국소적으로 발현되는 IGF2에 의한 폐 병변을 확인할 수 있어 폐기종과 같은 폐질환을 연구하기에 적합할 뿐 아니라, 폐기종의 치료 및 예방을 위한 새로운 분자지표로써, 폐기종 치료제를 스크리닝하기 위한 효과적인 모델 마우스로 이용할 수 있다. The present invention relates to a method of producing an emphysema-induced animal model, and more particularly to a method of producing an emphysematous animal model, comprising over-expressing insulin-like growth factor 2 (IGF2) will be. The transgenic animal produced by the method of the present invention is capable of confirming pulmonary lesions caused by IGF2 locally expressed only in pulmonary epithelial cells in lung tissues unlike the conventional model, As a new molecular marker for the treatment and prevention of emphysema, it can be used as an effective model mouse for screening for emphysema therapeutics.
Description
본 발명은 폐기종 동물 모델의 제조 방법 및 이를 이용한 폐기종 치료제 후보약물 스크리닝 방법에 관한 것이다.The present invention relates to a method for producing an emphysema animal model and a method for screening candidate drug candidates for emphysema using the same.
폐기종(emphysema)은 만성 폐쇄성 폐질환(chronic obstructive pulmonary disease; COPD) 중 하나로, 말단 세 기관지의 폐포 간 공간 벽이 탄성을 잃고 파괴되어 비정상적으로 영구 확장됨으로써, 폐의 산소-이산화탄소 교환 능력이 감소되어 호흡이 곤란해지는 질환이다. COPD는 흡연율 및 평균 수명의 연장으로 인하여 발병률 및 사망률이 증가하는 추세로서, 우리나라에서 10대 사망 원인 중 하나이다. 뿐만 아니라, 세계보건기구에서는 2020년 COPD가 전 세계 사망 원인 중 3위에 해당할 것으로 예측하였다. The emphysema is one of the chronic obstructive pulmonary disease (COPD), in which the space between the alveolar spaces of the distal bronchus loses elasticity and is destroyed, resulting in an abnormally permanent expansion, thereby reducing the oxygen-to-CO2 exchange capacity of the lung It is a disease that breathing becomes difficult. COPD is one of the leading causes of death in Korea, with an increasing incidence and mortality rate due to an increase in smoking rates and life expectancy. In addition, the World Health Organization predicted that COPD would be the third leading cause of death worldwide by 2020.
이러한 폐기종의 주요 원인은 흡연으로, 대기 오염이나 유해 작업 환경의 노출에 의해서도 생길 수 있으며, 호흡기 감염의 동반 시 진행 속도가 가속화된다. 폐기종의 증상인 조직 파괴로 인한 폐포 확장 증상은, matrix metalloproteinase (MMP), neutrophil elastase 등의 단백질 분해효소의 발현은 활성화되는 반면, 단백질 분해효소를 억제하는 α1-antitrypsin의 발현은 감소하기 때문인 것으로 알려졌다. 또한, 흡연에 의하여 폐포 내 대식세포 및 상피에서 여러 cytokine/chemokine의 분비가 유도되어 염증 반응이 활성화되면 염증 관련 세포들에 의한 cytokines/chemokines (TNF-α, IL-1β, IL-8, MCP-1, IFN-γ, IL-4, IL-13 등), reactive oxygen species (ROS) 등의 생성이 증가되어 단백질 분해효소의 발현 및 활성화로 인한 조직 손상이 가속화된다. The main causes of these emphysema are smoking, exposure to air pollution or exposure to harmful working conditions, and accelerated progression of respiratory infections. It is known that the alveolar expansion symptoms due to tissue destruction, which is a symptom of emphysema, are due to a decrease in the expression of α1-antitrypsin, which inhibits proteolytic enzymes, while the expression of proteolytic enzymes such as matrix metalloproteinase (MMP) and neutrophil elastase is activated . IL-1β, IL-8, MCP-1, IL-1β, IL-8, IL-8, and IL-8 in the alveolar macrophages and epithelium, respectively, 1, IFN-γ, IL-4, IL-13, etc.) and reactive oxygen species (ROS) are increased to accelerate tissue damage due to the expression and activation of proteolytic enzymes.
현재까지 폐기종에 대한 여러 병인이 알려지고 있으나, 아직까지 폐기종의 특성을 충분히 설명하지 못하고 있어 다른 요인에 의한 기전 연구가 필요한 실정이다.
To date, several etiologies of emphysema have been known, but the features of emphysema have yet to be fully explained.
한편, 인슐린 유사 성장인자(Insulin-like growth factor; IGF)는 아미노산의 조성 및 구조가 인슐린과 유사한 특성을 가지고 있으며, 정상 또는 암 세포에 대하여 성장을 촉진하고 사멸을 억제하는 인자로 작용한다. IGF는 70개의 아미노산으로 이루어진 IGF1과 67개의 아미노산으로 이루어진 IGF2로 나뉘며, IGF-1R, IGF-2R 또는 insulin receptor(IR)에 결합한다. IGF-1R 및 IR은 자체적으로 kinase 활성을 지니고 있어 IGF 결합에 의하여 autophosphorylation 되어 하위 신호를 전달하는 반면, IGF-2R은 이러한 활성을 지니고 있지 않아 IGF 매개 신호전달을 일으키지 않는다. IGF는 혈중 또는 세포 외액에서 IGF binding protein(IGFBP)에 결합하여 존재한다. 현재까지 6종의 IGFBP가 알려졌고 이들은 IGF를 다른 조직 또는 세포로 운반하거나 IGF가 수용체에 결합하는 것을 증가 또는 감소시켜 IGF의 작용을 조절하는 역할을 한다. Insulin-like growth factor (IGF), on the other hand, has an amino acid composition and structure similar to insulin, and promotes growth and suppresses the death of normal or cancer cells. IGF is composed of IGF1 consisting of 70 amino acids and IGF2 consisting of 67 amino acids and binds to IGF-1R, IGF-2R or insulin receptor (IR). IGF-1R and IR possess kinase activity by themselves and are autophosphorylated by IGF binding to transmit lower-level signals, whereas IGF-2R does not have such an activity and does not cause IGF-mediated signal transduction. IGF is bound to IGF binding protein (IGFBP) in blood or extracellular fluid. To date, six IGFBPs have been identified, which play a role in regulating the action of IGF by either transporting IGF to other tissues or cells or by increasing or decreasing the binding of IGF to the receptor.
IGF-1R signaling은 세포 성장, 분화를 조절하고 세포 사멸을 억제하고, 여러 암 종에서 IGF-1R signaling이 활성화되는 것으로 나타났다. 특히 IGF2는 암세포에 과발현되어 있는 IR의 isoform인 IR-A에 결합력이 높아 IR-A를 통하여 세포 성장 촉진 신호전달을 활성화시키는 것으로 나타났다. IGF-1R signaling regulates cell growth and differentiation, inhibits apoptosis, and IGF-1R signaling is activated in many cancer types. In particular, IGF2 has a high binding affinity to IR-A, an isoform of IR that is overexpressed in cancer cells, and thus activates cell growth promoting signaling through IR-A.
현재까지 폐기종 조직에서 IGFBP-3의 발현이 증가되어 있음이 알려져 있으나, IGF-1R signaling 또는 다른 IGF-1R signaling 관련 요소, 특히 IGF2와 폐기종과의 관련성에 대해서는 아직 보고된 바 없다. It is known that the expression of IGFBP-3 is increased in emphysema tissue, but the relationship between IGF-1R signaling or other IGF-1R signaling-related factors, especially IGF2, has not been reported.
본 발명자들은 기존 연구에서 IGF-1R signaling이 폐 암화 과정에서 활성화됨을 발표하였고(Kim et al., Cancer Res 2009;69:7439-7448), 예비 실험 결과 흡연 경력이 있는 폐암 환자의 조직에서 IGF-1R의 활성화 및 IGF1, IGF2의 발현이 증가하며, 특히 IGF1, IGF2와 IGF-1R의 활성화 정도가 유의적인 상관성이 있음을 확인하였다. 이는 담배 중 발암 성분에 의하여 IGF의 생성을 통하여 IGF-1R의 활성화를 유도할 수 있음을 나타내므로, 흡연에 의하여 유발되며 폐암 발병과도 관련이 있는 COPD에서도 적용될 수 있음을 시사한다. The present inventors have previously reported that IGF-1R signaling is activated in pulmonary carcinogenesis (Kim et al., Cancer Res 2009; 69: 7439-7448). As a result of preliminary experiments, 1R and the expression of IGF1 and IGF2 were increased. Especially, the degree of activation of IGF1, IGF2 and IGF-1R was significantly correlated. This suggests that it is possible to induce the activation of IGF-1R through the production of IGF by the carcinogenic component in tobacco smoke, so that it can be applied to COPD induced by smoking and also related to lung cancer.
기존 폐기종 마우스 모델은 담배 연기에 노출시키는 방법, 활성화된 염증 관련 세포를 주입하는 방법, elastase와 같은 폐기종 유발 효소를 처리하는 방법, 식이를 중단하거나 이를 elastase 처리와 병용하는 방법, LPS와 같은 염증유발 인자를 처리하는 방법, MMP-1, α1-antitrypsin 등 폐기종 관련 인자들의 발현을 조절한 유전자변형 마우스를 활용하는 방법 등이 알려져 있으나, protease 외 세포신호전달 관련 마커를 활용한 모델은 아직 제시되지 않았으며 특히 폐기종 관련 여러 모델에서 IGF, 특히 IGF2와의 상관성은 보고된 바 없다.
Existing emphysema mouse models include, but are not limited to, exposure to cigarette smoke, injection of activated inflammatory cells, treatment of emphysema-inducing enzymes such as elastase, stopping the diet or using it in combination with elastase treatment, A method of treating factors, and a method of utilizing a genetically modified mouse that regulates the expression of stress-related factors such as MMP-1 and α1-antitrypsin. However, a model utilizing a cell signaling-related marker outside of protease has not been proposed yet In particular, no correlation has been reported with IGF, especially IGF2, in various models of emphysema.
이에, 본 발명자들은 폐기종의 모델 동물을 제조하기 위해 노력하던 중 인슐린 유사 성장 인자 2(IGF2)가 과발현된 형질전환 마우스의 폐 조직에서 폐기종의 특징적인 현상이 현저히 나타남을 확인함으로써, 이를 통해, IGF2 과발현 형질전환 마우스를 폐기종 모델 마우스로 유용하게 이용할 수 있음을 밝힘으로써, 본 발명을 완성하였다. Accordingly, the inventors of the present invention have made efforts to produce model animals of emphysema, and confirmed that the characteristic phenomenon of emphysema appears in the lung tissue of transgenic mice overexpressing insulin-like growth factor 2 (IGF2) Overexpressing transgenic mice can be usefully used in emphysema model mice, thus completing the present invention.
본 발명은 상기와 같은 종래 기술상의 문제점을 해결하기 위해 안출된 것으로, 인슐린 유사 성장 인자 2(insuline-like growth factor 2; IGF2) 단백질을 과발현시키는 단계를 포함하는 폐기종 유발 동물 모델의 제조 방법; 및 상기 방법에 의해 제조된 동물 모델을 이용한 폐기종 치료제 스크리닝 방법을 제공하는 것을 그 목적으로 한다.Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a method of producing an emphysemotic animal model comprising over-expressing an insulin-like growth factor 2 (IGF2) protein. And an object of the present invention is to provide a method for screening for the treatment of emphysema using the animal model prepared by the above method.
상기 목적을 달성하기 위하여, 본 발명은 인슐린 유사 성장 인자 2(Insuline-like growth factor 2; IGF2) 단백질을 과발현시키는 단계를 포함하는 폐기종 유발 동물 모델의 제조 방법을 제공한다. In order to achieve the above object, the present invention provides a method for producing an emphysemotic animal model comprising over-expressing insulin-like growth factor 2 (IGF2) protein.
본 발명의 일 구현예로, 상기 IGF2 단백질을 과발현시키는 단계는 동물의 폐 상피세포에서 이루어지는 것을 특징으로 한다. In one embodiment of the present invention, the step of overexpressing the IGF2 protein is characterized in that it is carried out in lung epithelial cells of an animal.
본 발명의 다른 구현예로, 상기 IGF2 단백질은 서열번호 1로 기재되는 아미노산 서열을 포함하는 것을 특징으로 한다.In another embodiment of the present invention, the IGF2 protein comprises the amino acid sequence shown in SEQ ID NO: 1.
본 발명의 또 다른 구현예로, 상기 IGF2 단백질은 서열번호 2로 기재되는 염기 서열을 포함하는 핵산 서열을 통해 인코딩되는 것을 특징으로 한다.In another embodiment of the present invention, the IGF2 protein is encoded through a nucleic acid sequence comprising the nucleotide sequence shown in SEQ ID NO: 2.
본 발명의 또 다른 구현예로, 상기 IGF2 단백질은 surfactant protein C (SPC) 프로모터 유전자와 작동가능하게 연결된 것을 특징으로 한다.In another embodiment of the present invention, the IGF2 protein is operably linked to a surfactant protein C (SPC) promoter gene.
본 발명의 또 다른 구현예로, 상기 surfactant protein C (SPC) promoter 유전자는 서열번호 3의 폴리뉴클레오티드 서열을 포함하는 것을 특징으로 한다. In another embodiment of the present invention, the surfactant protein C (SPC) promoter gene comprises the polynucleotide sequence of SEQ ID NO: 3.
본 발명의 또 다른 구현예로, 상기 폐기종 유발 동물은 마우스인 것을 특징으로 한다.In another embodiment of the present invention, the emphysema-inducing animal is a mouse.
본 발명의 또 다른 구현예로, 상기 마우스는 IGF2 단백질을 hybrid C3H/C57B6 fertilized mouse egg에 microinjection하여 제조되는 것을 특징으로 한다.In another embodiment of the present invention, the mouse is characterized in that IGF2 protein is produced by microinjection into a hybrid C3H / C57B6 fertilized mouse egg.
아울러, 본 발명은 상기 방법에 의해 제조된 동물 모델을 이용한 폐기종 치료제 후보약물 스크리닝 방법을 제공한다. In addition, the present invention provides a method for screening candidate drug candidates for emphysema using the animal model produced by the above method.
본 발명의 일 구현예로, 상기 방법은 하기의 단계를 포함하는 것을 특징으로 한다:In one embodiment of the invention, the method is characterized in that it comprises the steps of:
1) 상기 본 발명의 방법에 의해 제조된 동물에 피검 화합물 또는 조성물을 투여하는 단계;1) administering the test compound or composition to the animal produced by the method of the present invention;
2) 상기 피검 화합물 또는 조성물이 처리된 동물(실험군)과 피검 화합물 또는 조성물을 미처리한 동물(대조군)로부터 각각 폐기종의 증상을 측정하는 단계; 및 2) measuring the symptoms of the emphysema from the animal (experimental group) treated with the test compound or the composition and the test compound or the animal (control group) not treated with the composition, respectively; And
3) 대조군에 비하여 실험군에서 폐기종의 증상이 감소된 피검 화합물 또는 조성물을 선별하는 단계. 3) Selecting the test compound or composition with reduced symptoms of emphysema in the experimental group compared to the control group.
본 발명의 다른 구현예로, 상기 단계 1)의 피검화합물 또는 조성물은 천연화합물, 합성화합물, RNA, DNA, 폴리펩티드, 효소, 단백질, 리간드, 항체, 항원, 박테리아 또는 진균의 대사 산물 및 생활성 분자로 이루어진 군으로부터 선택되는 어느 하나인 것을 특징으로 한다. In another embodiment of the present invention, the test compound or composition of step 1) is a natural compound, a synthetic compound, RNA, DNA, a polypeptide, an enzyme, a protein, a ligand, an antibody, an antigen, a bacterial or fungal metabolite, And the like.
본 발명의 방법에 의해 제조된 동물 모델은 기존 모델과 달리 IGF2를 폐 특이적으로 과발현되도록 유전자변형 마우스로 제작한 것이어서 기존의 담배 연기 노출, 활성화된 염증 관련 세포 주입, elastase 같은 폐기종 유발 효소 처리, LPS와 같은 염증유발 인자를 처리하는 방법 등에 의해 폐기종을 유발하는 모델에 비해 동물 개체 간 차이가 거의 없고, 단시간에 폐기종을 유발시킴으로써 비용이 저렴한 장점이 있다. 또한 폐기종의 원인으로 지목되는 담배의 특정 성분에 의해 폐 상피세포에서 발현이 증가되는 것으로 확인된 IGF2를 타겟으로 하고 있어서 폐기종의 병변 과정을 연구하는데 적합하다. 뿐만 아니라, 폐기종의 발생 및 진행 과정에서 IGF2의 발현조절에 관련된 기작 및 biomarker들을 규명함으로써 폐기종 및 COPD와 관련된 새로운 지표 발굴에도 활용될 수 있다. 또한 발굴된 biomarker들을 이용, 폐기종의 치료 및 예방을 대상으로 하는 신약 개발에 이용가능한 마우스 모델로도 이용할 수 있다.The animal model produced by the method of the present invention is prepared by genetically modified mouse to overexpress pulmonary-specific IGF2 unlike the conventional model, and it can be used for the treatment of exposure to tobacco smoke, activated inflammation-related cell injection, Compared with the model that induces emphysema by the method of treating inflammation inducers such as LPS, there is little difference between animals, and it is advantageous in cost because it induces emphysema in a short time. In addition, IGF2, which has been shown to increase expression in lung epithelial cells by specific components of tobacco, is identified as the cause of emphysema, and is suitable for studying the pathology of emphysema. In addition, identification of biomarkers and mechanisms involved in the regulation of IGF2 expression during the development and progression of emphysema can be exploited to identify new markers related to emphysema and COPD. It can also be used as a mouse model for the development of new drugs for the treatment and prevention of emphysema using excised biomarkers.
도 1은 본 발명의 IGF2 과발현 형질전환 마우스에서 적출된 폐의 폐기종 유발 여부를 확인하기 위한, 폐 조직의 사진 및 H&E 염색 결과이다.
도 2는 본 발명의 IGF2 과발현 형질전환 마우스에서 적출된 폐의 폐기종 유발 여부를 확인하기 위한, 폐 조직의 Masson's trichrome 염색 결과이다.
도 3은 본 발명의 IGF2 과발현 형질전환 마우스에서 적출된 폐의 폐기종 유발 여부를 확인하기 위한, 폐 조직의 사진이다.
도 4는 본 발명의 IGF2 과발현 형질전환 마우스의 폐조직에서 단백분해효소 MMP(matrix metalloproteinase)의 활성도 및 발현 정도를 확인하기 위한, in situ Zymography 및 gelatin Zymography 결과이다.
도 5는 본 발명의 IGF2 과발현 형질전환 마우스의 폐조직에서 α1-antitrypsin 및 MMP 단백질의 발현 변화를 확인하기 위한, 웨스턴 블랏팅 및 정량화 결과이다.
도 6은 폐 상피세포에서 IGF2 과발현에 의하여 유도된 폐 조직 손상 유발 효소인 MMP-1이 IGF2 발현 억제에 의하여 그 발현이 감소되는 것을 RT-PCR로 확인한 결과이다.
도 7은 폐 상피세포(BEAS-2B)에서 IGF2 발현을 Knock-down에 의해 저하(K/D)시키거나 과발현(O/E)시킨 후 α1-antitrypsin의 발현 변화를 확인하기 위한, RT-PCR 결과이다.
도 8은 담배 유래 발암원인 NNK의 장기간 동안의 처리(20, 30, 100일)에 의하여 사람 폐 상피세포인 HBEC 및 NHBE에서 폐기종과 연관된 것으로 알려진 IGFBP-3와 더불어 IGF2의 유전자 발현 변화를 RT-PCR로 확인한 결과이다.
도 9는 담배 유래 발암원인 NNK의 장기간 동안의 처리(7~150일)에 의하여 사람 폐 상피세포인 HBEC1과 BEAS-2B에서 IGF2의 processing에 관여하는 효소인 furin, PC-1 및 PC-7의 유전자 발현 변화를 RT-PCR로 확인한 결과이다.
도 10은 NNK 및 IGF2에 의하여 사람 폐 상피세포에서 furin의 발현이 증가하고, IGF2가 과발현된 조건에서 NNK를 처리하면 furin의 발현이 촉진됨을 RT-PCR로 확인한 결과이다.
도 11a 및 11b는 담배 유래 발암원인 NNK의 장기간 동안의 처리(20~150일)에 의하여 사람 폐 상피세포인 HBEC1과 BEAS-2B에서 폐 조직 손상을 유발하는 효소인 MMP-1, MMP-9, MMP-10, MMP-12, MMP-13의 유전자 발현 변화를 RT-PCR 및 웨스턴 블랏팅으로 확인한 결과이다.
도 12는 담배 유래 발암원인 NNK를 사람 폐 상피세포인 HBEC1과 BEAS-2B에 처리시, 항단백분해효소인 α1-antitrypsin의 발현이 감소되는 것을 RT-PCR로 확인한 결과이다.
도 13은 담배 유래 발암원인 NNK과 benzo[a]pyrene을 사람 폐 상피세포인 BEAS-2B에 병용처리시(NNK/B(a)P), α1-antitrypsin 발현 감소 및 MMP 발현 증가를 웨스턴 블랏팅으로 확인한 결과이다.
도 14는 마우스에 폐기종을 유발하는 담배 주요성분인 NNK과 benzo[a]pyrene를 병용투여(NNK/B(a)P)한 후, 폐조직을 적출하여 H&E 염색을 통하여 폐기종 유발 여부를 확인한 결과이다.
도 15는 마우스에 폐기종을 유발하는 담배 주요성분인 NNK과 benzo[a]pyrene를 병용투여(NNK/B(a)P)한 후, 폐조직을 적출하여 IGF2 단백질의 발현 변화를 웨스턴 블롯팅으로 확인한 결과이다.
도 16은 마우스에 폐기종을 유발하는 담배 주요성분인 NNK과 benzo[a]pyrene를 병용투여(NNK/B(a)P)한 후, 폐조직을 적출하여 α1-antitrypsin 및 MMP 단백질의 발현 변화를 RT-PCR, 웨스턴 블롯팅, 및 gelatin zymography로 확인한 결과이다.Brief Description of the Drawings Fig. 1 is a photograph of lung tissue and H & E staining results for confirming the induction of pulmonary emphysema induced by the IGF2 transgenic mouse of the present invention.
FIG. 2 is a result of masson's trichrome staining of lung tissue to confirm the induction of pulmonary emphysema extracted from the IGF2 transgenic mouse of the present invention.
Fig. 3 is a photograph of lung tissue for confirming the induction of emphysema of the lungs extracted from the IGF2 transgenic mouse of the present invention.
4 is to determine the degree of activity and expression of the protease MMP (matrix metalloproteinase) in the lung tissue of the transgenic mouse IGF2 expression of the invention, in situ Zymography and gelatin Zymography results.
FIG. 5 shows Western blotting and quantification results for confirming the expression changes of? 1-antitrypsin and MMP protein in the lung tissue of the IGF2-overexpressing mouse of the present invention.
FIG. 6 shows the results of RT-PCR showing that the expression of MMP-1, which is an inducer of pulmonary tissue damage induced by IGF2 overexpression in lung epithelial cells, is reduced by inhibition of IGF2 expression.
FIG. 7 shows the results of RT-PCR to confirm the expression of α1-antitrypsin after knockdown (K / D) or overexpression (O / E) of IGF2 expression in pulmonary epithelial cells (BEAS-2B) Results.
FIG. 8 shows changes in gene expression of IGF2 in addition to IGFBP-3, which is known to be associated with emphysema, in human lung epithelial cells, HBEC and NHBE, by long-term treatment of NNK (tobacco- PCR.
FIG. 9 is a graph showing the effect of furin, PC-1 and PC-7, which are enzymes involved in the processing of IGF2, in human lung epithelial cells HBEC1 and BEAS-2B by long-term treatment (7 to 150 days) of NNK, The changes in gene expression were confirmed by RT-PCR.
FIG. 10 shows the results of RT-PCR that furin expression was enhanced by NNK and IGF2, and furin expression was accelerated by NNK treatment when IGF2 was overexpressed.
FIGS. 11A and 11B are graphs showing changes in MMP-1, MMP-9, and MMP-9, enzymes inducing lung tissue damage in human lung epithelial cells, HBEC1 and BEAS-2B, by long-term treatment (20 to 150 days) of NNK, MMP-10, MMP-12 and MMP-13 gene expression by RT-PCR and Western blotting.
FIG. 12 shows the results of RT-PCR for the reduction of the expression of α1-antitrypsin, an anti-protease, in human lung epithelial cells, HBEC1 and BEAS-2B, which is a cancer-causing NNK.
FIG. 13 shows the results of a decrease in the expression of α1-antitrypsin and an increase in MMP expression in Western blotting (NNK / B (a) P) when NNK and benzo [a] pyrene which are tobacco- .
FIG. 14 shows the results of a combination of NNK and benzo [a] pyrene (NNK / B (a) P), a major component of tobacco that induces emphysema in mice, followed by H & E staining to be.
FIG. 15 shows the results of Western blotting of changes in IGF2 protein expression by extracting lung tissue (NNK / B (a) P) after the combination of NNK and benzo [a] pyrene, The result is confirmed.
FIG. 16 is a graph showing changes in expression of a1-antitrypsin and MMP protein after lung necrosis (NNK / B (a) P) in combination with NNK and benzo [a] pyrene, RT-PCR, Western blotting, and gelatin zymography.
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명은 인슐린 유사 성장 인자 2(Insuline-like growth factor 2; IGF2) 단백질을 과발현시키는 단계를 포함하는 폐기종 유발 동물 모델의 제조 방법을 제공한다. The present invention provides a method of producing an emphysematous animal model comprising over-expressing an Insuline-like growth factor 2 (IGF2) protein.
상기 IGF2 단백질을 과발현시키는 단계는 동물의 폐 상피세포에서 이루어지는 것이 바람직하나, 이에 한정되지 않는다. The step of overexpressing the IGF2 protein is preferably performed in lung epithelial cells of an animal, but is not limited thereto.
상기 IGF2 단백질은 서열번호 1로 기재되는 아미노산 서열을 포함하는 것이 바람직하나, 이에 한정되지 않는다. The IGF2 protein preferably comprises the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
상기 IGF2 단백질은 서열번호 2로 기재되는 염기 서열을 포함하는 핵산 서열을 통해 인코딩되는 것이 바람직하나, 이에 한정되지 않는다.The IGF2 protein is preferably encoded through a nucleic acid sequence comprising the nucleotide sequence shown in SEQ ID NO: 2, but is not limited thereto.
상기 IGF2 단백질은 surfactant protein C (SPC) 프로모터 유전자와 작동가능하게 연결된 것을 특징으로 한다.Wherein the IGF2 protein is operably linked to a surfactant protein C (SPC) promoter gene.
상기 surfactant protein C (SPC) promoter 유전자는 서열번호 3의 폴리뉴클레오티드 서열을 포함하는 것이 바람직하나, 이에 한정되지 않는다. The surfactant protein C (SPC) promoter gene preferably comprises the polynucleotide sequence of SEQ ID NO: 3, but is not limited thereto.
또한, 본 발명은 상기 방법에 의해 제조된 동물 모델을 제공한다. The present invention also provides an animal model produced by the above method.
상기 동물의 종류로는 마우스, 랫트(rat), 소, 말, 돼지, 원숭이, 오리, 개, 고양이 등이 될 수 있고, 마우스인 것이 바람직하나, 이에 한정되는 것은 아니다. The type of the animal may be a mouse, a rat, a cow, a horse, a pig, a monkey, a duck, a dog, a cat, and the like, but is not limited thereto.
또한, 상기 마우스는 IGF2 단백질을 hybrid C3H/C57B6 fertilized mouse egg에 microinjection하여 제조되는 것이 바람직하나, 이에 한정되는 것은 아니다.
In addition, the mouse is preferably prepared by microinjecting IGF2 protein into a hybrid C3H / C57B6 fertilized mouse egg, but is not limited thereto.
아울러, 본 발명은 상기 방법에 의해 제조된 동물 모델을 이용한 폐기종 동물 모델 스크리닝 방법을 제공한다. In addition, the present invention provides a method for screening an emphysema animal model using the animal model produced by the method.
상기 방법은 하기의 단계를 포함하는 것이 바람직하나, 이에 한정되는 것은 아니다:The method preferably includes, but is not limited to, the following steps:
1) 상기 본 발명의 방법에 의해 제조된 동물에 피검 화합물 또는 조성물을 투여하는 단계;1) administering the test compound or composition to the animal produced by the method of the present invention;
2) 상기 피검 화합물 또는 조성물이 처리된 동물(실험군)과 피검 화합물 또는 조성물을 미처리한 동물(대조군)로부터 각각 폐기종의 증상을 측정하는 단계; 및 2) measuring the symptoms of the emphysema from the animal (experimental group) treated with the test compound or the composition and the test compound or the animal (control group) not treated with the composition, respectively; And
3) 대조군에 비하여 실험군에서 폐기종의 증상이 감소된 피검 화합물 또는 조성물을 선별하는 단계. 3) Selecting the test compound or composition with reduced symptoms of emphysema in the experimental group compared to the control group.
상기 단계 1)의 피검화합물 또는 조성물은 천연화합물, 합성화합물, RNA, DNA, 폴리펩티드, 효소, 단백질, 리간드, 항체, 항원, 박테리아 또는 진균의 대사 산물 및 생활성 분자로 이루어진 군으로부터 선택되는 어느 하나인 것이 바람직하나, 이에 한정되지 않는다.
The test compound or composition of step 1) may be any one selected from the group consisting of a natural compound, a synthetic compound, RNA, DNA, a polypeptide, an enzyme, a protein, a ligand, an antibody, an antigen, a bacterial or fungal metabolite, But is not limited thereto.
본 발명의 구체적인 실시예에서, alveolar type II 상피세포에 특징적으로 발현하는 surfactant protein C (SPC) promoter를 사용하여 폐 상피세포에만 IGF2가 발현되도록 한 형질전환 마우스를 제작하였으며, 이를 통해 기존 모델과 달리 폐 선택적/국소적으로 발현되는 IGF2에 의한 폐기종을 확인할 수 있었다. In a specific example of the present invention, a transgenic mouse was produced by expressing IGF2 only in lung epithelial cells using a surfactant protein C (SPC) promoter that is expressed in alveolar type II epithelial cells. Pulmonary selective / localized IGF2-induced emphysema could be identified.
또한, IGF2 과발현 형질전환 마우스에서 폐기종의 특징적인 현상인 collagen 조직의 파괴, 단백분해효소인 MMP-9의 발현 증가 및 항단백분해효소인 α1-antitrypsin의 발현 감소가 나타남을 확인하였으며, 이러한 단백분해효소 및 항단백분해효소의 발현 변화를 IGF2의 발현을 조절한 폐 상피세포에서 재확인하였다. In addition, it was confirmed that the collagen tissue destruction, the expression of MMP-9 protein, and the expression of α1-antitrypsin, which is a proteolytic enzyme, decrease in IGF2 transgenic mice, Enzyme and antiproteinase expression were reaffirmed in lung epithelial cells regulated IGF2 expression.
또한, 담배 성분인 NNK를 장기간 처리한 폐 상피세포에서 IGF2, IGFBP-3 및 폐 조직 손상을 일으키는 MMP-1, MMP-10, MMP-12, MMP-13의 발현과 IGF2 processing에 관여하는 furin의 발현이 증가됨을 확인하였고, IGF2에 의하여 MMP-1의 발현이 증가됨을 확인하였다.In addition, the expression of MMP-1, MMP-10, MMP-12, and MMP-13, which are responsible for IGF2, IGFBP-3 and lung tissue damage, and furin involved in IGF2 processing in lung epithelial cells treated with NNK, Expression of MMP-1 was increased by IGF2.
또한, 담배 성분인 NNK와 benzo[a]pyrene를 마우스에 병용 투여시, 폐 조직에서 IGF2 과발현 형질전환 마우스와 유사한 폐기종 병변이 발생되고, IGF2 발현 증가, 단백분해효소 MMP-9의 발현 증가, 항단백분해효소인 α1-antitrypsin의 발현 감소 효과가 발생함을 확인하였다.In addition, when NNK and benzo [a] pyrene, a tobacco component, were administered to mice, similar pulmonary emphysema was observed in pulmonary tissues similar to that of IGF2 transgenic mice, and increased expression of IGF2, increased expression of proteolytic enzyme MMP-9, It was confirmed that the expression of α1-antitrypsin, a protease, was reduced.
이로써, 폐기종을 유발할 수 있는 신규 유전자 변형마우스 모델과 담배 발암원이 IGF2 발현을 증가시켜 폐기종을 유발할 수 있는 작용기전을 확인하였다. The new transgenic mouse model that can induce emphysema and the mechanism by which tobacco carcinogen could induce emphysema by increasing IGF2 expression were confirmed.
따라서, 본 발명의 방법에 의해 제조된 IGF2 과발현 형질전환 마우스는 폐기종의 치료 및 예방을 위한 새로운 분자지표와 폐기종 치료제를 스크리닝하기 위한 새로운 모델 동물로 유용하게 이용될 수 있다.
Therefore, the IGF2 transgenic mouse produced by the method of the present invention can be usefully used as a new model animal for screening new molecular markers and emphysema therapeutic agents for treatment and prevention of emphysema.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.
[[ 실시예Example ]]
실시예Example
1. IGF2 과발현 형질전환 마우스의 제작 1. Production of IGF2-overexpressing transgenic mice
1-1. 1-1. SPCSPC -- IGF2IGF2 transgenetransgene 의 발현벡터 제작Of expression vector
형질전환 마우스 제작을 위한 유전자 변형 벡터인 SPC-kbpA는 Dr. DeMayo (Baylor Medical School)로부터 분양받은 것을 이용하였다. 600 bp 길이의 인간 IGF2 cDNA를 PC-kbpA 벡터의 EcoRI 위치에 subcloning 하여 SPC-IGF2 plasmid를 제작하였다. 그러므로 IGF-2유전자 발현은 surfactant protein C (SPC) promoter의 조절 하에 있도록 제작되었다.
SPC-kbpA, a transgenic vector for transgenic mouse production, DeMayo (Baylor Medical School). A 600 bp human IGF2 cDNA was subcloned at the EcoRI site of the PC-kbpA vector to construct an SPC-IGF2 plasmid. Therefore, IGF-2 gene expression was designed to be controlled by the surfactant protein C (SPC) promoter.
1-2. 유전자 변형 마우스 제작 1-2. Genetically modified mouse production
SPC-IGF2 plasmid를 restriction enzyme인 NdeI 및 NotI을 처리하여 SPC/IGF2 transgene을 절단하였다. 6 kb의 insert를 gel electrophoresis를 통해 분리한 후 transgene을 hybrid C3H/C57B6 fertilized mouse egg에 microinjection하였다.The SPC-IGF2 plasmid was digested with restriction enzymes NdeI and NotI to cleave the SPC / IGF2 transgene. The 6 kb insert was separated by gel electrophoresis and the transgene was microinjected into a hybrid C3H / C57B6 fertilized mouse egg.
사람 IGF2 transgene을 포함하는 마우스는 PCR analysis로 확인하였다. 마우스 꼬리 일부를 절단한 후 genomic DNA를 분리한 다음 SPC promoter/IGF2 연결 부위 700 bp에 대한 primer를 이용하여 PCR을 수행하였다. 이때 사용한 primer 서열은 forward primer 5’- CATGGAATTCAGACACCAATGGGAATCC-3’, reverse primer 5’-CATGGAATTCTGCTCACTTCCGATTGCTG-3’이다. PCR은 95℃, 5분에서 initial denaturation을 실시한 뒤 95℃, 45초, 58℃, 45초, 72℃, 45초씩 총 35 cycles을 수행하였고, 72℃에서 7분간 final extension 반응을 실시하였다. 생성된 PCR product는 RedSafe dye가 포함된 2% agarose gel로 전기영동하여 분리하였고, UV transilluminator에서 관찰하여 IGF2 포함 여부를 확인하였다.
Mice containing human IGF2 transgene were identified by PCR analysis. Genomic DNA was isolated after cleavage of mouse tail and PCR was performed using a primer for 700 bp of SPC promoter / IGF2 junction. The primer sequence used here is the forward primer 5'-CATGGAATTCAGACACCAATGGGAATCC-3 'and the reverse primer 5'-CATGGAATTCTGCTCACTTCCGATTGCTG-3'. After initial denaturation at 95 ° C for 5 minutes, the PCR was performed at 95 ° C for 45 seconds, at 58 ° C for 45 seconds, at 72 ° C for 45 seconds, and for 35 cycles at 72 ° C for 7 minutes. The resulting PCR product was separated by electrophoresis on 2% agarose gel containing RedSafe dye, and the presence of IGF2 was observed by UV transilluminator.
실시예Example 2. 제작된 IGF2 과발현 형질전환 마우스의 폐기종 유발 확인 2. Identification of induction of emphysema in IGF2-overexpressing transgenic mice
상기 실시예 1에서 제작된 본 발명의 IGF2 과발현 형질전환 마우스에서 폐기종이 유발되었는지의 여부를 확인하기 위하여, 하기 실험을 수행하였다.
In order to confirm whether or not emphysema was induced in the IGF2 transgenic mouse of the present invention prepared in Example 1, the following experiment was conducted.
2-1. 실험방법2-1. Experimental Method
H&E 염색H & E dyeing
폐 선택적 IGF2 유전자변형 (과발현) 마우스의 폐를 분리하고, 분리한 폐를 10% formalin을 사용하여 24 시간 동안 고정한 뒤 파라핀 블록에 embedding하였다. 파라핀 블록은 4 μm의 두께로 박절하고 silane coating 슬라이드에 부착 후 건조하였다. 말린 slide glass를 65 ℃ dry-oven에 넣어 overnight으로 탈파라핀을 진행하였다. 그 후 slide glass를 xylene에 5 분씩 2회 넣어두었다. 탈파라핀 시킨 후, 100%, 95%, 70%, 50% 단계별 에탄올로 함수시키고 Harris hematoxylin 용액에 1 분 30 초간 염색한 뒤 1% HCl alcohol 용액과 ammonia로 침적시킨 다음, eosin 용액으로 30 초간 염색하였다. 그 후 95%, 100% 에탄올을 이용하여 순차적으로 탈수시킨 후 xylene으로 투명화한 뒤 봉입하여 현미경으로 관찰하고 사진을 촬영하였다.
The pulmonary selective IGF2 transgenic (overexpressing) mouse lungs were isolated and the isolated lungs were fixed in 10% formalin for 24 hours and embedded in paraffin blocks. The paraffin blocks were cut to a thickness of 4 μm and attached to silane coating slides and dried. The dried slide glass was placed in a 65 ° C dry-oven and deparaffinized overnight. The slide glass was then placed in xylene twice for 5 minutes each. After deparaffinization, the cells were treated with 100%, 95%, 70%, and 50% ethanol and stained with Harris hematoxylin for 1 minute and 30 seconds, immersed in 1% HCl alcohol solution and ammonia, stained with eosin for 30 seconds Respectively. After that, the cells were dehydrated sequentially with 95% and 100% ethanol, transparentized with xylene, sealed and observed under a microscope and photographed.
폐 상피세포 Lung epithelium RNARNA 추출 및 Extraction and RTRT -- PCRPCR
RT-PCR 분석을 위한 total RNA는 마우스에서 적출된 폐의 상피세포에서 분리하였다. 우선 Trizol Reagent를 첨가하여 세포를 균질화한 후, 여기에 chloroform을 첨가하여 13,000 rpm에서 20 분간 원심분리하였다. 상층액을 회수하여 동량의 isopropanol을 첨가하여 13,000 rpm에서 20 분간 원심분리하여 RNA를 침전시켰다. 침전된 RNA는 75% 에탄올로 세척한 후, 건조시켜 nuclease-free water에 용해시켰다. 260 nm의 흡광도를 측정하여 RNA를 정량하였고, A260/A280nm의 비율이 1.8~2.0 범위 내의 값을 갖는 RNA를 실험에 사용하였다. 모든 실험은 RNase-free한 조건하에서 이루어졌다.Total RNA for RT-PCR analysis was isolated from epithelial cells of lungs harvested from mice. The cells were homogenized by adding Trizol Reagent, and then chloroform was added thereto, followed by centrifugation at 13,000 rpm for 20 minutes. The supernatant was recovered and the same amount of isopropanol was added and the RNA was precipitated by centrifugation at 13,000 rpm for 20 minutes. The precipitated RNA was washed with 75% ethanol, dried and dissolved in nuclease-free water. RNA was quantitated by measuring absorbance at 260 nm, and RNA having a ratio of A260 / A280 nm within the range of 1.8 to 2.0 was used in the experiment. All experiments were performed under RNase-free conditions.
cDNA는 PrimeScriptTM 1st strand cDNA Synthesis Kit를 이용하여 2 ㎍의 total RNA, oligo dT primer (50 μM), dNTP mixture, 그리고 RNase free dH2O로 65 ℃ 5분간 반응하였고, 그 후 Template RNA primer mixture에 5×PrimeScript Buffer, RNase inhibitor, PrimeScript RTase, 및 RNase free dH2O를 넣어 42 ℃ 1 시간, 그리고 95 ℃에서 5 분간 heating 시킴으로써 반응을 중지시켜 합성하였다.The cDNA was reacted with 2 μg of total RNA, oligo dT primer (50 μM), dNTP mixture, and RNase-free dH 2 O at 65 ° C for 5 minutes using the PrimeScript ™ 1st strand cDNA synthesis kit. × PrimeScript Buffer, RNase inhibitor, PrimeScript RTase, and RNase-free dH 2 O, and incubated at 42 ° C for 1 hour and at 95 ° C for 5 minutes.
Polymerase chain reaction (PCR)은 2 μL cDNA, 4 μM primer sets, EconoTaq® 10 μL, 그리고 증류수를 가하여 반응액을 20 μL로 맞춘 다음 PCR 기기를 이용하여 수행하였다. PCR 조건은 95 ℃/10 초, 55~58 ℃/30 초, 72 ℃/1 분으로 하여 25~33회 증폭하였다. PCR 산물은 2% agarose gel에서 전기영동한 후 ethidium bromide (EtBr)로 염색하여 UV transilluminator에서 특정 밴드를 관찰하였다.
Polymerase chain reaction (PCR) was carried out using 2 μL cDNA, 4 μM primer sets, 10 μL of EconoTaq®, and distilled water. The PCR conditions were 25 to 33 times at 95 ° C / 10 seconds, 55 to 58 ° C / 30 seconds, and 72 ° C / 1 minute. The PCR products were electrophoresed on 2% agarose gel and stained with ethidium bromide (EtBr) to observe specific bands in the UV transilluminator.
WesternWestern blottingblotting
IGF2 과발현 형질전환 마우스 및 NNK/B(a)P를 처리한 마우스로부터 폐를 적출한 뒤, 폐 조직을 취하여 RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, protease inhibitor cocktail)에 넣고 TissueLyser를 이용하여 조직을 분쇄하여 단백질을 추출하였다. 추출된 단백질을 BCA법을 이용하여 정량한 뒤, 동일한 양의 단백질 (20∼50 μg)을 SDS-PAGE로 전기 영동하였다. 분리된 단백질을 PVDF membrane으로 transfer한 뒤, 단백질이 옮겨진 membrane을 TBST (20 mM Tris-HCl (pH 8), 137 mM NaCl, 0.1% Tween-20)에 희석한 3% bovine serum albumin (BSA) 용액으로 실온에서 1시간 동안 blocking하였다. Membrane을 1차 항체가 1:1,000의 비율로 희석된 3% BSA가 함유된 TBST에 넣고 4℃에서 밤새 배양하였다. Membrane을 TBST로 실온에서 1시간 동안 세척하였다. 세척한 membrane을 2차 항체가 1:5,000 ∼ 1:10,000의 비율로 희석된 3% skim milk가 함유된 TBST에 넣고 실온에서 1시간 동안 배양하였다. Membrane을 TBST로 실온에서 1시간 동안 세척한 뒤, ECL solution과 배양 후 X-ray film에 노출시켜 단백질 발현을 확인하였다.
The lungs were harvested from the mice treated with IGF2 overexpressing transgenic mice and NNK / B (a) P, and the lung tissues were harvested and resuspended in RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X -100, 0.25% sodium deoxycholate, 1 mM EDTA, 50 mM NaF, 1 mM Na 3 VO 4 , protease inhibitor cocktail) and the tissue was ground using a TissueLyser to extract proteins. The extracted protein was quantified using BCA method, and the same amount of protein (20 to 50 μg) was electrophoresed by SDS-PAGE. After transferring the separated proteins to PVDF membrane, 3% bovine serum albumin (BSA) solution in which proteins were transferred was diluted in TBST (20 mM Tris-HCl (pH 8), 137 mM NaCl, 0.1% Tween- At room temperature for 1 hour. Membranes were incubated overnight at 4 ° C in TBST containing 3% BSA diluted at a ratio of 1: 1,000 primary antibody. Membrane was washed with TBST at room temperature for 1 hour. The washed membrane was incubated in TBST containing 3% skim milk diluted at a ratio of 1: 5,000 to 1: 10,000 for 1 hour at room temperature. Membrane was washed with TBST for 1 hour at room temperature, and then exposed to X-ray film by ECL solution and incubated for protein expression.
IGF2IGF2 shRNAshRNA lentivirallentiviral vectorvector 처리 process
폐 상피세포인 HBEC1 세포를 2×105세포/well로 분주한 후 하루 동안 배양하였다. IGF2 shRNA lentiviral vector, Opti-MEM, FuGENE HD Transfection reagent를 혼합하여 실온에서 30분간 반응시키고, 세포에 처리하였다. 37℃, 5% CO2 배양기에서 48시간 배양한 후 실험에 이용하였다.
The lung epithelial cells, HBEC1 cells, were divided into 2 × 10 5 cells / well and cultured for one day. IGF2 shRNA lentiviral vector, Opti-MEM and FuGENE HD Transfection reagent were mixed and reacted at room temperature for 30 minutes and treated with cells. The cells were cultured in a 5% CO 2 incubator at 37 ° C for 48 hours.
IGF2IGF2 pBABEpBABE retroviralretroviral vectorvector 처리 process
폐 상피세포인 HBEC1 세포를 2×105/well로 6 well plate에 분주하고, 37℃, 5% CO2 incubator에서 24시간 동안 키운 후 well에 세포가 70~80%로 자랐을 때 lipofectamine 2000 4 μL와 DNA 1 ㎍ Opti-MEM에 희석하고 세포가 seeding되어 있는 dish에 넣고 37℃, 5% CO2 incubator에서 6시간 동안 배양하여 transfection 하였다. 배양 후 growth factor를 포함하는 complete media를 넣어준 다음 37℃, 5%, CO2 incubator에서 하루 동안 배양한 후 실험에 이용하였다.
Lung epithelial cells, HBEC1 cells, were plated at 6 × 10 5 / well in a 6-well plate and incubated for 24 h at 37 ° C in a 5% CO 2 incubator. Cells were grown in 70-80% And
Masson'sMasson's trichrometrichrome stainingstaining
muscle, collagen은 산성 염료인 Biebrich Scarlet acid fuchsin에 의해 적색으로 염색되고, phosphomolybdic, phosphotungstic acid는 collagen을 탈색시키며, 염기성 염료인 anillin blue와 collagen의 basic group을 결합시켜 blue로 염색시킴에 따라, 교원섬유(collagen fiber)의 양과 형태를 광학현미경으로 확인할 수 있는 방법이다.muscle, and collagen are stained red with acid dye Biebrich Scarlet acid fuchsin, phosphomolybdic and phosphotungstic acid decolorize collagen, and anillin blue, which is a basic dye, and basic group of collagen are combined and stained with blue, (collagen fiber) can be confirmed by optical microscope.
파라핀으로 고정된 폐 조직 절편을 100%, 95%, 70% alcohol로 탈파라핀화 및 수화시킨 다음 증류수로 세척하였다. 필요에 따라 Boulin's solution으로 56℃에서 1시간 동안 재 고정한 후 물로 5∼10분 동안 세척하였다. 조직을 Weigert's iron hematoxylin working soluion으로 10분간 염색한 뒤 온수 및 증류수로 세척하고, Biebrich scarlet-acid fuchsin solution으로 10∼15분간 염색하였다. 염색된 조직을 증류수로 세척한 뒤 phosphomolybdic-phosphotungstic acid solution에 10∼15분간 염색하고, aniline blue solution을 5∼10분 동안 처리한 뒤 증류수로 가볍게 세척하고 1% acetic acid solution을 2∼5분간 처리하였다. 증류수로 세척한 다음 염색된 조직을 95% ethanol과 absolute ethanol로 탈수시키고 xylene으로 세척하였다. Mounting solution으로 mounting 후 조직을 관찰하였다.
Paraffin-fixed lung tissue sections were de-paraffinized and hydrated in 100%, 95%, 70% alcohol and washed with distilled water. If necessary, it was remixed with Boulin's solution at 56 ° C for 1 hour and then washed with water for 5 to 10 minutes. The tissue was stained with Weigert's iron hematoxylin working solution for 10 minutes, washed with warm water and distilled water, and stained with Biebrich scarlet-acid fuchsin solution for 10-15 minutes. The stained tissue was washed with distilled water, stained with phosphomolybdic-phosphotungstic acid solution for 10 to 15 minutes, treated with aniline blue solution for 5 to 10 minutes, washed gently with distilled water and treated with 1% acetic acid solution for 2 to 5 minutes Respectively. After washing with distilled water, the stained tissue was dehydrated with 95% ethanol and absolute ethanol and washed with xylene. After mounting with mounting solution, tissues were observed.
inin situsitu ZymographyZymography
COPD를 일으키는 주된 인자로 알려진 단백분해효소 MMP(matrix metalloproteinase)의 활성도를 폐 조직 내에서 검색하는 방법으로, fluorescein isothiocyanate(FITC)가 결합된 DQ gelatin을 포함한 reaction buffer를 슬라이드에 적정량 가한 후, 37℃의 암실에서 12시간 동안 반응시켰다. 활성화된 MMP가 DQ gelatin을 분해하게 되고, 억제되었던 FITC가 형광을 내게 되므로, FITC의 형광 강도는 MMP의 활성과 비례한다.
The reaction buffer containing DQ gelatin with fluorescein isothiocyanate (FITC) was added to the slides in a suitable amount to detect the activity of proteolytic enzyme MMP (matrix metalloproteinase), which is known to be the main factor causing COPD, Lt; / RTI > for 12 hours. Since activated MMP degrades DQ gelatin and inhibited FITC fluoresces, the fluorescence intensity of FITC is proportional to the activity of MMP.
gelatingelatin zymographyzymography
실험동물의 기관지를 절개한 다음 PBS 3ml을 이용하여 시험동물의 폐를 총 4회 세척하고, 세척액을 3000 rpm에서 3분간 원심분리한 후 위의 상층액을 얻어 농축시켜 기관지 폐포 세척액을 얻었다. 이후, 기관지 폐포세척액 내의 단백질 농도를 측정한 후 각 샘플의 단백질 농도를 5× sample buffer를 이용하여 동량으로 혼합한 후 0.1% gelatin을 함유한 10% SDS-PAGE에 전기영동하였다. 전기영동 후 겔을 2.5% Triton X-100에 20분씩 2번 담그고 배양버퍼를 넣어 37℃에서 24시간 담근 후, 겔은 0.5% Coomassie brilliant blue 250 용액으로 착색시킨 후 탈색버퍼를 이용하여 탈색시킨 후 판독하였다.
Lungs of the test animals were washed 4 times with 3 ml of PBS, and the washings were centrifuged at 3000 rpm for 3 minutes. The supernatant was collected and concentrated to obtain bronchoalveolar lavage fluid. Then, protein concentration in bronchoalveolar lavage fluid was measured, and the protein concentration of each sample was mixed in the same amount using 5 × sample buffer, and then electrophoresed on 10% SDS-PAGE containing 0.1% gelatin. After electrophoresis, the gel was immersed twice in 2.5% Triton X-100 for 20 minutes, and incubated at 37 ° C for 24 hours. The gel was stained with 0.5% Coomassie brilliant blue 250 solution, discolored using decolorization buffer .
ImmunocytochemistryImmunocytochemistry
폐 조직 세포를 0.8×106이 되도록 6-well culture plate에 cover-slip을 넣어 분주한 후, 37℃, 5% CO2 incubator에서 밤새 배양하였다. 세포가 부착된 후 NNK/benzo[a]pyrene를 각각 10μM의 농도로 처리하고, 3일 후 배지를 제거한 후 PBS를 사용하여 5분씩 3회 세척하였다. 그 후 cold MeOH로 실온에서 30분 동안 세포를 고정시키고 다시 PBS를 사용하여 5분씩 3회 세척하고, 3% BSA solution을 사용하여 4℃에서 1시간 동안 blocking을 수행하였다. 그 후, blocking buffer에 α1-antitrypsin에 특이적으로 결합하는 1차 항체를 1:100으로 희석하여 4℃에서 밤새 반응시켰다. 슬라이드를 PBST(1:1000)로 10분씩 3회 세척한 후, 2차 항체인 Alexa 488(1:1000)을 사용하여 상온에서 1시간 동안 차광상태에서 반응시켰다. 그 후, PBST(1:000)를 사용하여 10분씩 4회 세척하였고, DAPI가 포함된 mounting solution을 사용하여 mounting을 진행한 후, 형광현미경으로 관찰하였다.
Lung tissue cells were plated on a 6-well culture plate at a density of 0.8 × 10 6 , and then cultured overnight in a 5% CO 2 incubator at 37 ° C. After the cells were attached, NNK / benzo [a] pyrene was treated at a concentration of 10 μM, and after 3 days, the medium was removed and washed three times with PBS for 5 minutes each. The cells were then fixed with cold MeOH for 30 minutes at room temperature, washed again three times for 5 minutes each with PBS, and blocked for 3 hours at 4 ° C with 3% BSA solution. Subsequently, the primary antibody specifically binding to α1-antitrypsin was diluted 1: 100 in blocking buffer and reacted overnight at 4 ° C. The slides were washed with PBST (1: 1000) three times for 10 minutes each, and then reacted with a secondary antibody, Alexa 488 (1: 1000), at room temperature for 1 hour in a shade state. Subsequently, PBST (1: 000) was used to wash four times for 10 minutes. Mounting was performed using a mounting solution containing DAPI, and then observed with a fluorescence microscope.
2-2. 실험결과2-2. Experiment result
1) 폐 조직 염색 및 적출된 폐 형태 확인1) Dissection of pulmonary tissues and identification of extracted lungs
H&E 염색H & E dyeing
상기 실시예 2-1의 H&E 염색을 통하여 생후 30일의 폐 선택적 IGF2 유전자변형(과발현) 마우스의 폐에서 폐기종으로의 조직 변화, 즉 폐 조직의 손상과 이로 인하여 폐포가 커지는 증상을 확인하였으며, 이를 도 1에 나타내었다. 도 1의 상단 사진 및 하단 좌측은 폐 조직 및 조직 단면에 대한 H&E 염색 결과를 나타낸 것으로, IGF2의 발현 정도가 다른 두 line(line 1, line 2)에서 동일하게 폐기종이 유발되었음을 확인하였다.
Through the H & E staining of Example 2-1, 30 days of lung-selective IGF2 gene mutation (overexpression) of the mouse was observed in the lung tissue to emphysema, that is, lung tissue damage and
Masson'sMasson's trichrometrichrome stainingstaining
상기 실시예 2-1의 Masson's trichrome staining 방법을 이용하여, 교원섬유(collagen fiber)의 양과 형태를 관찰하였다. Using the Masson's trichrome staining method of Example 2-1, the amount and shape of collagen fibers were observed.
그 결과, 도 2에 나타낸 바와 같이, 정상군(WT)의 폐조직은 교원섬유의 밀도가 조밀하고 배열이 규칙적인 반면, IGF2 유전자변형(과발현) 마우스의 폐조직은 교원섬유가 파괴되어 밀도가 엉성하고 배열이 불규칙적이며 양도 많이 줄어들어 있는 것을 확인할 수 있었다. 즉, COPD 환자들에서 발견되는 섬유화와 폐포벽이 무너지며 교원섬유가 파괴되어 콜라겐이 붕괴되는 현상을 확인하였다.
As a result, as shown in Fig. 2, lung tissue of the normal group (WT) has dense collagen fibers and regular arrangement, while the lung tissue of IGF2 transgenic (overexpressed) It was confirmed that the shape was loose, the arrangement was irregular, and the amount was greatly reduced. In other words, fibrosis and collapsing collapses, which are found in patients with COPD, collapsed due to destruction of collagen fibers.
적출된 폐 형태 확인Identify extracted lungs
또한, 19개월의 IGF2 과발현 형질전환 마우스의 폐에서, 폐기종으로의 조직 변화 중 하나인 폐 조직의 손상 현상을 육안으로 확인하였으며, 이를 도 3에 나타내었다.
In addition, damage of lung tissue, which is one of the tissue changes to emphysema, was visually confirmed in the lung of 19-month-old IGF2 transgenic transgenic mouse, which is shown in FIG.
2) 단백질분해효소(2) Protease ( MMPMMP -9) 및 -9) and 항단백분해효소(α1-antitrypsin)의Of the anti-protease (α1-antitrypsin) 발현/활성 확인 Identification of expression / activity
inin situsitu ZymographyZymography
COPD를 일으키는 주된 인자로 알려진 단백분해효소 MMP(matrix metalloproteinase)의 활성도를 폐조직 내에서 확인하기 위하여, 상기 실시예 2-1의 방법으로 in situ Zymography를 실시하였다.In order to confirm the activity of MMP protease known as a main factor causing the COPD (matrix metalloproteinase) in the lung tissue, in a method of the above Example 2-1 situ Zymography was performed.
그 결과, 도 4A에 나타낸 바와 같이, 정상군(b 참조)에 비하여 IGF2 과발현 형질전환 마우스의 폐 조직(d 참조)에서 MMP 활성이 증가됨을 알 수 있었다.
As a result, as shown in FIG. 4A, it was found that the MMP activity was increased in the lung tissue (see d) of IGF2 transgenic mouse compared to the normal group (see b).
gelatingelatin ZymographyZymography
기관지 폐포세척액(BAL) 내의 MMP-9의 단백질 분해능을 알아보기 위하여, 상기 실시예 2-1의 방법으로 gelatin zymography를 시행하였다. 기관지 폐포세척액(BAL)은 주로 염증세포들로 구성되어 있어 COPD 질환에서 염증반응의 증폭여부를 알아볼 수 있다. In order to examine the proteolytic activity of MMP-9 in bronchoalveolar lavage fluid (BAL), gelatin zymography was performed by the method of Example 2-1. Bronchoalveolar lavage fluid (BAL) is composed mainly of inflammatory cells, and it can be detected whether the inflammatory reaction is amplified in COPD disease.
그 결과, 도 4B에 나타낸 바와 같이, 정상군(WT)에 비하여 IGF2 과발현 형질전환 마우스의 폐 조직(IGF-2 TG)에서 MMP-9의 발현이 증가됨을 알 수 있었다.
As a result, as shown in FIG. 4B, the expression of MMP-9 was increased in the lung tissue (IGF-2 TG) of IGF2 transgenic mouse compared to the normal group (WT).
WesternWestern blottingblotting
COPD 환자에서는 항단백분해효소인 α1-antitrypsin의 발현이 감소되고 단백분해효소인 MMP-9의 발현이 증가되기 때문에, IGF2 과발현 형질전환 마우스에서도 이러한 현상이 일어나는지 확인하기 위하여, 적출된 폐에 대하여 상기 실시예 2-1의 방법으로 Western blotting을 수행하였다.In the COPD patients, the expression of α1-antitrypsin, an anti-protease, is decreased and the expression of MMP-9, a protease, is increased. Therefore, in order to confirm whether this phenomenon occurs in IGF2 transgenic mice, Western blotting was performed by the method of Example 2-1.
그 결과, 도 5에 나타낸 바와 같이, 군 당 4마리의 마우스로부터 확보된 폐 조직에 대하여 실시한 결과, 정상군(WT)에 비하여 IGF2 과발현 형질전환 마우스(IGF2 TG)에서 α1-antitrypsin의 발현이 감소되고 MMP-9의 발현이 증가됨을 확인하였는 바, 이는 항단백분해효소와 단백분해효소간의 불균형에 의해 폐포 확장 증상이 나타날 수 있음을 시사하는 것이다.
As a result, as shown in Fig. 5, the expression of α1-antitrypsin was decreased in IGF2 transgenic mice (IGF2 TG) compared to the normal group (WT) And that the expression of MMP-9 is increased. This suggests that the symptoms of alveolar expansion may be caused by an imbalance between the protease and the protease.
RTRT -- PCRPCR
IGF2 발현이 증가됨에 따라 폐 조직 손상을 유발하는 MMP-1의 발현이 증가되고, IGF2 발현 억제에 의하여 basal level 또는 IGF2 발현 증가에 의해 유도된 MMP-1 발현이 감소됨을 확인하였다(도 6 참조). As the IGF2 expression was increased, MMP-1 expression was increased and MMP-1 expression induced by basal level or IGF2 expression was decreased by inhibition of IGF2 expression (see FIG. 6) .
또한, 폐 상피세포 BEAS-2B에서 IGF2 발현을 Knock-down에 의해 저하(K/D)시키거나 과발현(O/E)시킨 결과, 도 7에 나타낸 바와 같이, IGF2의 발현이 저하(K/D)되면 대조군(EV; empty vector)에 비하여 항단백분해효소인 α1-antitrypsin의 발현이 증가된 반면 IGF2이 과발현(O/E)되면 대조군에 비하여 α1-antitrypsin의 발현이 감소함을 알 수 있었다.
As shown in FIG. 7, when IGF2 expression was decreased (K / D) or overexpressed (O / E) by knockdown in lung epithelial cell BEAS-2B, the expression of IGF2 decreased ), The expression of α1-antitrypsin, an anti-proteolytic enzyme, was increased in comparison with the control (EV) vector, whereas the expression of α1-antitrypsin was decreased when IGF2 was overexpressed (O / E).
3) 담배 유래 3) Tobacco origin 발암원(NNK)과Carcinogen (NNK) and IGF2IGF2 , , MMPMMP 유전자 발현의 상관관계 확인 Correlation of gene expression
담배 유래 발암원인 NNK의 장기간 동안의 처리(20, 30, 100일)에 의하여 사람 폐 상피세포인 HBEC 및 NHBE에서 폐기종과 연관된 것으로 알려진 IGFBP-3와 더불어 IGF2의 유전자 발현이 증가됨을 확인하였다(도 8), The long-term treatment of NNK (20, 30, and 100 days), a tobacco-derived carcinogen, was found to increase IGF2 gene expression in addition to IGFBP-3, which is known to be associated with emphysema, in human lung epithelial cells HBEC and NHBE 8),
또한, 담배 유래 발암원인 NNK의 장기간 동안의 처리 (7∼150일)에 의하여 사람 폐 상피세포인 HBEC1과 BEAS-2B에서 IGF2의 processing에 관여하는 효소인 furin, PC-1 및 PC-7의 유전자 발현이 증가되고(도 9), NNK 및 IGF2에 의하여 사람 폐 상피세포에서 furin의 발현이 증가하며, IGF2가 과발현된 조건에서 NNK를 처리하면 furin 발현 증가 효과가 강화됨을 확인하였다(도 10). In addition, the long-term treatment (7 to 150 days) of NNK, a tobacco-derived carcinogen, was used to detect the genes of furin, PC-1 and PC-7, enzymes involved in the processing of IGF2 in human lung epithelial cells HBEC1 and BEAS- (Fig. 9). The expression of furin in human lung epithelial cells was increased by NNK and IGF2, and NNK treatment with IGF2 overexpression enhanced the effect of increasing furin expression (Fig. 10).
또한, NNK의 장기간 동안의 처리 (20∼150일)에 의하여 사람 폐 상피세포인 HBEC1과 BEAS-2B에서 폐 조직 손상을 유발하는 단백질 분해효소인 MMP-1, MMP-9, MMP-10, MMP-12, MMP-13의 유전자 발현이 증가됨을 확인하였다(도 11A 및 11B).MMP-1, MMP-9, MMP-10, and MMP-8, which cause lung tissue damage in human lung epithelial cells HBEC1 and BEAS-2B, were treated with NNK for a long period of time (20 to 150 days) -12, and MMP-13 gene expression was increased (Figs. 11A and 11B).
또한, 담배 유래 발암원인 NNK을 사람 폐 상피세포인 HBEC1과 BEAS-2B에 처리시, 항단백분해효소인 α1-antitrypsin의 발현이 감소됨을 확인하였다(도 12).In addition, it was confirmed that the expression of α1-antitrypsin, which is an anti-protease, was reduced when NNK, a tobacco-derived cancer cell, was treated with human lung epithelial cells HBEC1 and BEAS-2B (FIG.
아울러, 담배 유래 발암원인 NNK과 benzo[a]pyrene을 사람 폐 상피세포인 BEAS-2B에 병용처리시, α1-antitrypsin 발현 감소 및 MMP 발현 증가 효과가 더욱 강화됨을 확인하였고, 상기 실시예 2-1의 방법으로 immunocytochemistry를 수행한 결과 NNK와 benzo[a]pyrene을 병용 투여한 폐 상피세포에서 α1-antitrypsin의 발현이 감소되는 것을 재확인하였다(도 13).In addition, it was confirmed that α1-antitrypsin expression and MMP expression increasing effect were further enhanced when the NNK and benzo [a] pyrene, which are tobacco-derived cancer cells, were administered to human lung epithelial cell BEAS-2B, Immunocytochemistry revealed that α1-antitrypsin expression was decreased in lung epithelial cells treated with NNK and benzo [a] pyrene (FIG. 13).
이러한 결과는, 담배 발암원에 의하여 발현 및 활성화된 IGF2는 폐 조직 손상을 유발하는 MMP의 발현을 유도하고, α1-antitrypsin의 발현을 억제함으로서 폐기종을 유발하는 것으로 종합할 수 있다. 본 연구 결과는 담배 발암원에 의한 MMP의 활성화에 따른 폐 조직 손상 과정에 있어 IGF2가 관여될 수 있음을 새롭게 규명한 것이다.
These results suggest that IGF2 expressed and activated by tobacco cancer can induce the expression of MMP that induces lung tissue damage and induce emphysema by suppressing the expression of α1-antitrypsin. The results of this study confirm that IGF2 may be involved in lung tissue damage induced by activation of MMP by tobacco oncogenes.
4) 폐기종 유발 담배 성분인 NNK와 benzo[a]pyrene의 마우스(4) NNK and benzo [a] pyrene of the tobacco-induced tobacco in vivoin vivo ) 병용투여 효과 확인) Confirmation of concurrent administration effect
H&E 염색H & E dyeing
마우스에 폐기종을 유발하는 담배 주요성분인 NNK과 benzo[a]pyrene(이하, B(a)P)를 5개월 (5M) 및 7개월 (7M) 동안 병용투여한 후 폐조직을 적출하여, 상기 실시예 2-1의 방법으로 H&E 염색을 실시한 결과, 도 14에 나타낸 바와 같이, 정상군(WT)에 비하여 NNK/B(a)P 병용처리된 마우스 군에서, IGF2 과발현 형질전환 마우스와 유사하게 밀도가 엉성하고 배열이 불규칙한 폐기종 병변이 발생됨을 알 수 있었다.
NNK and benzo [a] pyrene (hereinafter referred to as B (a) P), which are major components of tobacco that induces emphysema in mice, were administered for 5 months (5M) and 7 months (7M) As shown in Fig. 14, H & E staining was carried out by the method of Example 2-1. As shown in Fig. 14, in the mouse group treated with NNK / B (a) P in comparison with the normal group (WT) It was found that lesions with a dense density and irregular arrangement occurred.
in vivoin vivo 상에서 IGF2 발현 증가 확인 IGF2 < / RTI >
폐기종을 유발하는 담배 주요성분인 NNK과 benzo[a]pyrene를 마우스에 병용투여시, in vivo 상에서도 IGF2의 발현이 증가되었는지 확인하기 위하여, 적출된 폐조직을 대상으로 real-time PCR 및 웨스턴 블랏팅을 수행하였다.In order to confirm the increased expression of IGF2 in vivo when NNK and benzo [a] pyrene, which are the main components of tobacco inducing emphysema, were administered to mice, the extracted lung tissues were subjected to real-time PCR and Western blotting Respectively.
그 결과, 도 15에 나타낸 바와 같이, 대조군(VEH; vehicle (corn oil) 처리군)에 비하여 NNK/B(a)P 병용처리된 군에서 IGF2의 발현이 증가됨을 확인하였다.
As a result, as shown in Fig. 15, it was confirmed that the expression of IGF2 was increased in the group treated with NNK / B (a) P compared to the control (VEH; vehicle (corn oil) treated group).
inin vivovivo 상에서 α1- Lt; RTI ID = antitrypsinantitrypsin 및 And MMPMMP 발현 변화 확인 Identification of expression changes
COPD 환자에서는 항단백분해효소인 α1-antitrypsin의 발현이 감소되고 단백분해효소인 MMP의 발현이 증가되기 때문에, 폐기종을 유발하는 담배 주요성분인 NNK과 benzo[a]pyrene를 마우스에 병용 투여 시에도 이러한 현상이 일어나는지 확인하기 위하여, 폐 조직을 적출하여 상기 실시예 2-1의 방법으로 RT-PCR, 웨스턴 블롯팅, 및 gelatin zymography를 수행하였다.In COPD patients, the expression of α1-antitrypsin, which is an anti-protease, is decreased and the expression of MMP, a protease, is increased. Therefore, when co-administered with NNK and benzo [a] pyrene, In order to confirm whether this phenomenon occurred, lung tissues were extracted and subjected to RT-PCR, Western blotting and gelatin zymography by the method of Example 2-1.
그 결과, 도 16에 나타낸 바와 같이, 대조군(VEH; vehicle (corn oil) 처리군)에 비하여 NNK/B(a)P 병용처리된 군에서 α1-antitrypsin의 mRNA 및 단백질 발현양이 감소되고, MMP-9의 mRNA 및 단백질 발현양이 증가됨을 확인하였다. 또한, gelatin Zymography 결과, MMP-9 및 MMP-12의 발현이 증가되었다.
As a result, as shown in FIG. 16, the amount of α1-antitrypsin mRNA and protein expression was decreased in the group treated with NNK / B (a) P compared with the control group (VEH -9 and mRNA and protein expression levels were increased. Gelatin Zymography also increased the expression of MMP-9 and MMP-12.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.
<110> SNU R&DB FOUNDATION <120> Development of a novel emphysema-related animal model using insulin-like growth factor2 (IGF2) <130> PB14-12106 <150> KR 13/083697 <151> 2013-07-16 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 180 <212> PRT <213> Homo sapiens <400> 1 Met Gly Ile Pro Met Gly Lys Ser Met Leu Val Leu Leu Thr Phe Leu 1 5 10 15 Ala Phe Ala Ser Cys Cys Ile Ala Ala Tyr Arg Pro Ser Glu Thr Leu 20 25 30 Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys Gly Asp Arg 35 40 45 Gly Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Ser Arg Arg Ser Arg 50 55 60 Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu Leu 65 70 75 80 Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu Arg Asp Val Ser Thr 85 90 95 Pro Pro Thr Val Leu Pro Asp Asn Phe Pro Arg Tyr Pro Val Gly Lys 100 105 110 Phe Phe Gln Tyr Asp Thr Trp Lys Gln Ser Thr Gln Arg Leu Arg Arg 115 120 125 Gly Leu Pro Ala Leu Leu Arg Ala Arg Arg Gly His Val Leu Ala Lys 130 135 140 Glu Leu Glu Ala Phe Arg Glu Ala Lys Arg His Arg Pro Leu Ile Ala 145 150 155 160 Leu Pro Thr Gln Asp Pro Ala His Gly Gly Ala Pro Pro Glu Met Ala 165 170 175 Ser Asn Arg Lys 180 <210> 2 <211> 5182 <212> DNA <213> Homo sapiens <400> 2 cgcctgtccc cctcccgagg cccgggctcg cgacggcaga gggctccgtc ggcccaaacc 60 gagctgggcg cccgcggtcc gggtgcagcc tccactccgc cccccagtca ccgcctcccc 120 cggcccctcg acgtggcgcc cttccctccg cttctctgtg ctccccgcgc ccctcttggc 180 gtctggcccc ggcccccgct ctttctcccg caaccttccc ttcgctccct cccgtccccc 240 ccagctccta gcctccgact ccctcccccc ctcacgcccg ccctctcgcc ttcgccgaac 300 caaagtggat taattacacg ctttctgttt ctctccgtgc tgttctctcc cgctgtgcgc 360 ctgcccgcct ctcgctgtcc tctctccccc tcgccctctc ttcggccccc ccctttcacg 420 ttcactctgt ctctcccact atctctgccc ccctctatcc ttgatacaac agctgacctc 480 atttcccgat accttttccc ccccgaaaag tacaacatct ggcccgcccc agcccgaaga 540 cagcccgtcc tccctggaca atcagacgaa ttctcccccc ccccccaaaa aaaagccatc 600 cccccgctct gccccgtcgc acattcggcc cccgcgactc ggccagagcg gcgctggcag 660 aggagtgtcc ggcaggaggg ccaacgcccg ctgttcggtt tgcgacacgc agcagggagg 720 tgggcggcag cgtcgccggc ttccagacac caatgggaat cccaatgggg aagtcgatgc 780 tggtgcttct caccttcttg gccttcgcct cgtgctgcat tgctgcttac cgccccagtg 840 agaccctgtg cggcggggag ctggtggaca ccctccagtt cgtctgtggg gaccgcggct 900 tctacttcag caggcccgca agccgtgtga gccgtcgcag ccgtggcatc gttgaggagt 960 gctgtttccg cagctgtgac ctggccctcc tggagacgta ctgtgctacc cccgccaagt 1020 ccgagaggga cgtgtcgacc cctccgaccg tgcttccgga caacttcccc agataccccg 1080 tgggcaagtt cttccaatat gacacctgga agcagtccac ccagcgcctg cgcaggggcc 1140 tgcctgccct cctgcgtgcc cgccggggtc acgtgctcgc caaggagctc gaggcgttca 1200 gggaggccaa acgtcaccgt cccctgattg ctctacccac ccaagacccc gcccacgggg 1260 gcgccccccc agagatggcc agcaatcgga agtgagcaaa actgccgcaa gtctgcagcc 1320 cggcgccacc atcctgcagc ctcctcctga ccacggacgt ttccatcagg ttccatcccg 1380 aaaatctctc ggttccacgt ccccctgggg cttctcctga cccagtcccc gtgccccgcc 1440 tccccgaaac aggctactct cctcggcccc ctccatcggg ctgaggaagc acagcagcat 1500 cttcaaacat gtacaaaatc gattggcttt aaacaccctt cacataccct ccccccaaat 1560 tatccccaat tatccccaca cataaaaaat caaaacatta aactaacccc cttccccccc 1620 ccccacaaca accctcttaa aactaattgg ctttttagaa acaccccaca aaagctcaga 1680 aattggcttt aaaaaaaaca accaccaaaa aaaatcaatt ggctaaaaaa aaaaagtatt 1740 aaaaacgaat tggctgagaa acaattggca aaataaagga atttggcact ccccaccccc 1800 ctctttctct tctcccttgg actttgagtc aaattggcct ggacttgagt ccctgaacca 1860 gcaaagagaa aagaaggacc ccagaaatca caggtgggca cgtcgctgct accgccatct 1920 cccttctcac gggaattttc agggtaaact ggccatccga aaatagcaac aacccagact 1980 ggctcctcac tcccttttcc atcactaaaa atcacagagc agtcagaggg acccagtaag 2040 accaaaggag gggaggacag agcatgaaaa ccaaaatcca tgcaaatgaa atgtaattgg 2100 cacgaccctc acccccaaat cttacatctc aattcccatc ctaaaaagca ctcatacttt 2160 atgcatcccc gcagctacac acacacaaca cacagcacac gcatgaacac agcacacaca 2220 cgagcacagc acacacacaa acgcacagca cacacagcac acagatgagc acacagcaca 2280 cacacaaacg cacagcacac acacgcacac acatgcacac acagcacaca aacgcacggc 2340 acacacacgc acacacatgc acacacagca cacacacaaa cgcacagcac acacaaacgc 2400 acagcacaca cgcacacaca gcacacacac gagcacacag cacacaaacg cacagcacac 2460 gcacacacat gcacacacag cacacacact agcacacagc acacacacaa agacacagca 2520 cacacatgca cacacagcac acacacgcga acacagcaca cacgaacaca gcacacacag 2580 cacacacaca aacacagcac acacatgcac acagcacacg cacacacagc acacacatga 2640 acacagcaca cagcacacac atgcacacac agcacacacg catgcacagc acacatgaac 2700 acagcacaca cacaaacaca cagcacacac atgcacacac agcacacaca ctcatgcgca 2760 gcacatacat gaacacagct cacagcacac aaacacgcag cacacacgtt gcacacgcaa 2820 gcacccacct gcacacacac atgcgcacac acacgcacac ccccacaaaa ttggatgaaa 2880 acaataagca tatctaagca actacgatat ctgtatggat caggccaaag tcccgctaag 2940 attctccaat gttttcatgg tctgagcccc gctcctgttc ccatctccac tgcccctcgg 3000 ccctgtctgt gccctgcctc tcagaggagg gggctcagat ggtgcggcct gagtgtgcgg 3060 ccggcggcat ttgggataca cccgtagggt gggcggggtg tgtcccaggc ctaattccat 3120 ctttccacca tgacagagat gcccttgtga ggctggcctc cttggcgcct gtccccacgg 3180 cccccgcagc gtgagccacg atgctcccca taccccaccc attcccgata caccttactt 3240 actgtgtgtt ggcccagcca gagtgaggaa ggagtttggc cacattggag atggcggtag 3300 ctgagcagac atgcccccac gagtagcctg actccctggt gtgctcctgg aaggaagatc 3360 ttggggaccc ccccaccgga gcacacctag ggatcatctt tgcccgtctc ctggggaccc 3420 cccaagaaat gtggagtcct cgggggccgt gcactgatgc ggggagtgtg ggaagtctgg 3480 cggttggagg ggtgggtggg gggcagtggg ggctgggcgg ggggagttct ggggtaggaa 3540 gtggtcccgg gagattttgg atggaaaagt caggaggatt gacagcagac ttgcagaatt 3600 acatagagaa attaggaacc cccaaatttc atgtcaattg atctattccc cctctttgtt 3660 tcttggggca tttttccttt tttttttttt tttgtttttt ttttacccct ccttagcttt 3720 atgcgctcag aaaccaaatt aaaccccccc cccatgtaac aggggggcag tgacaaaagc 3780 aagaacgcac gaagccagcc tggagaccac cacgtcctgc cccccgccat ttatcgccct 3840 gattggattt tgtttttcat ctgtccctgt tgcttgggtt gagttgaggg tggagcctcc 3900 tggggggcac tggccactga gcccccttgg agaagtcaga ggggagtgga gaaggccact 3960 gtccggcctg gcttctgggg acagtggctg gtccccagaa gtcctgaggg cggagggggg 4020 ggttgggcag ggtctcctca ggtgtcagga gggtgctcgg aggccacagg agggggctcc 4080 tggctggcct gaggctggcc ggaggggaag gggctagcag gtgtgtaaac agagggttcc 4140 atcaggctgg ggcagggtgg ccgccttccg cacacttgag gaaccctccc ctctccctcg 4200 gtgacatctt gcccgcccct cagcaccctg ccttgtctcc aggaggtccg aagctctgtg 4260 ggacctcttg ggggcaaggt ggggtgaggc cggggagtag ggaggtcagg cgggtctgag 4320 cccacagagc aggagagctg ccaggtctgc ccatcgacca ggttgcttgg gccccggagc 4380 ccacgggtct ggtgatgcca tagcagccac caccgcggcg cctagggctg cggcagggac 4440 tcggcctctg ggaggtttac ctcgccccca cttgtgcccc cagctcagcc cccctgcacg 4500 cagcccgact agcagtctag aggcctgagg cttctgggtc ctggtgacgg ggctggcatg 4560 accccggggg tcgtccatgc cagtccgcct cagtcgcaga gggtccctcg gcaagcgccc 4620 tgtgagtggg ccattcggaa cattggacag aagcccaaag agccaaattg tcacaattgt 4680 ggaacccaca ttggcctgag atccaaaacg cttcgaggca ccccaaatta cctgcccatt 4740 cgtcaggaca cccacccacc cagtgttata ttctgcctcg ccggagtggg tgttcccggg 4800 ggcacttgcc gaccagcccc ttgcgtcccc aggtttgcag ctctcccctg ggccactaac 4860 catcctggcc cgggctgcct gtctgacctc cgtgcctagt cgtggctctc catcttgtct 4920 cctccccgtg tccccaatgt cttcagtggg gggccccctc ttgggtcccc tcctctgcca 4980 tcacctgaag acccccacgc caaacactga atgtcacctg tgcctgccgc ctcggtccac 5040 cttgcggccc gtgtttgact caactcaact cctttaacgc taatatttcc ggcaaaatcc 5100 catgcttggg ttttgtcttt aaccttgtaa cgcttgcaat cccaataaag cattaaaagt 5160 catgaaaaaa aaaaaaaaaa aa 5182 <210> 3 <211> 3814 <212> DNA <213> Artificial Sequence <220> <223> human surfactant protein C promoter sequence <400> 3 aagcttgaag actgctgctc tctaccacgt tagctcccct gtggcggagg atgactgtcc 60 taagagctca taggcacggg gcaaggggag cctggctgtc agctgcctgg gcttctagtc 120 ttgtgctttt tgcacaccag tccagggaac gaagaccact ggctttaaga tgcttcccca 180 gctgtcccca gactctgcca gcaggggatt ctctggtctg agcttaagtt gtgttctccc 240 agccagggat gcccctgccc tttgatgtct ccttgctgcc acacatttag ccgccctccc 300 catgccagct tggggggagg gaagcagtgg gggtagggag gtggctgggg cagctgggca 360 actgtcccca cccgtccctg gcacggctct gcccagtaca caaagagcaa agtgaatctt 420 gtccccaccc ctgcagctga ggggctggag gaggaaacgg ggaggcccac acagaagggg 480 tggccaccgt ggggctgtcc atcactcagg gctctcagag ggagtcaacc cagaaacaga 540 caaagagggt gagtctgggc tgtgttctta gctagtgaga ggtcccctag aggatgaagt 600 agatgatgct aatgaggatg actggatgtc acacccatga tgctattagg tcctcataat 660 agcatagtga ggtggacagc tagtacctga cccatctcac agatgaacat actaatgcct 720 aacaaagcag aacgactcac gctgggtccc agagctggcc agtggaagca ctgagacctc 780 cacatactga aggcatggac tattgaccgc tgttggtatt ggtctcatca ttgactatca 840 ttaagtgttg gctgtttgca tgcttcctgc ccagtggcag gttcaaagaa gcccgcagga 900 agcgtgctcc tttctttccc agggcccgca attgggctgg aagatagagc aacaaaaagc 960 gcccatgtaa ctcatgggaa cattcatgtg tgctgaatgg caggtgaagg tgccacagag 1020 aggctgagga tttcagaggg caccatgaac tggagtgagg tcgcagagca ggtgccattg 1080 gctcttggcc tgtttgggtg ggtggcattc agagaggtgg aaggtcagat gcactgttca 1140 cgcctgtaat ctcagcactt tggaaggcca aggtaggagg atcacacgag gccaggagat 1200 caacgctgca gtgagctatg aagctgtgat tgcaccactg cactgcagct tgggtaacag 1260 agtgagaccc tgtctctaaa taattaaata aataaaataa aaataaaacc ggagaagtgg 1320 agagggattg gaggtgggct ttcacagagg gagaaacggc ttaagtacag gccaaaaagt 1380 gagaaggctg cagacagggc tggtaggggg agggggaaat ttggcacacc cagctaaagg 1440 tccttctgtg tctccttctc caaggaaccc aagaccttca cttggttggt gtgagcactc 1500 caggaggcag gcaccctccc tcagccctca agcaagcaaa aatgggttta aaaaaagaag 1560 gagaagaagc agcagcagca gccgccacag agcttgtgac agctacagcc taagggcaac 1620 aggcagggga gaccaaggac cagaaagagc agaggctttt tcaaagaaag agatccctct 1680 cccagcaccc agcgatggcg gcaaacccca cccacagtgc ctgctaagaa caagtcccac 1740 gtgagaacaa catggccccc cgagatgccc acagggaccc cgagatgcct gcagtgcttg 1800 gctctcccgc tggccagctg cccacctggc tcaggcccag tactcgtgag tcagccgatc 1860 aatcccaatg ttgccaggat gatgggggcg ggagtaaggg ccctggggga gggcaggggt 1920 gggcactgca ggcgagctgt ctcccacatc tggcacctgc acacagctga ggccgagctg 1980 agaggatgct tctgtgggct ccccctcctc ccggcacccc tcccctcctt tcactgtcct 2040 aggacactct ctggctgctg gagtcttagg caaatattta aaggggcagc aagggggtga 2100 ggagggtggt gggagcaaac acttccctcc ctttcttcct ccctgcgctt ctcaggggct 2160 ctcagttcag atgccatgct gttatgcaac cttggggctg aaggccctcc agatggagag 2220 ggggacaggg gaacctgcca gctcatgacc gaagggcagg gcccaggtgg gagggggctg 2280 gggcagggga caggaactgg ggtggcatgt taaaggacag gaggctggtt gggcatggtg 2340 gctcacacct gtaatcctag cactttagga ggccgagatc acttgagccc aggagttcaa 2400 gaccagcctg ggcaacatgg tgaaaactca tctctataaa acaagcaaaa attagctggg 2460 cacaatggca tgcactggta gtcccagcta cttgggatgc tgaggtgtga ggatcaccgg 2520 agtccaggag gtcaacgctg cagtgagcag tgatctcgct actgcatgcc agcctgggtg 2580 ataaagtgag accctgtctc acaacaaaac aaaacaaaac aaaacaaaag gataggaggt 2640 taagggagcg agcccaggcc tggactctgc cacagtcacc tgagtttaga ggtgaaggga 2700 ctttagagac cacctggccc aaggagtgaa agggaacctg agagaaaggg tgagccagcc 2760 caaagtcatt ggcagatttg acctcgtaaa tacatagaga tggctttggg aaggcactag 2820 gaaagacaga gaaaagagaa ggagacagtc ctcaaagctg atcgtatttg ggtgagtatt 2880 attctcaggg caaatttagg atcagaggat gcagaaaggg gagtctagag gggtagagtg 2940 tagaccacag ggtgagtgag ctgattcgag gatggggaga ctgggagccc accagtgacc 3000 agagccagcc ctgttcaggg ctgtccgggc agaagaaagc agtgtcagac ctggaatctg 3060 ccatcagcac agcctgcaat tgacagacaa gcccagagca aagaaggaag cactgcacat 3120 gagtaagagc ttgccaccag tggggacaga gtttccagaa ttaggaaaat aatcactggg 3180 ggcaagtttg aggttggtac cagatatgtg ggaggaggca aggtaaggga aagagtactt 3240 gaagttggaa ctggtccttg cagggaaatg cacatttatg aaaccccgaa aactgatgtc 3300 aaagcacctc ctgccttggg cagagtcctc tcagagtcta caggtgctgc ctccagaacc 3360 ctcttcctgg agcgcatccc tatgtatcta gaaattctgc tgggaaatat gatggtcaga 3420 cccttggcca cctgaaaggt tcagggtggt agaagaaaaa ggaaagccac agggcagcag 3480 gggcaggtgc cagcaaggaa ggcaggcacg ccaggaagac acccatggtg agaagtgcag 3540 atggcccgag ggcaagtttg ctcaactcac ccaggtttgc tcttgctggg gccaagagga 3600 ctcatgtgcc agggccaagg gcccttgggg gctctcacag ggggcttatc tgggcttcgg 3660 ttctggaggg ccaggaacaa acaggcttca aagccaaggg cttggctggc acacaggggg 3720 cttggtcctt cacctctgtc ccctctccct acggacacat ataagaccct ggtcacacct 3780 gggagaggag gagaggagag catagcacct gcag 3814 <110> SNU R & DB FOUNDATION <120> Development of a novel emphysema-related animal model using insulin-like growth factor 2 (IGF2) <130> PB14-12106 <150> KR 13/083697 <151> 2013-07-16 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 180 <212> PRT <213> Homo sapiens <400> 1 Met Gly Ile Pro Met Gly Lys Ser Met Leu Val Leu Leu Thr Phe Leu 1 5 10 15 Ala Phe Ala Ser Cys Cys Ile Ala Ala Tyr Arg Pro Ser Glu Thr Leu 20 25 30 Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys Gly Asp Arg 35 40 45 Gly Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Ser Arg Arg Ser Arg 50 55 60 Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu Leu 65 70 75 80 Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu Arg Asp Val Ser Thr 85 90 95 Pro Pro Thr Val Leu Pro Asp Phe Pro Arg Tyr Pro Val Gly Lys 100 105 110 Phe Phe Gln Tyr Asp Thr Trp Lys Gln Ser Thr Gln Arg Leu Arg Arg 115 120 125 Gly Leu Pro Ala Leu Leu Arg Ala Arg Arg Gly His Val Leu Ala Lys 130 135 140 Glu Leu Glu Ala Phe Arg Glu Ala Lys Arg His Arg Pro Leu Ile Ala 145 150 155 160 Leu Pro Thr Gln Asp Pro Ala His Gly Gly Ala Pro Pro Glu Met Ala 165 170 175 Ser Asn Arg Lys 180 <210> 2 <211> 5182 <212> DNA <213> Homo sapiens <400> 2 cgcctgtccc cctcccgagg cccgggctcg cgacggcaga gggctccgtc ggcccaaacc 60 gagctgggcg cccgcggtcc gggtgcagcc tccactccgc cccccagtca ccgcctcccc 120 cggcccctcg acgtggcgcc cttccctccg cttctctgtg ctccccgcgc ccctcttggc 180 gtctggcccc ggcccccgct ctttctcccg caaccttccc ttcgctccct cccgtccccc 240 ccagctccta gcctccgact ccctcccccc ctcacgcccg ccctctcgcc ttcgccgaac 300 caaagtggat taattacacg ctttctgttt ctctccgtgc tgttctctcc cgctgtgcgc 360 ctgcccgcct ctcgctgtcc tctctccccc tcgccctctc ttcggccccc ccctttcacg 420 ttcactctgt ctctcccact atctctgccc ccctctatcc ttgatacaac agctgacctc 480 atttcccgat accttttccc ccccgaaaag tacaacatct ggcccgcccc agcccgaaga 540 cagcccgtcc tccctggaca atcagacgaa ttctcccccc ccccccaaaa aaaagccatc 600 cccccgctct gccccgtcgc acattcggcc cccgcgactc ggccagagcg gcgctggcag 660 aggagtgtcc ggcaggaggg ccaacgcccg ctgttcggtt tgcgacacgc agcagggagg 720 tgggcggcag cgtcgccggc ttccagacac caatgggaat cccaatgggg aagtcgatgc 780 tggtgcttct caccttcttg gccttcgcct cgtgctgcat tgctgcttac cgccccagtg 840 agaccctgtg cggcggggag ctggtggaca ccctccagtt cgtctgtggg gaccgcggct 900 tctacttcag caggcccgca agccgtgtga gccgtcgcag ccgtggcatc gttgaggagt 960 gctgtttccg cagctgtgac ctggccctcc tggagacgta ctgtgctacc cccgccaagt 1020 ccgagaggga cgtgtcgacc cctccgaccg tgcttccgga caacttcccc agataccccg 1080 tgggcaagtt cttccaatat gacacctgga agcagtccac ccagcgcctg cgcaggggcc 1140 tgcctgccct cctgcgtgcc cgccggggtc acgtgctcgc caaggagctc gaggcgttca 1200 gggaggccaa acgtcaccgt cccctgattg ctctacccac ccaagacccc gcccacgggg 1260 gcgccccccc agagatggcc agcaatcgga agtgagcaaa actgccgcaa gtctgcagcc 1320 cggcgccacc atcctgcagc ctcctcctga ccacggacgt ttccatcagg ttccatcccg 1380 aaaatctctc ggttccacgt ccccctgggg cttctcctga cccagtcccc gtgccccgcc 1440 tccccgaaac aggctactct cctcggcccc ctccatcggg ctgaggaagc acagcagcat 1500 cttcaaacat gtacaaaatc gattggcttt aaacaccctt cacataccct ccccccaaat 1560 tatccccaat tatccccaca cataaaaaat caaaacatta aactaacccc cttccccccc 1620 ccccacaaca accctcttaa aactaattgg ctttttagaa acaccccaca aaagctcaga 1680 aattggcttt aaaaaaaaca accaccaaaa aaaatcaatt ggctaaaaaa aaaaagtatt 1740 aaaaacgaat tggctgagaa acaattggca aaataaagga atttggcact ccccaccccc 1800 ctctttctct tctcccttgg actttgagtc aaattggcct ggacttgagt ccctgaacca 1860 gcaaagagaa aagaaggacc ccagaaatca caggtgggca cgtcgctgct accgccatct 1920 cccttctcac gggaattttc agggtaaact ggccatccga aaatagcaac aacccagact 1980 ggctcctcac tcccttttcc atcactaaaa atcacagagc agtcagaggg acccagtaag 2040 accaaaggag gggaggacag agcatgaaaa ccaaaatcca tgcaaatgaa atgtaattgg 2100 cacgaccctc acccccaaat cttacatctc aattcccatc ctaaaaagca ctcatacttt 2160 atgcatcccc gcagctacac acacacaaca cacagcacac gcatgaacac agcacacaca 2220 cgagcacagc acacacacaa acgcacagca cacacagcac acagatgagc acacagcaca 2280 cacacaaacg cacagcacac acacgcacac acatgcacac acagcacaca aacgcacggc 2340 acacacacgc acacacatgc acacacagca cacacacaaa cgcacagcac acacaaacgc 2400 acagcacaca cgcacacaca gcacacacac gagcacacag cacacaaacg cacagcacac 2460 gcacacacat gcacacacag cacacacact agcacacagc acacacacaa agacacagca 2520 cacacatgca cacacagcac acacacgcga acacaca cacgaacaca gcacacacag 2580 cacacacaca aacacagcac acacatgcac acagcacacg cacacacagc acacacatga 2640 acaccacca cagcacacac atgcacacac agcacacacg catgcacagc acacatgaac 2700 acagcacaca cacaaacaca cagcacacac atgcacacac agcacacaca ctcatgcgca 2760 gcacatacat gaacacagct cacagcacac aaacacgcag cacacacgtt gcacacgcaa 2820 gcacccacct gcacacacac atgcgcacac acacgcacac ccccacaaaa ttggatgaaa 2880 acaataagca tatctaagca actacgatat ctgtatggat caggccaaag tcccgctaag 2940 attctccaat gttttcatgg tctgagcccc gctcctgttc ccatctccac tgcccctcgg 3000 ccctgtctgt gccctgcctc tcagaggagg gggctcagat ggtgcggcct gagtgtgcgg 3060 ccggcggcat ttgggataca cccgtagggt gggcggggtg tgtcccaggc ctaattccat 3120 ctttccacca tgacagagat gcccttgtga ggctggcctc cttggcgcct gtccccacgg 3180 cccccgcagc gtgagccacg atgctcccca taccccaccc attcccgata caccttactt 3240 actgtgtgtt ggcccagcca gagtgaggaa ggagtttggc cacattggag atggcggtag 3300 ctgagcagac atgcccccac gagtagcctg actccctggt gtgctcctgg aaggaagatc 3360 ttggggaccc ccccaccgga gcacacctag ggatcatctt tgcccgtctc ctggggaccc 3420 cccaagaaat gtggagtcct cgggggccgt gcactgatgc ggggagtgtg ggaagtctgg 3480 cggttggagg ggtgggtggg gggcagtggg ggctgggcgg ggggagttct ggggtaggaa 3540 gtggtcccgg gagattttgg atggaaaagt caggaggatt gacagcagac ttgcagaatt 3600 acatagagaa attaggaacc cccaaatttc atgtcaattg atctattccc cctctttgtt 3660 tcttggggca tttttccttt tttttttttt tttgtttttt ttttacccct ccttagcttt 3720 atgcgctcag aaaccaaatt aaaccccccc cccatgtaac aggggggcag tgacaaaagc 3780 aagaacgcac gaagccagcc tggagaccac cacgtcctgc cccccgccat ttatcgccct 3840 gattggattt tgtttttcat ctgtccctgt tgcttgggtt gagttgaggg tggagcctcc 3900 tggggggcac tggccactga gcccccttgg agaagtcaga ggggagtgga gaaggccact 3960 gtccggcctg gcttctgggg acagtggctg gtccccagaa gtcctgaggg cggagggggg 4020 ggttgggcag ggtctcctca ggtgtcagga gggtgctcgg aggccacagg agggggctcc 4080 tggctggcct gaggctggcc ggaggggaag gggctagcag gtgtgtaaac agagggttcc 4140 atcaggctgg ggcagggtgg ccgccttccg cacacttgag gaaccctccc ctctccctcg 4200 gtgacatctt gcccgcccct cagcaccctg ccttgtctcc aggaggtccg aagctctgtg 4260 ggacctcttg ggggcaaggt ggggtgaggc cggggagtag ggaggtcagg cgggtctgag 4320 cccacagagc aggagagctg ccaggtctgc ccatcgacca ggttgcttgg gccccggagc 4380 ccacgggtct ggtgatgcca tagcagccac caccgcggcg cctagggctg cggcagggac 4440 tcggcctctg ggaggtttac ctcgccccca cttgtgcccc cagctcagcc cccctgcacg 4500 cagcccgact agcagtctag aggcctgagg cttctgggtc ctggtgacgg ggctggcatg 4560 accccggggg tcgtccatgc cagtccgcct cagtcgcaga gggtccctcg gcaagcgccc 4620 tgtgagtggg ccattcggaa cattggacag aagcccaaag agccaaattg tcacaattgt 4680 ggaacccaca ttggcctgag atccaaaacg cttcgaggca ccccaaatta cctgcccatt 4740 cgtcaggaca cccacccacc cagtgttata ttctgcctcg ccggagtggg tgttcccggg 4800 ggcacttgcc gaccagcccc ttgcgtcccc aggtttgcag ctctcccctg ggccactaac 4860 catcctggcc cgggctgcct gtctgacctc cgtgcctagt cgtggctctc catcttgtct 4920 cctccccgtg tccccaatgt cttcagtggg gggccccctc ttgggtcccc tcctctgcca 4980 tcacctgaag acccccacgc caaacactga atgtcacctg tgcctgccgc ctcggtccac 5040 cttgcggccc gtgtttgact caactcaact cctttaacgc taatatttcc ggcaaaatcc 5100 catgcttggg ttttgtcttt aaccttgtaa cgcttgcaat cccaataaag cattaaaagt 5160 catgaaaaaa aaaaaaaaaa aa 5182 <210> 3 <211> 3814 <212> DNA <213> Artificial Sequence <220> <223> human surfactant protein C promoter sequence <400> 3 aagcttgaag actgctgctc tctaccacgt tagctcccct gtggcggagg atgactgtcc 60 taagagctca taggcacggg gcaaggggag cctggctgtc agctgcctgg gcttctagtc 120 ttgtgctttt tgcacaccag tccagggaac gaagaccact ggctttaaga tgcttcccca 180 gctgtcccca gactctgcca gcaggggatt ctctggtctg agcttaagtt gtgttctccc 240 agccagggat gcccctgccc tttgatgtct ccttgctgcc acacatttag ccgccctccc 300 catgccagct tggggggagg gaagcagtgg gggtagggag gtggctgggg cagctgggca 360 actgtcccca cccgtccctg gcacggctct gcccagtaca caaagagcaa agtgaatctt 420 gtccccaccc ctgcagctga ggggctggag gaggaaacgg ggaggcccac acagaagggg 480 tggccaccgt ggggctgtcc atcactcagg gctctcagag ggagtcaacc cagaaacaga 540 caaagagggt gagtctgggc tgtgttctta gctagtgaga ggtcccctag aggatgaagt 600 agatgatgct aatgaggatg actggatgtc acacccatga tgctattagg tcctcataat 660 agcatagtga ggtggacagc tagtacctga cccatctcac agatgaacat actaatgcct 720 aacaaagcag aacgactcac gctgggtccc agagctggcc agtggaagca ctgagacctc 780 cacatactga aggcatggac tattgaccgc tgttggtatt ggtctcatca ttgactatca 840 ttaagtgttg gctgtttgca tgcttcctgc ccagtggcag gttcaaagaa gcccgcagga 900 agcgtgctcc tttctttccc agggcccgca attgggctgg aagatagagc aacaaaaagc 960 gcccatgtaa ctcatgggaa cattcatgtg tgctgaatgg caggtgaagg tgccacagag 1020 aggctgagga tttcagaggg caccatgaac tggagtgagg tcgcagagca ggtgccattg 1080 gctcttggcc tgtttgggtg ggtggcattc agagaggtgg aaggtcagat gcactgttca 1140 cgcctgtaat ctcagcactt tggaaggcca aggtaggagg atcacacgag gccaggagat 1200 caacgctgca gtgagctatg aagctgtgat tgcaccactg cactgcagct tgggtaacag 1260 agtgagaccc tgtctctaaa taattaaata aataaaataa aaataaaacc ggagaagtgg 1320 agagggattg gaggtgggct ttcacagagg gagaaacggc ttaagtacag gccaaaaagt 1380 gagaaggctg cagacagggc tggtaggggg agggggaaat ttggcacacc cagctaaagg 1440 tccttctgtg tctccttctc caaggaaccc aagaccttca cttggttggt gtgagcactc 1500 caggaggcag gcaccctccc tcagccctca agcaagcaaa aatgggttta aaaaaagaag 1560 gagaagaagc agcagcagca gccgccacag agcttgtgac agctacagcc taagggcaac 1620 aggcagggga gaccaaggac cagaaagagc agaggctttt tcaaagaaag agatccctct 1680 cccagcaccc agcgatggcg gcaaacccca cccacagtgc ctgctaagaa caagtcccac 1740 gtgagaacaa catggccccc cgagatgccc acagggaccc cgagatgcct gcagtgcttg 1800 gctctcccgc tggccagctg cccacctggc tcaggcccag tactcgtgag tcagccgatc 1860 aatcccaatg ttgccaggat gatgggggcg ggagtaaggg ccctggggga gggcaggggt 1920 gggcactgca ggcgagctgt ctcccacatc tggcacctgc acacagctga ggccgagctg 1980 agaggatgct tctgtgggct ccccctcctc ccggcacccc tcccctcctt tcactgtcct 2040 aggacactct ctggctgctg gagtcttagg caaatattta aaggggcagc aagggggtga 2100 ggagggtggt gggagcaaac acttccctcc ctttcttcct ccctgcgctt ctcaggggct 2160 ctcagttcag atgccatgct gttatgcaac cttggggctg aaggccctcc agatggagag 2220 ggggacaggg gaacctgcca gctcatgacc gaagggcagg gcccaggtgg gagggggctg 2280 gggcagggga caggaactgg ggtggcatgt taaaggacag gaggctggtt gggcatggtg 2340 gctcacacct gtaatcctag cactttagga ggccgagatc acttgagccc aggagttcaa 2400 gaccagcctg ggcaacatgg tgaaaactca tctctataaa acaagcaaaa attagctggg 2460 cacaatggca tgcactggta gtcccagcta cttgggatgc tgaggtgtga ggatcaccgg 2520 agtccaggag gtcaacgctg cagtgagcag tgatctcgct actgcatgcc agcctgggtg 2580 ataaagtgag accctgtctc acaacaaaac aaaacaaaac aaaacaaaag gataggaggt 2640 taagggagcg agcccaggcc tggactctgc cacagtcacc tgagtttaga ggtgaaggga 2700 ctttagagac cacctggccc aaggagtgaa agggaacctg agagaaaggg tgagccagcc 2760 caaagtcatt ggcagatttg acctcgtaaa tacatagaga tggctttggg aaggcactag 2820 gaaagacaga gaaaagagaa ggagacagtc ctcaaagctg atcgtatttg ggtgagtatt 2880 attctcaggg caaatttagg atcagaggat gcagaaaggg gagtctagag gggtagagtg 2940 tagaccacag ggtgagtgag ctgattcgag gatggggaga ctgggagccc accagtgacc 3000 agagccagcc ctgttcaggg ctgtccgggc agaagaaagc agtgtcagac ctggaatctg 3060 ccatcagcac agcctgcaat tgacagacaa gcccagagca aagaaggaag cactgcacat 3120 gagtaagagc ttgccaccag tggggacaga gtttccagaa ttaggaaaat aatcactggg 3180 ggcaagtttg aggttggtac cagatatgtg ggaggaggca aggtaaggga aagagtactt 3240 gaagttggaa ctggtccttg cagggaaatg cacatttatg aaaccccgaa aactgatgtc 3300 aaagcacctc ctgccttggg cagagtcctc tcagagtcta caggtgctgc ctccagaacc 3360 ctcttcctgg agcgcatccc tatgtatcta gaaattctgc tgggaaatat gatggtcaga 3420 cccttggcca cctgaaaggt tcagggtggt agaagaaaaa ggaaagccac agggcagcag 3480 gggcaggtgc cagcaaggaa ggcaggcacg ccaggaagac acccatggtg agaagtgcag 3540 atggcccgag ggcaagtttg ctcaactcac ccaggtttgc tcttgctggg gccaagagga 3600 ctcatgtgcc agggccaagg gcccttgggg gctctcacag ggggcttatc tgggcttcgg 3660 ttctggaggg ccaggaacaa acaggcttca aagccaaggg cttggctggc acacaggggg 3720 cttggtcctt cacctctgtc ccctctccct acggacacat ataagaccct ggtcacacct 3780 gggagaggag gagaggagag catagcacct gcag 3814
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