KR101987851B1 - Animal model for age-related macular degeneration using shiga toxin type 1 B-subunit derived from Enterohamorrhagic Escherichia coli. and the screening method using the same - Google Patents
Animal model for age-related macular degeneration using shiga toxin type 1 B-subunit derived from Enterohamorrhagic Escherichia coli. and the screening method using the same Download PDFInfo
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- KR101987851B1 KR101987851B1 KR1020170055168A KR20170055168A KR101987851B1 KR 101987851 B1 KR101987851 B1 KR 101987851B1 KR 1020170055168 A KR1020170055168 A KR 1020170055168A KR 20170055168 A KR20170055168 A KR 20170055168A KR 101987851 B1 KR101987851 B1 KR 101987851B1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
본 발명은 시가톡신 타입 1 B-서브유닛이 RPE세포막 표면상에 발현하는 Gb3수용체를 통하여 RPE 세포내로 전달되고 이를 통하여 망막색소상피세포(RPE)가 세포괴사 또는 부착성상실이 됨으로써 황반변성이 발생되는 점을 이용한 동물모델 제조방법 및 이를 이용한 스크리닝 방법에 관한 것이다. The present invention is based on the finding that the cytotoxin type 1 B-subunit is transferred into the RPE cells through the Gb3 receptor expressed on the surface of the RPE cell membrane, thereby causing retinal pigment epithelial cells (RPE) to become necrotic or adherent, And a screening method using the same.
Description
본 발명은 노인성 황반변성에 대한 치료제 또는 치료방법의 효과를 검증하기 위한 동물모델, 이의 제조방법 및 이를 이용한 스크리닝 방법에 관한 것이다.The present invention relates to an animal model for verifying the effect of a therapeutic agent or therapeutic method on senile AMD, a method for producing the same, and a screening method using the same.
노인성 황반변성(AMD ; age-related macular degeneration)이라고도 하는 황반변성은 시력 상실을 일으키는 질병이다. Cleveland Clinic에 따르면, AMD는 50세가 넘는 다수의 사람에게서 나타나는 흔한 시력 상실의 원인중 하나이다. AMD에는 건성(atrophic), 습성(exudative) 두 가지 유형으로 구분할 수 있으며, 건성 노인성 황반변성은 시력변화가 초기에 발생할 수 있으나 단순히 노화로 인한 증상으로 알려져 있다. 습성 노인성 황반변성은 중증으로 진행된 형태의 황반변성이며 비정상적 혈관이 황반과 망막 아래에서 성장하여 건강한 세포들을 파괴시켜 결국 시력상실로 이어질 수 있다. AMD는 눈의 광원들을 구분하여 분명하게 볼 수 있게 도와주는 망막의 중앙부분의 황반 내부의 세포들이 괴사되거나 파괴될 때 발생한다. AMD의 경우 주로 망막색소상피(RPE; Retinal pigmental epithelium)의 손상으로 기인한 퇴화성 병태(degeneration), 변성(alteration)이나 괴사(apoptosis) 등으로 특징지어 왔다. 아직까지 노인성 황반변성에 대한 치료법이 개발되어 있지 않으며 대부분 증상이 없는 상황으로 비정상적 혈관을 파괴하는 레이저 수술과 항혈관 내피세포성장인자(VEGF)주사를 통해 시력상실 속도를 늦추는 효과 정도만 보고 있는 실정이다. 황반변성은 영구적인 병태로 AMD 환자는 인구의 노령화와 맞물려 2030년 정도까지 630만명에 달할 것으로 예상되며 노인들의 황반변성에 통증이 없으므로 양쪽눈의 시력이 완전히 상실될 때까지 알 수 없는 문제가 있다. 현재까지 인간 망막색소상피세포에 특이적으로 병태를 야기하여 괴사시킴으로써 유도되는 AMD모델에 대한 기법이 개발되고 있지 않아 정확한 병인을 타겟으로 하여 치료되는 치료물질이나 치료방법을 개발하는데 어려움이 있다. 따라서 RPE세포 괴사 유도법을 활용한 AMD 평가모델의 개발은 매우 요구되는 기술이라 할 수 있다. AMD, also known as age-related macular degeneration (AMD), is a disease that causes blindness. According to the Cleveland Clinic, AMD is one of the common causes of vision loss in many people over the age of 50. AMD can be divided into two types: atrophic and exudative. Dry AMD can be caused by early changes in vision, but is known to be simply a symptom of aging. Mature senile AMD is a progressive form of macular degeneration and abnormal blood vessels may grow below the macula and retina, destroying healthy cells and eventually leading to loss of vision. AMD occurs when cells inside the macula in the central part of the retina, which help distinguish and clearly distinguish the light sources of the eye, are either necrotic or destroyed. AMD has been characterized primarily by degeneration, alteration, or apoptosis caused by damage to the retinal pigment epithelium (RPE). There is no treatment for senile AMD. Most of the cases have no symptoms and the effect of laser surgery to destroy abnormal blood vessels and vascular endothelial growth factor (VEGF) injections slows down the rate of visual loss. Macular degeneration is a permanent condition and AMD patients are expected to reach 6.3 million by 2030 in conjunction with the aging of the population, and there is no pain in the macular degeneration of the elderly, so there is an unknown problem until both eyes are completely lost sight. There has been no development of a technique for an AMD model induced by necrosis caused by specific pathological conditions in human retinal pigment epithelial cells and thus it is difficult to develop a therapeutic substance or a therapeutic method for treating a precise pathogen. Therefore, development of AMD evaluation model using RPE cell necrosis induction method is a very demanding technology.
한편, 장출혈성 대장균으로부터 분비되어 지는 시가독소(Shiga toxin)는 정교한 숙주세포 수용체 Gb3타깃과 세포막 투과침투능력을 보유하고 있다. 장출혈성 대장균(EHEC)은 인체 치사성 변종 시가독소 생성 대장균(Shiga toxin-producing E.coli;STEC) 발생에 종종 관여되는데, 독소의 전신성 감염능이 위협적이다. 특히나 이러한 치사성 독소인 시가독소는 감염 후 점막 상피를 투과해 혈류를 타고 신장기능을 파괴하여 급성 신부전증과 뇌혈관막을 용혈 침투하여 중추 뇌신경계통 마비의 원인이 되는 것으로 알려져 있다. 대부분의 병원성 세균의 기전처럼 변종 시가독소 생성균 역시 인체세포에 치사성 독소배출 및 감염 후 숙주세포의 정상과정에 관여하여 병원성 발병에 적대적인 숙주 환경을 감염균에 우호적인 환경으로 전환시킨다. 독소 단백질에 대한 명확한 기능 및 기전이 현재까지 활발히 연구가 진행중이나, 인체 내 핵심타깃 및 경로 등의 연구 미흡으로 만족할 만한 효과적인 백신이 개발되지 못하고 있는 실정이다. 이 미생물 유래 단백질 시가독소(Shiga toxin)는 AB5구조 형태이며, A-subunit의 N-glycosidase 단백질 합성저해 효소능을 보유한 독성부분으로 인해 급성 신장기능장애 용혈성 요독증후군 및 신경독성 합병증을 유발하는 것으로 알려져 왔으며, 지난 20년간 숙주세포 병리기전 연구가 상당부분 검증되어 왔다. 그러나 비독성 StxB-서브유닛 단백질 부분은 숙주세포막을 Gb3수용체 매개로 통과하여 세포 내 전달능이 뛰어나므로 암세포 살상을 위한 약물 conjugation를 통한 항암치료제 개발 등에 이용되는 사례가 보고되고 있을 뿐이다. 신변종 장출혈성균에서 배출되는 시가독소(Shiga toxin)가 숙주의 생체 필수 단백질 합성 및 전반적 번역 (translation)과정을 저해함으로써 면역력을 저하시키거나, 괴사시킨다는 연구 보고가 있다 (Johaness Nature review 2010). 또한 이러한 미생물유래 비독성 시가독소 B-서브유닛(StxB-sub)은 이미 숙주세포내 독성을 나타내지 않으며 MAPK, NF-kB나 염증조절 복합체활성(inflammasome)에 전혀 영향을 주지 않아, 과염증 반응유발이 없는 단지 세포내부전달(Intracellular trafficking)기능만을 담당하는 것이 정립된 연구결과이므로 안전한 재조합 단백질 효능개선 물질로 사용이 가능하다고 보여진다. On the other hand, Shiga toxin secreted from intestinal hemorrhagic Escherichia coli possesses elaborate host cell receptor Gb3 target and transmembrane permeability. Enterohemorrhagic Escherichia coli (EHEC) is often involved in the pathogenesis of Shiga toxin-producing E. coli (STEC), a systemic lethal variant. Systemic infectivity of toxins is a threat. In particular, these toxic toxins, which are lethal toxins, are transmitted through the mucosal epithelium after infection and destroy the kidney function in the bloodstream, causing hemolytic infiltration of acute renal failure and cerebral vascular membrane, which is known to cause central nervous system paralysis. Like most pathogenic bacterial pathogens, the variant cytotoxin-producing bacteria also participate in the normal processes of host cells after lethal toxin release and infection in human cells, transforming the host environment hostile to host pathogenic bacteria into a favorable environment for the pathogens. Although a definite function and mechanism of toxin protein has been actively researched so far, research on key target and pathway in the human body is insufficient, so that a satisfactory effective vaccine has not been developed. This microorganism-derived protein, Shiga toxin, is in the form of AB 5 , which is responsible for the acute kidney dysfunction, hemolytic uremic toxicity and neurotoxic complications due to the toxic part of the A-subunit that inhibits N-glycosidase protein synthesis Has been known, and research on host cell pathology has been largely verified for the last 20 years. However, since the non-toxic StxB-subunit protein moiety passes through the Gb3 receptor mediator of the host cell membrane and is superior in intracellular delivery capability, there have been reported cases in which the drug conjugation for cancer cell death is used for the development of anticancer drugs. There are reports that Shiga toxin released from the genitourinary elongation haemorrhagic germs reduces immunity or necrosis by inhibiting the host's essential protein synthesis and translation process (Johaness Nature review 2010). In addition, these microorganism-derived nontoxic cytotoxic B-subunits (StxB-sub) do not already exhibit intracellular toxicity and have no effect on MAPK, NF-kB or inflammatory mediators, And it is believed that it can be used as a safe recombinant protein efficacy improving material since it is established that it is only responsible for intracellular trafficking function.
반면, 시가 독소의 B 서브유닛을 이용한 기술로는 항원 특이적 CD8 면역 반응을 증가시키는 백신에 적용한 기술이 공지되어 있으며, 노인성 황반변성 동물모델을 제조하는 방법의 선행기술로는 엡스타인 바 바이러스(Epstein Barr virus; EBV) 감염 인간망막색소상피세포(human retinal pigment epithelial cells; ARPE)를 이용한 혈관신생 질환 스크린용 세포모델을 제조하는 방법, HTRA1 폴리펩티드 또는 HTRA1 유도체를 암호화하는 유전자를 포함하는 DNA 및 발현벡터를 이용한 방법, NPC 유전자 돌연변이로 인해 유도된 노인성 황반변성 모델 제조방법, actr1b, dynactin3, 또는 dynactin4 유전자 기능 결손으로 유도하는 방법을 이용하여 동물모델을 준비하는 기술들이 공지되어 있다. 그러나 이러한 동물모델 제조기술은 장기간의 제조과정이 요구되며 유전자 조작에 의한 동물모델이므로 신규 치료제를 평가할 때 망막색소상피세포의 손상에 대한 효과의 지표로 보기에는 한계가 있는 문제점이 있었다.On the other hand, techniques employing the B subunit of the cigarette toxin are known to be applied to a vaccine that increases the antigen-specific CD8 immune response, and prior art methods for producing the senescent macular degeneration model include Epstein A method for producing a cell model for angiogenesis screen using Barr virus (EBV) infected human retinal pigment epithelial cells (ARPE), a DNA comprising a gene encoding HTRA1 polypeptide or HTRA1 derivative, and an expression vector There are known techniques for preparing an animal model using a method using an NPC gene mutation, a method for preparing a senile AMD model induced by NPC gene mutation, and a method for deriving actrlb, dynactin3, or dynactin4 gene function deficiency. However, since such an animal model manufacturing technique requires a long-term manufacturing process and is an animal model by gene manipulation, there is a limit in view of the effect of the new therapeutic agent on the damage of retinal pigment epithelial cells.
현재까지 시가독도 B-subunit은 눈을 제외한 조직내 세포들 (신장, 장관, 뇌 등)에서는 독성을 나타낸다는 보고가 없다. 또한 동물실험에서 조차 wild-type Shiga toxin (20ng/kg)을 투여한 마우스에서는 4일 이후 생존하지 못했으나, B-subunit만을 투여한 마우스는 생존에 전혀 영향을 주지 못하였다. Until now, there has been no report that poisonous Dokdo B-subunit shows toxicity in tissues other than eyes (kidney, liver, brain, etc.). Furthermore, even in animal studies, mice that received wild-type Shiga toxin (20 ng / kg) did not survive after 4 days, but mice administered only B-subunit did not affect survival at all.
그러나 흥미롭게도 인간 망막 황반변성에 병리적으로 연관된 망막색소상피의 RPE세포에 시가독소타입 1 B-서브유닛(Stx1B-subunit)을 처리하였을 때, 24, 36 및 48 시간까지 지속적으로 인간 망막 색소상피의 손상과 세포 괴사 인지분자지표인 caspase-3/7 활성을 유의적 수치로 유도되는 것을 확인하였고, 이를 기반으로 본 발명인 황반변성모델 및 이를 이용한 스크리닝 방법을 완성하게 되었다.Interestingly, when RPE cells of the retinal pigment epithelium pathologically associated with human retinal macular degeneration were treated with a ciguatoxin-type 1 B-subunit (Stx1B-subunit), human RPE cells were continuously exposed for 24, 36 and 48 hours Caspase-3/7 activity, which is a molecular marker of damage and cell necrosis, was induced to a significant level. Based on this, the present invention's macular deformation model and screening method using the same were completed.
본 발명은 노인성 황반변성 치료제 또는 치료방법을 보다 효율적으로 개발하기 위해 시가독소 타입 1 B-서브유닛을 이용한 노인성 황반변성 동물모델 및 이의 제조방법, 이를 이용한 스크리닝 방법을 제공하는데 목적이 있다.The present invention provides an age-matched macular degeneration animal model using a cigarette toxin type 1 B-subunit, a method for producing the same, and a screening method using the same, in order to more effectively develop a therapeutic or therapeutic agent for senile AMD.
본 발명은 a) 인간을 제외한 동물의 망막에 시가독소 타입 1 B-서브유닛(Stx1B-subunit)를 주입하여 처리하는 단계; b) 상기 시가독소 타입 1 B-서브유닛이 망막색소상피세포(RPE)를 손상시켜 황반변성이 유도되는 단계;를 포함하는 황반변성 동물모델 제조방법을 제공한다. The present invention provides a method of treating a retina of an animal other than a human by injecting a ciguatoxin type 1 B-subunit (Stx1B-subunit) into the retina; b) the above-described cigarette toxin type 1 B-subunit damages RPE to induce macular degeneration.
또한 상기 제조방법에서 황반변성은 24 내지 48시간 내에 유도되고, 상기 황반변성은 건성 또는 습성일 수 있다. Also, in this method, the macular degeneration is induced within 24 to 48 hours, and the macular degeneration can be dry or wet.
또 다른 측면에서 본 발명은 상기 황반변성이 노인성인 동물모델 제조방법을 제공한다.In another aspect, the present invention provides a method for preparing an animal model in which the macular degeneration is senile.
여기서, 상기 동물은 원숭이, 개, 고양이, 토끼, 모르모트, 랫트, 마우스, 소, 양, 돼지 및 염소로 이루어진 군에서 선택된 비인간 척추동물을 포함한다.Herein, the animal includes a non-human vertebrate selected from the group consisting of a monkey, a dog, a cat, a rabbit, a guinea pig, a rat, a mouse, a cattle, a sheep, a pig and chlorine.
본 발명의 제조방법에 사용되는 시가독소 타입 1 B-서브유닛은 RPE세포막 표면상에 발현하는 Gb3수용체를 통하여 RPE 세포내로 전달되어 황반변성을 유도하는 것을 특징으로 한다. 상기 손상은 RPE세포괴사 또는 부착성상실에 의한 것이다.The cigarette toxin type 1 B-subunit used in the production method of the present invention is characterized in that it is transferred into RPE cells through a Gb3 receptor expressed on the surface of the RPE cell membrane to induce macular degeneration. The damage is due to RPE cell necrosis or loss of adhesion.
또한 본 발명은 상기 제조방법에 의해 제조된 황반변성 동물모델을 제공하며, 이에 따른 황반변성 동물모델에 황반변성 치료제 또는 치료제 후보 물질을 투여하여 황반변성의 치유 정도를 지표로 하여, 상기 황반변성 치료제 또는 치료제 후보물질을 스크리닝하는 방법도 제공한다.In addition, the present invention provides a macroreticular animal model prepared by the above-described method, wherein the macular degeneration animal model is administered with a macular degeneration therapeutic agent or a therapeutic agent, and the degree of healing of macular degeneration is used as an index, Methods of screening candidate therapeutic agents are also provided.
본 발명은 시가독소 타입 1 B-서브유닛을 이용한 노인성 황반변성 동물모델 및 이의 제조방법, 이를 이용한 스크리닝 방법을 제공함으로써, 황반변성 치료제 및 치료방법을 효과적으로 개발하는데 적절한 지표로 활용될 수 있다.The present invention can be used as an appropriate index for effectively developing a macular degeneration therapeutic agent and a therapeutic method by providing a senile AMD-like animal model using a cigarette toxin type 1 B-subunit, a method for producing the same, and a screening method using the same.
또한, 본 발명에서 제공하는 시가독소 타입 1 B-서브유닛을 이용하여 황반변성을 유도하는 방법을 통하여 황반변성 동물모델을 신속하게 공급할 수 있으며, 스크리닝 기간을 단축하여 치료제 개발에 소요되는 비용을 감소시킬 수 있다.In addition, it is possible to rapidly supply a macular degeneration model through the method of inducing the macular degeneration using the cigarette toxin type 1 B-subunit provided by the present invention, and it is possible to reduce the cost for development of the therapeutic agent by shortening the screening period .
도 1은 망막상피세포막표면(RPE membrane)에 발현하는 Gb3 수용체를 나타낸다.
도 2는 망막색소상피세포(RPE) 표면의 Gb3 수용체 매개로 세포내 전달된 타입 1 B-subunit [(alexa 488 형광접합); DAPI세포핵 염색]을 나타낸다.
도 3은 인간 망막색소상피세포(RPE)에서의 시가독소 타입 1 B-서브유닛 (Stx1 B-subunit)에 의한 세포괴사 정도의 지표인 caspase-3/7활성 측정 결과를 나타낸다.
도 4는 타입 1 B-서브유닛(Stx1B-subunit)이 처리된 인간망막색소상피(RPE) 세포에서 생존지표인 Bcl-2 단백질의 유의적 활성감소를 나타낸다.
도 5는 Gb3 수용체가 발현하는 세포주(7종)와 RPE 세포에 대한 B-subunit 독성능 시험 결과를 나타낸다.
도 6은 망막색소상피세포(RPE)의 타입 1 B-subunit(36시간)에 의한 손상 결과를 나타낸다.
도 7은 망막색소상피세포 RPE의 타입 1 B-subunit(Stx1 B subunit)에 의해 유도된 DNA N말단 라벨링 이용한 세포괴사측정 (Apoptosis TUNEL staining) 결과를 나타낸다.
도 8은 마우스의 망막색소상피세포에서 Gb3 expression을 나타낸다.
도 9는 마우스의 망막에서 Gb3 expression을 나타낸다.
도 10은 PBS가 주사된 망막색소상피세포에서의 염색질 절편을 나타낸다.
도 11은 타입 1 B-서브유닛가 주사된 망막색소상피세포에서의 염색질 절편을 나타낸다.
도 12는 타입 1 시가독소가 주사된 망막색소상피세포에서의 염색질 절편을 나타낸다.Figure 1 shows Gb3 receptors expressed on the RPE membrane of the retinal epithelium.
Figure 2 shows the Gb3 receptor mediated intracellular delivery of type 1 B-subunit (alexa 488 fluorescent conjugate) on the surface of retinal pigment epithelial cells (RPE); DAPI nucleus staining].
FIG. 3 shows the results of measurement of caspase-3/7 activity, which is an index of degree of cytotoxicity by the cytotoxin type 1 B-subunit (Stx1 B-subunit) in human retinal pigment epithelial cells (RPE).
Figure 4 shows a significant decrease in the activity of the Bcl-2 protein, a survival indicator in human RPE cells treated with type 1 B-subunit (StxlB-subunit).
FIG. 5 shows the results of B-subunit toxicity test on Gb3 receptor expressing cell lines (7 species) and RPE cells.
Figure 6 shows the results of damage by type 1 B-subunit (36 hours) of retinal pigment epithelial cells (RPE).
Figure 7 shows the results of apoptosis TUNEL staining using DNA N-terminal labeling induced by type 1 B-subunit (Stx1 B subunit) of RPE RPE cells.
Figure 8 shows Gb3 expression in retinal pigment epithelial cells of mice.
Figure 9 shows Gb3 expression in the mouse retina.
Figure 10 shows the chromatin sections in PBS-injected retinal pigment epithelial cells.
Figure 11 shows a chromatin slice in a retinal pigment epithelial cell in which Type 1 B-subunit was injected.
Figure 12 shows a chromatin slice in retinal pigment epithelial cells in which
이하, 본 발명의 실시예를 참조하여 상세하게 설명한다. 본 발명을 설명함에 있어, 관련된 공지 구성 또는 기능에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명은 생략한다.Hereinafter, embodiments of the present invention will be described in detail. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.
시가독소의 B-subunit의 숙주 인지 수용체 Gb3가 발현하는 세포에서 유의적인 독성을 보이지 않았으나, 황반변성에 관계있는 망막색소상피(RPE) 세포에서 Gb3 수용체가 발현함에도 불구하고 특이적으로 타입 1B-subunit에 의한 유의적인 독성활성을 보였다. 황반변성에 특성을 나타내는 RPE의 세포괴사와 부착성 상실과 같은 현상이 B-subunit에 의해 유도됨을 확인한 것으로 보아, 본 RPE 세포막 표면 Gb3수용체 타깃 B-subunit에 의한 AMD 병태모델 신규 평가법으로 사용될 수 있음을 확인하였다. Although there was no significant toxicity in the cells expressing the receptor of the B-subunit of the cytotoxin or of the receptor Gb3, the expression of the Gb3 receptor in the RPE cells related to the macular degeneration was specifically expressed in the type 1B-subunit Showed significant toxic activity. It is confirmed that the phenomenon such as apoptosis and adherence loss of RPE which is characteristic of macular degeneration is induced by B-subunit, and it can be used as a new evaluation method of the AMD condition model by the RPE cell surface Gb3 receptor target B-subunit Respectively.
이미 알려진 시가독소 특이수용체 Gb3 발현세포들 대부분에서 B-subunit의 처리가 괴사유도나 독성이 전혀 나타나지 않았으며, 망막상피에만 유일하게 약 24 시간 이후 특이적으로 세포독성을 표현적으로 보였고, 약 12 시간 이후 세포괴사 마커 caspase-3/7 활성도에 유의적으로 차이를 보였다. 따라서 시가독소 타입 1 B-subunit을 활용하여 황반변성유도를 24시간 안에 신속하게 유도하여 AMD평가모델로 활용할 수 있음을 알 수 있었다. In most of the known cytotoxin-specific Gb3-expressing cells, B-subunit treatment did not induce necrosis induction or toxicity at all. Only the retinal epithelium showed specific cytotoxicity after about 24 hours, and about 12 And the cell necrosis marker caspase-3/7 activity was significantly different after the time. Thus, we found that the use of cigarette toxin type 1 B-subunit can induce AMD within 24 hours and can be used as an AMD evaluation model.
망막상피에서만 특이적으로 손상을 줄 수 있는 시가독소 타입 1 B-서브유닛을 AMD 평가에 활용할 경우 눈이 아닌 다른 장기나 조직에 손상과 염증성 반응을 유도하지 않을 것이므로, 신뢰성이 높고 부작용없는 유효한 모델로 사용가능하다.Using the ciguatoxin type 1 B-subunit, which can specifically damage only the retinal epithelium, for AMD evaluation will not induce damage or inflammatory response to organs or tissues other than the eye, so a validated model with no side effects .
<실시예 1> 시험에 사용된 시가독소 타입 1 B-서브유닛 정제Example 1: Cigarette toxin type 1 B-subunit purification used in the test
재조합 균주 E.coli DH5α(pCKS112)는 Stx1(시가독소 타입 1)과 시가독소 타입 1 B-서브유닛(stx1B) operon의 과발현을 하고 이 균쥬를 약 48 시간 동안 37℃에서 배양하였다(Texas A&M 대학 Vernon Tesh 연구실). Crude bacterial 파쇄물은 연속이온치환법과, 크로마토크래피, immunoaffinity chromatography 과정을 거쳐 SDS-PAGE를 silver staining한 후 최종적으로 순도측정과 함께 정제되었다. 최종적으로 시가독소 타입 1을 감지할 수 있는 bovine polyclonal antibody를 이용한 Western blot분석에 의해 동정되었다. 추가적으로 내제독소인 LPS를 제거하기 위해 정제물은 ActiClean Etox칼럼을 거쳐 Limulus amebocyte lysate(LAL assay) 분석에 의해 0.1ng/ml endotoxin수준으로 LPS제거율을 보였다.The recombinant strain E. coli DH5α (pCKS112) overexpresses Stx1 (cigarette toxin type 1) and the cigarette toxin type 1 B-subunit (stx1B) operon and the strain was incubated at 37 ° C for about 48 hours Vernon Tesh Laboratory). Crude bacterial lysates were purified by SDS-PAGE after serial staining, chromatofocusing, immunoaffinity chromatography and finally staining. Finally, it was identified by Western blot analysis using a bovine polyclonal antibody capable of detecting
<실시예 2> FACS 을 이용한 GB3 세포막 발현 분석<Example 2> Analysis of GB3 cell membrane expression using FACS
인체 망막색소상피세포주(ARPE-19)를 2x106을 12-well plate에 옮겨 분주한 후 배양액은 basal Dulbecco’s modified Eagle’s medium과 Ham’s F12 medium의 1:1 혼합배양액(DMEM/F-12)으로 fetal bovine serum 10%와 penicillin (100 U/mL), streptomycin (100 μg/mL)을 함유하고 있으며 5%의 이산화탄소 환경의 습윤 배양기에서 37℃의 온도로 배양하였다. antibiotics가 없고 0.5% FBS가 함유된 DMEM:F12 배지에 시가독소 타입 1(Stx1) (100 ng/ml), Stx2(10 ng/ml)농도로 각 well 당 1 ml씩 투여한 후 30분 또는 1시간 배양한 다음 1 x PBS로 세척한 후 trypsin-EDTA로 떼어 원심분리(5분 동안 4℃ 1500 rpm)하여 세포를 가라앉힌다. Anti-human Gb3/CD77 monoclonal 또는 Mouse IgM를 넣은 FACS buffer(1 x PBS with 2 % FBS)로 세포를 풀어준 후 4℃에서 30분간 염색한다. 세포막에서 Gb3의 발현은 FACS로 분석한다. 시가독소(Stx)을 처리하지 않은 세포의 Gb3 세포막 발현을 대조군으로 사용하였다.The culture medium was transferred to a 12-well plate containing 2x10 6 cells of human retinal pigment epithelium (ARPE-19) and cultured in a 1: 1 mixture of basal Dulbecco's modified Eagle's medium and Ham's F12 medium (DMEM / F-12)
<실시예 3> Caspase 3/7 activity(세포괴사 마커를 활용한 활성도 측정)≪ Example 3 > Caspase 3/7 activity (activity measurement using cell necrosis marker)
인체 망막색소상피세포주(ARPE-19)를 5 x 106을 6-well plate에 옮겨 분주한 후 세포사멸을 유도하기 위해 antibiotics가 없고 0.5% FBS가 함유된 DMEM:F12 배지에 시가독소 타입 1(Stx1) (100 ng/ml), 시가독소 타입 2(Stx2)(10 ng/ml)농도로 각 well당 1 ml씩 투여한 후 24hr, 36hr, 그리고 48hr 동안 배양한 후 1 x PBS로 세척한 후 protease inhibitor를 포함하는 lysis buffer를 이용하여 세포를 용해하고 단백질을 정량 후 각 sample 당 동일양의 단백질을 caspase 3/7 activity reagent와 혼합하여 white plate에서 1시간 30분 동안 37℃ 반응시킨다. 반응시킨 샘플을 luminometer를 이용하여 분석한다.5 × 10 6 of human retinal pigment epithelium (ARPE-19) was transferred to a 6-well plate and incubated in DMEM: F12 medium without antibiotics and 0.5% FBS to induce apoptosis. Stx1) (100 ng / ml) and cigarette toxin type 2 (Stx2) (10 ng / ml), and incubated for 24 hours, 36 hours, and 48 hours Protease is dissolved in the lysis buffer containing protease inhibitor. Protein is quantified. The same amount of protein is mixed with caspase 3/7 activity reagent for each sample and reacted at 37 ℃ for 1 hour and 30 minutes on white plate. The reacted sample is analyzed using a luminometer.
<실시예 4> 세포형광 이미지 분석Example 4 Cell fluorescence image analysis
ARPE-19 세포에서 시가독소 타입 1 B-서브유닛(Stx1B)의 세포 내 trafficking을 평가하기 위해서 푸른색 형광의 핵염색 마커를 염색 후 Alexa Flour 488을 부착시킨 시가독소 타입 1 B 서브유닛(Stx1B)를 처리하였다. 처리 후 PBS로 비특이적 물질들을 제거하기 위해 부착세포를 washing 하였다. 라이브 세포 이미지 장비를 이용하여 실시간으로 Stx1B의 위치 변화를 확인하였다. Stx1B를 처리 후 10분 이내에는 세포막 주위에 존재하던 Stx1B가 시간이 지남에 따라 핵마커와 주위에 존재하는 것이 관찰되었다. 그리고 Stx1B를 처리 후 130 분 부터는 세포의 모양이 수축되는 것을 확인하였다. To evaluate the intracellular trafficking of the cigarette-toxin type 1 B-subunit (Stx1B) in ARPE-19 cells, a blue fluorescent nuclear staining marker was stained followed by Alexa Flour 488-conjugated ciguatoxin type 1 B subunit (Stx1B) Lt; / RTI > After the treatment, the adherent cells were washed to remove non-specific substances with PBS. The location of Stx1B was confirmed in real time using live cell imaging equipment. Within 10 minutes after treatment with Stx1B, Stx1B, which was present around the cell membrane, was observed to be present around the nuclear markers over time. After 130 minutes of treatment with Stx1B, the shape of the cells contracted.
<실시예 5> 웨스턴 블랏팅<Example 5> Western blotting
사용하고자 하는 세포 ARPE를 처리 약 24 시간 전에 5x106 cells/well 농도로 DMEM/F12배지에 배양하여 생착시킨다. 세포를 48 시간동안 처리한 후 harvest를 수행하여 파쇄용 버퍼 lysis buffer (조성;50 mM Tris-HCl (pH 7.4), 7.5% glycerol, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 1.0% sodium deoxycholate, 0.25 mM sodium pyrophosphate, 2.0 mM sodium vandate, 2.0 mM sodium fluoride, 10 μg/ml aprotinin, 1.0 ug/ml leupeptin, 1.0 μg/ml pepstatin and 200 mM phenylmethylsulfonyl fluoride)를 첨가하여 세포를 rocking 방식으로 분쇄하였다. 분쇄된 세포 lysates에 약 30 분간 반응시킨 후 초고속 원심분리기에서 약 16,000g에서 20분간 분리하였다. 침전된 세포불순물은 제외한 상층액만을 새로운 effendorf tube에 옮겨 DC 단백질 정량법을 통해 농도를 결정하였다. 약 50 μg정도 단백질(total protein)을 SDS-PAGE에 loading 한 후 MES(morpholine ethanesulfonic acid)-SDS running 버퍼를 채운 후 30 분간 전기영동을 실시한다. PVDF로 transfer 시킨 후 TBST(100 mM Tris-HCl (pH 7.0), 150 mM NaCl, 0.1% Tween20)로 washing 시행한다. 단백질이 부착된 membrane은 Bcl-2 primary 항체를 4℃에서 16시간 동안 incubation 시킨 후 5분간의 washing 단계를 거쳐 horseradish peroxidase-라벨된 2차 항체를 incubation 상온에서 1시간 동안 수행하여 Chemidoc 장비를 활용해 시각화시킨다.Cell ARPE to be used is cultured in DMEM / F12 medium at a concentration of 5 × 10 6 cells / well for about 24 hours before treatment. After the cells were treated for 48 hours, they were harvested and lysed in a lysis buffer (composition: 50 mM Tris-HCl (pH 7.4), 7.5% glycerol, 150 mM NaCl, 1 mM EDTA, 0.1% NP- ml of leupeptin, 1.0 μg / ml of pepstatin and 200 mM of phenylmethylsulfonyl fluoride) was added to the cells to effect pulverization in a rocking manner Respectively. The cell lysates were reacted for about 30 minutes and then separated at about 16,000 g for 20 minutes in a high-speed centrifuge. Only the supernatant, except for precipitated cell impurities, was transferred to a new effendorf tube and its concentration was determined by DC protein determination method. Approximately 50 μg of total protein is loaded on SDS-PAGE, filled with MES (morpholine ethanesulfonic acid) -SDS running buffer, and subjected to electrophoresis for 30 minutes. Transfer to PVDF and wash with TBST (100 mM Tris-HCl (pH 7.0), 150 mM NaCl, 0.1% Tween 20). Protein-bound membranes were incubated with Bcl-2 primary antibody for 16 h at 4 ° C, followed by washing for 5 min. The secondary antibody, horseradish peroxidase-labeled, was incubated at room temperature for 1 h. Visualize.
<실시예 6> TUNEL assay≪ Example 6 > TUNEL assay
시가독소 타입 1B-subunit에 의해 망막색소상피의 손상에 대한 괴사기전에 대한 실험적 증거를 위해 세포 핵내 DNA말단을 형광 라벨링하여 apoptosis의 특징인 DNA fragmentation을 감지하는 TUNEL(Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay를 수행하였다. Stx에 의해 유도되는 세포사멸을 Tunel assay를 이용하여 측정하였다. ARPE-19를 1x105으로 cover glass가 들어있는 24well에 분주 후 antibiotics가 없고 0.5% FBS가 함유된 DMEM:F12 배지에 Stx1 (100ng/ml), Stx2(10ng/ml)농도로 각 well당 500 ㎕씩 투여한 후 12시간 그리고 24hr동안 처리하였다. 1 x PBS로 세척한 후 4% paraformaldehyde로 10분간 세포를 고정 후 다시 세척 후, 0.2% triton X-10m으로 5분간 실온에서 세포를 permeabilization한다. 다음 과정은 DeadEnd colorimetric TUNEL analysis Kit 매뉴얼을 통해 진행하였다.(TUNEL) (terminal deoxynucleotidyl transferase dUTP nick end labeling) that detects DNA fragmentation, which is characteristic of apoptosis, by fluorescence labeling of the DNA end in the nucleus for experimental evidence of necrosis mechanism for damage to retinal pigment epithelium by cytotoxin type 1B-subunit assay. Stx - induced apoptosis was measured using the Tunel assay. ARPE-19 for the 1x10 5 cover glass is no antibiotics and then dispensed into 24well containing 0.5% of FBS-containing DMEM: in F12 medium Stx1 (100ng / ml), Stx2 (10ng / ml) 500 ㎕ each well at a concentration And then treated for 12 hours and 24 hours. After washing with 1 × PBS, cells are fixed with 4% paraformaldehyde for 10 minutes, and then cells are permeabilized with 0.2% triton X-10m for 5 minutes at room temperature. The following procedure was performed through the DeadEnd colorimetric TUNEL analysis Kit manual.
<실시예 7> Real-time PCRExample 7 Real-time PCR
ARPE-19에 Stx에 의한 ER stress에 의해 유도되는 유전자 발현에 미치는 영향을 알아보기 위해 real-time pcr을 시행하였다. ARPE-19를 2x105수준으로 12wells 분주 후 배양하였다. antibiotics가 없고 0.5% FBS가 함유된 DMEM:F12 배지에 Stx1 (100ng/ml), Stx2(10ng/ml)농도로 각 well당 1ml씩 투여한 후 배양시간을 달리하여 배양한 후 RNA는 RNeasy mini kit를 이용하여 분리하였다. 분리한 RNA는 정량하여 ReverTra Ace -α- ® (TOYOBO)를 이용해 cDNA로 합성하였다. 합성한 cDNA와 각 유전자의 primer 그리고 Thunderbird SYBR qPCR Mix (TOYOBO)를 혼합하여 ViiA7을 통해 Real-time PCR을 수행하였다. Real-time PCR was performed to investigate the effect of ARPE-19 on ER stress-induced gene expression by Stx. ARPE-19 was cultured at 2 × 10 5 level after 12 wells. The cells were incubated with different concentrations of Stx1 (100 ng / ml) and Stx2 (10 ng / ml) in DMEM: F12 medium supplemented with 0.5% FBS . The isolated RNA was quantified and synthesized with cDNA using ReverTra Ace-α- ® (TOYOBO). Real-time PCR was performed on ViiA7 by mixing the synthesized cDNA with primers of each gene and Thunderbird SYBR qPCR Mix (TOYOBO).
<실시예 8> 동물실험≪ Example 8 > Animal experiment
Male B6 mice(6 주령)에 유리체강 내 주사 (Intravitreal injection)로 Stx1(50 ng/1 ㎕), Stx1B (50 ng/1 ㎕), PBS을 주입하였다. 안구 조직 내 Stx1B 단백의 분포를 확인하기 위해 6 mouse(대조군 3마리, 치료군 3마리)를 사용하였다. 안구 획득은 개체사 이전 1시간 이내에 적출하였으며, 분석방법은 Retinal/RPE flat-mount로 실시하였다. 이에 대한 결과는 도 8 내지 도 12에 나타난 바와 같으며, Gb3는 retinal vessel과 RPE에 전반적인 발현되는 것을 관찰할 수 있었고, Stx1B는 RPE만 영향을 주고 전체적인 독소는 낮다는 것을 알 수 있었다.Stx1 (50 ng / 1 μl), Stx1B (50 ng / 1 μl) and PBS were injected into male B6 mice (6 weeks old) by intravitreal injection. Six mice (3 control groups, 3 treatment groups) were used to determine the distribution of Stx1B protein in the eye tissues. The ocular acquisition was performed within 1 hour before the individual's death, and the analytical method was performed with Retinal / RPE flat-mount. The results are shown in FIG. 8 to FIG. 12. Gb3 was observed to be expressed entirely in retinal vessels and RPE. Stx1B affects only RPE and overall toxin was low.
이하, 상기 실험에 의한 결과에 대하여 설명한다.Hereinafter, results of the above experiment will be described.
이전 연구들에 따르면 shiga toxin는 세포 표면에 붙기 위해 특정한 glycolipid, 특히 Globotriaosylceramide (Gb3)의 세포 표면에서의 발현 여부가 중요하다. kidney에서의 특히 몇몇의 세포유형들이 높은 Gb3의 발현을 보이고 있어 상대적으로 Gb3의 발현이 낮은 Human brain endothelial cell에 비해 Stx에 대한 효과가 더 민감하다는 것을 보여준다. 이 Stx에 대한 ARPE-19에 대한 세포독성을 가지는지 알아보기 위해서 먼저 ARPE-19에서 Gb3의 발현이 되는지 확인하였다. 세포표면에서의 Gb3의 발현은 monoclonal anti-CD77 antibody를 이용하여 FACS를 통해 분석하였다. 정상 상태에서의 ARPE-19 세포는 높은 Gb3의 발현을 보여주며 Stx를 처리하고 이른 시간에서의 ARPE-19에서의 Gb3의 발현 변화는 정상상태에서의 세포와 비슷한 Gb3의 발현을 보여준다. ARPE-19의 세포 표면에서 Gb3는 발현을 하며 Stx처리에 의한 Gb3의 발현은 변하지 않았다 ( 도 1 참고). 망막상피세포내(RPE) B-subunit이 결합할 수 있는 수용체 Gb3발현유무 및 세포내 전달능시험을 수행하였을 때 도 1과 같이 Gb3발현을 알 수 있다. Previous studies have shown that shiga toxin is expressed on the cell surface of a specific glycolipid, especially Globotriaosylceramide (Gb3), to attach to the cell surface. In particular, some cell types in kidney show high Gb3 expression, indicating that the effect on Stx is more sensitive than that of human brain endothelial cells, which have relatively low Gb3 expression. To determine whether this Stx has cytotoxicity against ARPE-19, we first confirmed the expression of Gb3 in ARPE-19. Expression of Gb3 on the cell surface was analyzed by FACS using monoclonal anti-CD77 antibody. In steady state, ARPE-19 cells show high Gb3 expression and Stx-treated and early-stage changes in Gb3 expression in ARPE-19 show similar Gb3 expression in normal cells. Gb3 was expressed on the cell surface of ARPE-19, and expression of Gb3 by Stx treatment did not change (see Fig. 1). Gb3 expression can be detected in the presence or absence of Gb3 receptors capable of binding to RPE B-subunit and in the intracellular transport function as shown in Fig.
또한 ARPE-19 세포에서 시가독소 타입 1 B-subunit의 세포 내 trafficking을 결정하기 위해 본 발명에서는 면역형광현미경을 사용하여 Alexa Flour 488을 부착시킨 시가독소 타입 1 B-subunit를 세포 내 trafficking을 확인하였다. ARPE-19 세포에서타입 시가독소 타입 1 B-subunit를 처리한 30분 이후부터 세포 내에서 형광이 증가되는 것을 관찰하였다. 그리고 이러한 형광은 핵 주위에서 존재하는 것으로 관찰되었다. 라이브 세포 이미지에서 Alexa Flour 488을 부착시킨 시가독소 타입 1 B-subunit와 적색 형광의 ER마커가 같은 위치에 존재하는 것을 확인하였다. 정제된 시가독소 타입 1 B-subunit에 Alexas-488의 형광을 접합(conjugation)수행 후 30분 동안 형광접합 시가독소 타입 1 B-subunit을 incubation시켰다. 30분 후 비특이적 결합을 제거한 후 형광현미경에서 관찰하였을 때 RPE세포 내부로 Gb3수용체 매개로 endocytosis에 의해 전달되어 cytosol에서 488파장의 초록색 형광을 보이는 시가독소 타입 1 B-subunit을 보였다 (도 2 참고). In order to determine the intracellular trafficking of the cytotoxin type 1 B-subunit in ARPE-19 cells, intracellular trafficking of the cytotoxin type 1 B-subunit in which Alexa Flour 488 was attached using immunofluorescence microscopy was confirmed in the present invention . In ARPE-19 cells, fluorescence was observed to increase in the cells from 30 minutes after the treatment with type-CsA toxin type 1 B-subunit. These fluoresces were observed to be present around the nucleus. In the live cell image, we confirmed that the cigarette-toxin type 1 B-subunit with Alexa Flour 488 attached and the ER marker of red fluorescence were in the same position. After conjugation of Alexas-488 to the purified cigarette-toxin type 1 B-subunit, the fluorescent conjugated cytotoxic type 1 B-subunit was incubated for 30 minutes. After 30 minutes, the nonspecific binding was removed and the fluorescence microscope revealed a ciguatoxin-type 1 B-subunit that was transferred by Gb3 receptor mediated endocytosis into RPE cells and showed 488 wavelength green fluorescence in the cytosol (see Figure 2) .
이전 연구에 따르면 Stx는 여러 세포주에서 세포사멸을 유도하는데 ARPE-19경우 역시 Stx에 의한 ribotoxic stress와 ER stress를 활성화시키는 것을 보여주었다. ER stress경우는 caspase-8를 활성화시키고 mitochondrial membrane potential을 감소시킴으로써 하위 caspase signaling에 의한 세포사멸을 유도한다고 알려져 있다. 세포괴사 활성지표인 caspase 3/7 효소 활성측정법을 시행한 결과 정상 Stx의 경우 24hr, 48hr 모두에서 mutant를 가진 Stx에 비해 월등히 많은 apoptotic cell이 관찰되는 것을 볼 수 있다. 뿐만 아니라 caspase 8에 활성화에 의한 mitochondrial membrane potential을 감소가 caspase3/7을 활성화 시키는지 확인하기 위해 caspase3/7 activity를 측정한 결과 역시 정상 Stx를 처리한 세포에서 mutant(시가독소 독성부분 A 서브유닛의 167번 아미노산 E를 Q로, 170번 아미노산 R을 L로 site-directed mutagenesis시켜 독성을 제거한 돌연변이 독소)를 처리한 세포에 비해 상당한 activity를 보여준다. 결론적으로 시가독소 타입 1 B-서브유닛(Stx1B-subunit) 의 RPE 세포막 표면상에 발현하는 Gb3매개 endocytosis 통한 세포내 전달이후 도 3에서 보여지는 바와 같이, RPE 세포 손상테스트에서 Stx1B-subunit(Stx1B)은 wild type 형태의 시가독소 타입 1(Stx1)과 타입 2(Stx2)에서와 대등한 수준의 세포괴사 활성지표인 caspase-3/7 activity 측정도를 보였다. 그러나 N-glycosidase 효소 비활성된 mutant 시가독소(Stx1X, Stx2X)와 타입 2 B-서브유닛(Stx2B)은 대조군(con)과 같은 수치를 보임으로써 RPE에 손상을 유도하지 못하였다 (도 3 참고).Previous studies have shown that Stx induces apoptosis in several cell lines, while ARPE-19 also activates Stx-induced ribotoxic and ER stress. It is known that ER stress induces apoptosis by lower caspase signaling by activating caspase-8 and decreasing mitochondrial membrane potential. As a result of the measurement of caspase 3/7 enzyme activity, which is an indicator of cell necrosis activity, it can be seen that there are much more apoptotic cells in normal Stx than in Stx with mutant in both 24hr and 48hr. In addition, caspase-3 activity was measured by caspase-8 in order to determine whether activation of caspase-3/7 activates mitochondrial membrane potential. Compared to cells treated with a mutant toxin that has been cleared of toxicity by site-directed mutagenesis of amino acid E at position 167 with R at position 170 and amino acid R at position 170). In conclusion, intracellular delivery through the Gb3-mediated endocytosis on the surface of the RPE cell membrane of the cigarette toxin type 1 B-subunit (Stx1B-subunit). As shown in Figure 3, the Stx1B-subunit (Stx1B) Showed activity levels of caspase-3/7 activity, which is an indicator of cell necrosis activity equivalent to that of wild-type cigarette toxin type 1 (Stx1) and type 2 (Stx2). However, the N-glycosidase enzyme-inactivated mutant cigarette toxin (Stx1X, Stx2X) and type 2 B-subunit (Stx2B) did not induce damage to the RPE by the same number as the control (con).
추가적으로, 도 4에서 보여지는 바와 같이 Stx1B-서브유닛에서는 wild-type Stx1과 Stx2와 마찬가지로 RPE세포의 생존성을 나타내는 pro-survival factor Bcl-2의 활성감소를 보임으로써 인간망막색소상피에 상당한 손상을 야기했다. 그러나 효소비활성된 mutant 시가독소(Stx1X, Stx2X)와 타입 2 B-서브유닛(Stx2B)은 대조군(con)과 마찬가지 수준의 Bcl-2 활성을 보였으므로 RPE 손상이 미미할 것으로 예측된다(도 4 참고). In addition, as shown in FIG. 4, in the Stx1B-subunit, similar to the wild-type Stx1 and Stx2, the activity of the pro-survival factor Bcl-2 indicating the survival of the RPE cells was decreased, thereby causing considerable damage to the human retinal pigment epithelium It caused. However, the enzyme-inactivated mutant cytotoxin (Stx1X, Stx2X) and type 2 B-subunit (Stx2B) showed the same level of Bcl-2 activity as the control (con) .
시가독소 B-subunit이 인지하여 결합하는 Gb3수용체를 발현하는 8종류의 숙주세포(UD-THP1, DTHP1, Monocyte, Macrophage, Hela, Caco2, HK2(Kidney), RPE)에 시가독소 타입 1 B-subunit과 타입 2 B-subunit을 처리하였을 시, 타입 1 B-subunit에서 유의적으로 RPE세포 특이적으로 괴사 유도현상을 보였다 (도 5 참고)The cytotoxin type 1 B-subunit (B-subunit) was introduced into eight host cells (UD-THP1, DTHP1, Monocyte, Macrophage, Hela, Caco2 and HK2 (Kidney), RPE) expressing the Gb3 receptor that the cytotoxin B- And type 2 B-subunit, RPE cell-specific necrosis induction was observed in type 1 B-subunit (see FIG. 5)
타입 1 B-subunit을 처리하였을 때 망막상피세포 형태적 변화(morphology)를 관찰하였을 때 대조군에 비해 타입 1 B-subunit 처리된 RPE세포가 타입 2 B-subunit보다 심각한 세포손상을 보였다(도 6 참고). 따라서 타입 1 B-subunit에 의해 망막상피세포 RPE에서 심각한 괴사가 wild-type 시가독소 타입 1과 같은 수준으로 유도되었다고 판단되며 이는 황반변성 AMD 질환의 특징적 형태라고 볼 수 있다. When morphology of retinal epithelial cells was observed upon treatment with type 1 B-subunit, RPE cells treated with type 1 B-subunit showed more severe cell damage than those treated with type 2 B-subunit (see FIG. 6) ). Thus, severe necrosis in RPE of the retinal epithelium by Type 1 B-subunit is thought to be induced to the same level as wild-
시가독소 타입 1 B-subunit에 의해 망막색소상피의 손상에 대한 괴사기전에 대한 실험적증거를 위해 세포 핵내 DNA말단을 형광 라벨링하여 apoptosis의 특징인 DNA fragmentation을 감지하는 TUNEL(Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay를 수행한 결과, 도 7에서 보이는 바와 같이 wild-type 시가독소 타입 1과 2와 마찬가지로 타입 1 B-서브유닛에서도 유의적인 fragmentation TUNEL이 나타난 것을 관찰하였다. 그러나 타입 2 B-subunit에서는 세포사멸에 대한 관찰을 할 수 없었다.For experimental evidence of necrotizing mechanisms of retinal pigment epithelium damage by cytotoxin-type 1 B-subunit, fluorescence labeling of the intracellular DNA terminus was used to detect DNA fragmentation, a characteristic of apoptosis. Terminal deoxynucleotidyl transferase dUTP nick end labeling ) assay, a significant fragmentation TUNEL was observed in the type 1 B-subunit as well as wild-type
이상의 설명은 본 발명을 예시적으로 설명한 것에 불과한 것으로, 본 발명에 속하는 기술분야에서 통상의 지식을 가지는 자라면 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 다양한 변형이 가능할 것이다. 따라서, 본 명세서에 개시된 실시예들은 본 발명을 한정하기 위한 것이 아니라 설명하기 위한 것이고, 이러한 실시예에 의하여 본 발명의 사상과 범위가 한정되는 것은 아니다. 본 발명의 보호범위는 아래의 청구범위에 의하여 해석되어야 하며, 그와 동등한 범위내에 있는 모든 기술은 본 발명의 권리범위에 포함하는 것으로 해석되어야 한다.The foregoing description is merely illustrative of the present invention, and various modifications may be made without departing from the essential characteristics of the present invention. Accordingly, the embodiments disclosed herein are intended to be illustrative rather than limiting, and the spirit and scope of the present invention is not limited by these embodiments. The scope of protection of the present invention should be construed according to the following claims, and all techniques within the scope of the same should be construed as being included in the scope of the present invention.
Claims (9)
b) 상기 시가독소 타입 1 B-서브유닛이 망막색소상피세포(RPE)를 손상시켜 황반변성이 유도되는 단계;를 포함하는 황반변성 동물모델 제조방법
a) injecting a cigarette toxin type 1 B-subunit (Stx1B-subunit) into the retina of an animal other than a human, and treating the retina;
b) the step of inducing macular degeneration by damaging the retinal pigment epithelial cells (RPE) of said cytotoxin type 1 B-subunit;
The method of claim 1, wherein the macular degeneration is induced within 24 to 48 hours
The method of claim 1, wherein the macular degeneration is dry or wet.
The method according to claim 1, wherein the macular degeneration is senile
The method of claim 1, wherein the animal is a non-human vertebrate animal selected from the group consisting of a monkey, a dog, a cat, a rabbit, a guinea pig, a rat, a mouse, a cattle, .
2. The method of claim 1, wherein said cytotoxin type 1 B-subunit is delivered into RPE cells via a Gb3 receptor expressed on the RPE cell membrane surface.
2. The method of claim 1, wherein the injury is RPE cell necrosis or adhesion loss.
A macular degeneration animal model prepared according to any one of claims 1 to 7
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010104326A (en) | 2008-10-31 | 2010-05-13 | Japan Health Science Foundation | Model animal for age-related macular degeneration, and method for preparing the same |
Non-Patent Citations (2)
| Title |
|---|
| Moo-Seung Lee and Vernon L. Tesh, 'Roles of Shiga Toxins in Immunopathology', Toxins 11, 212(2019) |
| Optom Vis Sci. vol. 91(8), pp.878-886(2014 Aug) |
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| KR20180121026A (en) | 2018-11-07 |
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