KR20230130275A - Animal model for diabetic peripheral neuropathy using zebrafish and use thereof - Google Patents
Animal model for diabetic peripheral neuropathy using zebrafish and use thereof Download PDFInfo
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
본 발명은 제브라피쉬를 이용한 당뇨병성 말초신경병증 동물모델 및 이의 용도에 관한 것으로서, 보다 상세하게는 화학유전학 기법 중 하나인 강화된 효능 니트로리덕테이즈(Enhanced potency nitroreductase; epNTR)/메트로니다졸(metronidazole; MTZ) 시스템을 이용한 제브라피쉬 췌장 베타세포 선택적 사멸 동물모델 제조방법 및 말초신경병증 분석방법에 관한 것이다. 본 발명을 통해 epNTR/MTZ 시스템을 이용한 당뇨병성 말초신경병증 제브라피쉬 동물모델을 제조 및 분석방법을 제공함으로써 신약후보물질의 유효성을 평가할 수 있는 효과가 있다. 또한, 제브라피쉬의 배아가 포유동물보다 투명하고 발생속도가 빠르기 때문에 신속 정확하게 다수의 당뇨병성 말초신경병증 신약후보물질을 초기에 스크리닝이 가능한 신약후보물질 스크리닝 기술로 적용이 가능할 것으로 예상된다.The present invention relates to a diabetic peripheral neuropathy animal model using zebrafish and its use, and more specifically, to enhanced potency nitroreductase (epNTR)/metronidazole, one of the chemical genetic techniques. This relates to a method for manufacturing a zebrafish pancreatic beta cell selective killing animal model using the MTZ) system and a method for analyzing peripheral neuropathy. The present invention has the effect of evaluating the effectiveness of new drug candidates by providing a manufacturing and analysis method for a diabetic peripheral neuropathy zebrafish animal model using the epNTR/MTZ system. In addition, because zebrafish embryos are more transparent and develop faster than mammals, it is expected that it can be applied as a new drug candidate screening technology that can quickly and accurately screen a large number of diabetic peripheral neuropathy drug candidates at an early stage.
Description
본 발명은 제브라피쉬를 이용한 당뇨병성 말초신경병증 동물모델 및 이의 용도에 관한 것이다.The present invention relates to an animal model of diabetic peripheral neuropathy using zebrafish and its use.
당뇨병은 인슐린 분비 저해 또는 인슐린 저항성으로 인하여 체내 혈당치 수준이 비정상적으로 높아지는 고혈당(Hyperglycemia) 증상이 장기간 지속되어 나타나는 질환을 말한다. 이러한 고혈당 증상이 지속되면 단순히 당뇨병에만 국한되지 않고 체내의 다양한 조직에 손상을 주어 합병증을 유발한다. 특히, 당뇨병성 말초신경병증은 당뇨병의 3대 합병증 중의 하나로 서구 사회에서 장애를 일으키는 선두 질환 중 하나이다. 그러나 높은 유병율에 비해 당뇨병성 말초신경병증의 발생기전에 대한 연구가 미비한 실정이며, 또한 치료법이 혈당조절에 국한되어 있으며 아직까지 근본적인 치료제가 없기에 치료법 및 치료약물의 개발이 필요한 실정이다.Diabetes refers to a disease in which symptoms of hyperglycemia (hyperglycemia) persist for a long time, in which the blood sugar level in the body increases abnormally due to inhibition of insulin secretion or insulin resistance. If these symptoms of high blood sugar persist, it is not limited to diabetes alone but damages various tissues in the body, causing complications. In particular, diabetic peripheral neuropathy is one of the three major complications of diabetes and one of the leading diseases causing disability in Western society. However, compared to the high prevalence, research on the pathogenesis of diabetic peripheral neuropathy is insufficient, and treatment is limited to blood sugar control and there is no fundamental treatment yet, so the development of treatments and drugs is necessary.
제브라피쉬는 인간 유전체와 높은 유전학적 및 기능적 유사성을 가진 척추동물로서, 신경계 및 췌장을 비롯한 각종 기관 형성과정이 인간과 매우 유사한 동물모델이다. 이와 함께 제브라피쉬는 유전자 조작이 용이해 형질전환모델 개발이 가능하며, 배아가 투명하기 때문에 생체 내에서 말초신경섬유 손상 및 신경병증 형성과정을 쉽게 관찰할 수 있고, 대량의 배아 확보가 가능하기 때문에 약물 후보물질의 스크리닝이 가능하다는 장점이 있다. 이러한 장점에도 불구하고 제브라피쉬를 활용한 당뇨병성 말초신경병증 동물모델 제조 연구와 말초신경병증 분석방법에 대한 연구는 거의 이루어져 있지 않다.Zebrafish is a vertebrate with high genetic and functional similarity to the human genome, and is an animal model whose process of forming various organs, including the nervous system and pancreas, is very similar to that of humans. In addition, zebrafish is easy to genetically manipulate, making it possible to develop transgenic models, and because the embryos are transparent, peripheral nerve fiber damage and neuropathy formation processes can be easily observed in vivo, and large quantities of embryos can be secured. It has the advantage of being able to screen drug candidates. Despite these advantages, little research has been done on manufacturing diabetic peripheral neuropathy animal models using zebrafish and methods for analyzing peripheral neuropathy.
화학유전학 기법 중 하나인 니트로리덕테이즈(Nitroreductase; NTR)/메트로니다졸(Metronidazole; MTZ) 시스템은 박테리아에서 유래된 NTR이 전구약물(pro-drug) 상태의 메트로니다졸을 환원(reduction)하여 세포독성 상태로 전환시킴으로써 세포사멸을 유발한다. 이러한 시스템을 이용하여 제브라피쉬 췌장베타세포를 선택적으로 사멸시킬 수 있는 모델이 존재하지만, 효율적인 세포사멸을 위해서는 장시간 고농도의 메트로니다졸 처리가 요구된다. 강화된 효능 니트로리덕테이즈(Enhanced potency nitroreductase; epNTR)은 NTR의 단점을 보완함과 동시에 제브라피쉬 동물모델에 최적화되어 개량된 형태로써, 단시간 저농도의 메트로니다졸 처리만으로 세포사멸이 가능하다. 이러한 시스템을 이용하여 현재 췌장베타세포를 선택적으로 사멸시킬 수 있는 제브라피쉬 동물모델은 없는 실정이다.The Nitroreductase (NTR)/Metronidazole (MTZ) system, one of the chemical genetic techniques, uses NTR derived from bacteria to reduce pro-drug metronidazole to a cytotoxic state. It causes cell death by conversion. There is a model that can selectively kill zebrafish pancreatic beta cells using this system, but treatment with high concentrations of metronidazole for a long period of time is required for efficient cell death. Enhanced potency nitroreductase (epNTR) is an improved form that complements the shortcomings of NTR and is optimized for the zebrafish animal model, enabling cell death with only low-concentration metronidazole treatment for a short period of time. There is currently no zebrafish animal model that can selectively kill pancreatic beta cells using this system.
본 발명의 목적은 췌장 베타세포 특이적으로 강화된 효능 니트로리덕테이즈(Enhanced potency nitroreductase; epNTR)을 발현하는 형질전환 제브라피쉬를 제조하는 단계; 및 상기 제조된 형질전환 제브라피쉬에 메트로니다졸(Metronidazole, MTZ)을 처리하는 단계를 포함하는 당뇨병성 말초신경병증 제브라피쉬 동물모델 제조방법, 상기 방법으로 제조된 당뇨병성 말초신경병증 제브라피쉬 동물모델 및 이를 이용한 당뇨병성 말초신경병증 치료제의 스크리닝 방법을 제공하는데 있다.The object of the present invention is to prepare transgenic zebrafish expressing enhanced potency nitroreductase (epNTR) specifically for pancreatic beta cells; and a method for producing a diabetic peripheral neuropathy zebrafish animal model comprising the step of treating the transgenic zebrafish prepared above with metronidazole (MTZ), a diabetic peripheral neuropathy zebrafish animal model prepared by the above method, and the same. The purpose is to provide a screening method for diabetic peripheral neuropathy treatment using the present invention.
본 발명은 췌장 베타세포 특이적으로 강화된 효능 니트로리덕테이즈(Enhanced potency nitroreductase; epNTR)을 발현하는 형질전환 제브라피쉬를 제조하는 단계; 및 상기 제조된 형질전환 제브라피쉬에 메트로니다졸(Metronidazole, MTZ)을 처리하는 단계를 포함하는 당뇨병성 말초신경병증 제브라피쉬 동물모델 제조방법을 제공한다.The present invention includes the steps of producing transgenic zebrafish expressing enhanced potency nitroreductase (epNTR) specifically for pancreatic beta cells; and treating the prepared transgenic zebrafish with metronidazole (MTZ). A method for producing a diabetic peripheral neuropathy zebrafish animal model is provided.
또한, 본 발명은 췌장 베타세포 특이적으로 epNTR을 발현하는 Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) 형질전환 제브라피쉬에 MTZ를 처리한, 당뇨병성 말초신경병증 제브라피쉬 동물모델을 제공한다.In addition, the present invention provides a diabetic peripheral neuropathy zebrafish animal model in which Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) transgenic zebrafish that specifically express epNTR in pancreatic beta cells are treated with MTZ. do.
또한, 본 발명은 상기 당뇨병성 말초신경병증 제브라피쉬 동물모델에 약물 후보 물질을 처리하는 단계; 및 상기 약물 후보 물질을 처리한 동물 모델에서 고혈당으로 인해 말초신경손상 또는 섬모세포손상 수준을 측정하는 단계; 및 대조군과 비교하여 상기 동물 모델의 말초신경손상 또는 섬모세포손상 수준이 감소된 약물 후보 물질을 선별하는 단계를 포함하는 당뇨병성 말초신경병증 치료제 스크리닝 방법을 제공한다.In addition, the present invention includes the steps of treating the diabetic peripheral neuropathy zebrafish animal model with a drug candidate; And measuring the level of peripheral nerve damage or ciliated cell damage due to hyperglycemia in an animal model treated with the drug candidate; and selecting a drug candidate with reduced levels of peripheral nerve damage or ciliated cell damage in the animal model compared to the control group.
본 발명은 제브라피쉬를 이용한 당뇨병성 말초신경병증 동물모델 및 이의 용도에 관한 것으로서, 보다 상세하게는 화학유전학 기법 중 하나인 강화된 효능 니트로리덕테이즈(Enhanced potency nitroreductase; epNTR)/메트로니다졸(metronidazole; MTZ) 시스템을 이용한 제브라피쉬 췌장 베타세포 선택적 사멸 동물모델 제조방법 및 말초신경병증 분석방법에 관한 것이다. 본 발명을 통해 epNTR/MTZ 시스템을 이용한 당뇨병성 말초신경병증 제브라피쉬 동물모델을 제조 및 분석방법을 제공함으로써 신약후보물질의 유효성을 평가할 수 있는 효과가 있다. 또한, 제브라피쉬의 배아가 포유동물보다 투명하고 발생속도가 빠르기 때문에 신속 정확하게 다수의 당뇨병성 말초신경병증 신약후보물질을 초기에 스크리닝이 가능한 신약후보물질 스크리닝 기술로 적용이 가능할 것으로 예상된다.The present invention relates to a diabetic peripheral neuropathy animal model using zebrafish and its use, and more specifically, to enhanced potency nitroreductase (epNTR)/metronidazole, one of the chemical genetic techniques. This relates to a method for manufacturing a zebrafish pancreatic beta cell selective killing animal model using the MTZ) system and a method for analyzing peripheral neuropathy. The present invention has the effect of evaluating the effectiveness of new drug candidates by providing a manufacturing and analysis method for a diabetic peripheral neuropathy zebrafish animal model using the epNTR/MTZ system. In addition, because zebrafish embryos are more transparent and develop faster than mammals, it is expected that it can be applied as a new drug candidate screening technology that can quickly and accurately screen a large number of diabetic peripheral neuropathy drug candidates at an early stage.
도 1은 본 발명의 실험 계획 모식도(A) 및 메트로니다졸에 의한 췌장 베타세포의 사멸을 확인하기 위하여 췌장 베타세포 및 적색 형광단백질의 강도를 분석한 결과(B-E)를 나타낸다.
도 2는 고혈당으로 인한 말초신경손상을 확인하기 위하여 감각신경에서 발현되는 형광리포터 형질전환 제브라피쉬 Tg(nbt:dsred)를 Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry)와 교배하였고, 이로부터 얻은 치어의 말초 측면 신경(Periphreal lateral line nerve)과 섬모세포(hair cell)의 손상정도를 분석한 결과를 나타낸다.
도 3은 NTR/MTZ 시스템과 epNTR/MTZ 시스템의 당뇨모델에서 말초신경손상을 비교분석하기 위하여 감각신경에서 발현되는 형광리포터 형질전환 제브라피쉬 Tg(nbt:dsred)를 Tg(ins:NTR-mcherry), Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry)와 각각 교배하였고, 이로부터 얻은 치어의 말초 측면 신경의 손상정도를 분석한 결과를 나타낸다.
도 4는 말초 측면 신경손상에 따른 제브라피쉬의 주류성 변화를 분석한 결과를 나타낸다. Figure 1 shows a schematic diagram of the experimental plan of the present invention (A) and the results (BE) of analyzing the intensity of pancreatic beta cells and red fluorescent protein to confirm the death of pancreatic beta cells by metronidazole.
Figure 2 shows that in order to confirm peripheral nerve damage caused by hyperglycemia, a fluorescent reporter transgenic zebrafish Tg(nbt:dsred) expressed in sensory nerves was crossed with Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry). This shows the results of analyzing the degree of damage to the peripheral lateral line nerve and hair cells of fry obtained from .
Figure 3 shows the fluorescence reporter transgenic zebrafish Tg(nbt:dsred) expressed in sensory nerves compared to Tg(ins:NTR-mcherry) for comparative analysis of peripheral nerve damage in diabetes models of the NTR/MTZ system and the epNTR/MTZ system. , Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry), respectively, and the results of analyzing the degree of damage to the peripheral lateral nerves of the resulting fry are shown.
Figure 4 shows the results of analyzing changes in mainstreaming in zebrafish due to peripheral lateral nerve damage.
본 발명에서는 epNTR/MTZ 시스템을 이용하여 당뇨병 제브라피쉬 동물모델을 제조하고 당뇨로 인한 말초신경손상과 이로 인한 제브라피쉬 주류성 행동양상을 분석하였다. 먼저, 유전자 클로닝 기술을 이용해 ins:gal4vp16 및 5uas:epNTR-p2a-mcherry 플라스미드 DNA를 만들고, 유전자전이효소(Transposase)를 이용하여 Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) 형질전환 제브라피쉬를 제조하였다. 만들어진 동물모델에 메트로니다졸을 처리하여 췌장 베타세포 사멸 및 혈당 수치를 분석하였고, 이로 인해 나타나는 말초 측면 신경(Peripheral lateral line nerve) 손상 및 제브라피쉬 주류성(Rheotaxis) 행동양상을 분석함으로써, 제조된 모델을 당뇨병성 말초신경병증 동물모델로서 검증하고, 본 발명을 완성하였다.In the present invention, a diabetic zebrafish animal model was prepared using the epNTR/MTZ system and the peripheral nerve damage caused by diabetes and the resulting behavioral patterns of zebrafish were analyzed. First, create ins:gal4vp16 and 5uas:epNTR-p2a-mcherry plasmid DNA using gene cloning technology, and then use transposase to create Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) transgenic zebrafish. was manufactured. The created animal model was treated with metronidazole to analyze pancreatic beta cell death and blood sugar levels. By analyzing the resulting peripheral lateral line nerve damage and zebrafish rheotaxis behavior, the manufactured model was diagnosed as diabetic. It was verified as an animal model for sexual peripheral neuropathy, and the present invention was completed.
본 발명은 췌장 베타세포 특이적으로 강화된 효능 니트로리덕테이즈(Enhanced potency nitroreductase; epNTR)을 발현하는 형질전환 제브라피쉬를 제조하는 단계; 및 상기 제조된 형질전환 제브라피쉬에 메트로니다졸(Metronidazole, MTZ)을 처리하는 단계를 포함하는 당뇨병성 말초신경병증 제브라피쉬 동물모델 제조방법을 제공한다. The present invention includes the steps of producing transgenic zebrafish expressing enhanced potency nitroreductase (epNTR) specifically for pancreatic beta cells; and treating the prepared transgenic zebrafish with metronidazole (MTZ). A method for producing a diabetic peripheral neuropathy zebrafish animal model is provided.
바람직하게는, 상기 형질전환 제브라피쉬를 제조하는 단계는 1) 인슐린 프로모터 유전자, gal4vp16 유전자 및 polyA 서열을 포함하는 ins:gal4vp16pA 플라스미드를 제조하는 단계; 2) 5uas 프로모터 유전자, epNTR 유전자 및 P2A 펩타이드가 결합된 적색 형광단백질(mCherry) 유전자를 포함하는 5uas:epNTR-p2a-mCherry 플라스미드를 제조하는 단계; 3) 상기 1) 단계 및 2) 단계에서 제조된 플라스미드를 유전자전이효소(Transposase)와 함께 제브라피쉬 수정란에 미세주입(microinjection)하는 단계; 및 4) 상기 미세주입된 수정란의 성체(F0)와 야생형 제브라피쉬를 교배하여, 적색 형광단백질을 발현하는 형질전환 제브라피쉬(F1)를 선별하는 단계를 포함할 수 있으나, 이에 제한되는 것은 아니다. Preferably, the step of preparing the transgenic zebrafish includes 1) preparing an ins:gal4vp16pA plasmid containing the insulin promoter gene, gal4vp16 gene, and polyA sequence; 2) preparing a 5uas:epNTR-p2a-mCherry plasmid containing the 5uas promoter gene, the epNTR gene, and the red fluorescent protein (mCherry) gene combined with the P2A peptide; 3) microinjecting the plasmids prepared in steps 1) and 2) together with a gene transferase into a zebrafish fertilized egg; and 4) crossing the adult (F0) of the microinjected fertilized egg with a wild-type zebrafish to select a transgenic zebrafish (F1) expressing a red fluorescent protein, but is not limited thereto.
보다 바람직하게는, 상기 형질전환 제브라피쉬(F1)는 Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) 형질전환 제브라피쉬 일 수 있으나, 이에 제한되는 것은 아니다.More preferably, the transgenic zebrafish (F1) may be a Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) transgenic zebrafish, but is not limited thereto.
보다 바람직하게는, 상기 메트로니다졸(Metronidazole, MTZ)을 처리하는 단계는 형질전환 제브라피쉬 수정 후 3일 치어 단계부터 6일 동안, 5mM MTZ를 처리할 수 있으나, 이에 제한되는 것은 아니다.More preferably, the step of treating the transgenic zebrafish with metronidazole (MTZ) may be performed with 5mM MTZ for 6 days from the fry stage on the 3rd day after fertilization of the transgenic zebrafish, but is not limited thereto.
또한, 본 발명은 췌장 베타세포 특이적으로 epNTR을 발현하는 Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) 형질전환 제브라피쉬에 MTZ를 처리한, 당뇨병성 말초신경병증 제브라피쉬 동물모델을 제공한다. In addition, the present invention provides a diabetic peripheral neuropathy zebrafish animal model in which Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) transgenic zebrafish that specifically express epNTR in pancreatic beta cells are treated with MTZ. do.
바람직하게는, 상기 당뇨병성 말초신경병증 제브라피쉬 동물모델은 고혈당으로 인해 말초신경손상 및 섬모세포손상이 유발될 수 있으나, 이에 제한되는 것은 아니다.Preferably, the diabetic peripheral neuropathy zebrafish animal model may cause peripheral nerve damage and ciliated cell damage due to hyperglycemia, but is not limited thereto.
또한, 본 발명은 상기 당뇨병성 말초신경병증 제브라피쉬 동물모델에 약물 후보 물질을 처리하는 단계; 및 상기 약물 후보 물질을 처리한 동물 모델에서 고혈당으로 인해 말초신경손상 또는 섬모세포손상 수준을 측정하는 단계; 및 대조군과 비교하여 상기 동물 모델의 말초신경손상 또는 섬모세포손상 수준이 감소된 약물 후보 물질을 선별하는 단계를 포함하는 당뇨병성 말초신경병증 치료제 스크리닝 방법을 제공한다. In addition, the present invention includes the steps of treating the diabetic peripheral neuropathy zebrafish animal model with a drug candidate; And measuring the level of peripheral nerve damage or ciliated cell damage due to hyperglycemia in an animal model treated with the drug candidate; and selecting a drug candidate with reduced levels of peripheral nerve damage or ciliated cell damage in the animal model compared to the control group.
본 발명의 용어 “대조군”이란 약물 후보 물질을 처리하지 않은 상태의 당뇨병성 말초신경병증 제브라피쉬 동물모델을 의미할 수 있다.The term “control group” in the present invention may refer to a zebrafish animal model of diabetic peripheral neuropathy in which the drug candidate is not treated.
상기 약물 후보 물질이란 당뇨병성 말초신경병증에 대한 치료효과가 있는 것으로 예상되어, 치료 효과를 측정하기 위한 실험대상으로 사용될 수 있는 모든 물질을 의미하며, 실험동물에 적용할 수 있고 치료 효과를 측정할 수 있으면, 그 종류가 특별히 제한되지 않으며, 바람직하게는 천연물, 합성화합물, RNA, DNA, 폴리펩티드, 효소, 단백질, 리간드, 항체, 박테리아 또는 진균의 대사물 및 생활성 분자로 이루어진 군에서 선택될 수 있다. The drug candidate refers to any substance that is expected to have a therapeutic effect on diabetic peripheral neuropathy and can be used as an experimental subject to measure the therapeutic effect. It can be applied to experimental animals and the therapeutic effect can be measured. If possible, the type is not particularly limited, and is preferably selected from the group consisting of natural products, synthetic compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, metabolites of bacteria or fungi, and bioactive molecules. there is.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.
<< 실험예Experiment example >>
하기의 실험예들은 본 발명에 따른 각각의 실시예에 공통적으로 적용되는 실험예를 제공하기 위한 것이다. The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.
1. One. 제브라피쉬의of zebrafish 사육 및 관리 Breeding and management
본 발명의 모든 실험 과정은 고려대학교 의과대학 동물실험 윤리위원회 (Korea University Institutional Animal Care & Use Committee (IACUC))의 승인을 받았으며, 국립수의과학 검역원의 동물 실험 규정에 따라 실험을 수행하였다. 본 발명에서는 야생형 제브라피쉬, Tg(ins:NTR-mcherry), Tg(ins:gal4vp16), Tg(5uas:epNTR-p2a-mcherry), Tg(nbt:dsred) 형질전환 제브라피쉬가 사용되었으며, 성체(adult) 및 배아(embryo) 제브라피쉬는 섭씨 28.5도씨 온도에서 14시간 빛과 10시간 어둠 일주기를 가지는 사육실에서 사육되었다. 형태학적 특징을 정의하기 위하여 제브라피쉬 배아와 치어(larva)의 단계는 수정 후 며칠 (days post-fertilization, dpf)로 사용되었다. 제브라피쉬 배아의 색소 형성을 막기 위하여 배아 배지 (embryo medium)로 희석된 0.003% PTU (1-phenyl-2-thiourea)가 수정 후 1일 배아 단계에 처리되었다.All experimental procedures of the present invention were approved by the Korea University Institutional Animal Care & Use Committee (IACUC), and experiments were conducted in accordance with the animal experiment regulations of the National Veterinary Science and Quarantine Service. In the present invention, wild-type zebrafish, Tg(ins:NTR-mcherry), Tg(ins:gal4vp16), Tg(5uas:epNTR-p2a-mcherry), and Tg(nbt:dsred) transgenic zebrafish were used, and adult ( Adult and embryonic zebrafish were reared in a breeding room with a circadian cycle of 14 hours of light and 10 hours of darkness at a temperature of 28.5 degrees Celsius. To define morphological characteristics, zebrafish embryo and larva stages were used as days post-fertilization (dpf). To prevent pigment formation in zebrafish embryos, 0.003% PTU (1-phenyl-2-thiourea) diluted in embryo medium was treated at the 1-day embryonic stage after fertilization.
2. 2. 플라스미스plasmid DNA 및 형질전환 DNA and transformation 제브라피쉬zebrafish 제조 manufacturing
췌장 베타세포 특이적으로 epNTR을 발현하는 형질전환 제브라피쉬를 제조하기 위하여 attB1과 attB2 시퀀스가 포함된 epNTR 유전자 특이적 프라이머를 제조 및 이용하여 중합효소 연쇄반응을 수행하였다. 이를 통해 확보된 중합효소 연쇄반응 산물은 BP 클론반응 효소(BP clonase, Invitrogen)로부터 중간 진입 벡터(middle entry vector)에 클로닝 되었다. 다음으로, 인슐린 프로모터가 포함된 5'-진입 벡터, gal4vp16 유전자가 포함된 중간 진입 벡터, polyA 서열이 포함된 3'-진입 벡터 그리고 목적 벡터(Destination vector)들을 LR 클론반응 효소(LRⅡ Clonase, Invitrogen)를 이용하여 ins:gal4vp16pA 플라스미드를 제조하였다. 또한, 5uas 프로모터가 삽입된 5'-진입 벡터, epNTR 유전자가 삽입된 중간벡터, P2A 펩타이드가 결합된 적색 형광단백질(mCherry) 유전자가 삽입된 3'-진입 벡터 그리고 목적 벡터들을 LR 클론반응 효소(LRⅡ Clonase, Invitrogen)를 이용하여 5uas:epNTR-p2a-mCherry 플라스미드를 제조하였다. 이렇게 만들어진 플라스미드 DNA는 제브라피쉬 수정란 1세포기에 유전자전이효소(Transposase)와 함께 미세주입(microinjection)하여 성체(Founder, F0)로 키웠고, 야생형 제브라피쉬와 교배하여 적색 형광단백질을 발현하는 제브라피쉬(F1)를 선별하여 형질전환 제브라피쉬를 제조하였다. 플라스미드 DNA 제조에 사용된 프라이머는 다음과 같다.To prepare transgenic zebrafish that specifically express epNTR in pancreatic beta cells, polymerase chain reaction was performed using epNTR gene-specific primers containing attB1 and attB2 sequences. The polymerase chain reaction product obtained through this was cloned into a middle entry vector from BP clonase (Invitrogen). Next, the 5'-entry vector containing the insulin promoter, the intermediate entry vector containing the gal4vp16 gene, the 3'-entry vector containing the polyA sequence, and the destination vector were incubated with LR clone reaction enzyme (LRⅡ Clonase, Invitrogen). ) was used to prepare the ins:gal4vp16pA plasmid. In addition, the 5'-entry vector into which the 5uas promoter was inserted, the intermediate vector into which the epNTR gene was inserted, the 3'-entry vector into which the P2A peptide-conjugated red fluorescent protein (mCherry) gene was inserted, and the target vector were incubated with the LR clone reaction enzyme ( 5uas:epNTR-p2a-mCherry plasmid was prepared using LRⅡ Clonase, Invitrogen). The plasmid DNA created in this way was grown into adults (Founder, F0) by microinjection along with gene transferase into the 1-cell stage of zebrafish fertilized eggs, and then crossed with wild-type zebrafish to produce zebrafish (F1) that express red fluorescent protein. ) were selected to produce transgenic zebrafish. Primers used to prepare plasmid DNA are as follows.
attB1_epNTR_F: GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGGATATTATTAGTGTGGCCCTGAAGAGattB1_epNTR_F: GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGGATATTATTAGTGTGGCCCTGAAGAG
attB1_epNTR_R: GGGGACCACTTTGTACAAGAAAGCTGGGTCCGCCACCTCTGTCAGTGTGATGTTCTGAattB1_epNTR_R: GGGGACCACTTTGTACAAGAAAGCTGGGTCCGCCACCTCTGTCAGTGTGATGTTTCTGA
3. 메트로니다졸 처리3. Metronidazole treatment
메트로니다졸은 0.2% 다이메틸 설폭사이드(dimethyl sulfoxide, DMSO)가 포함된 배아 배지에 메트로니다졸(MTZ, Sigma, MO, USA; 5mM)을 용해시켜 제조되었고, 형질전환 제브라피쉬 수정 후 3일 치어 단계부터 6일간 처리하여 췌장 베타세포를 사멸시켰다.Metronidazole was prepared by dissolving metronidazole (MTZ, Sigma, MO, USA; 5mM) in embryo medium containing 0.2% dimethyl sulfoxide (DMSO), and incubated in transgenic zebrafish from the fry stage on day 3 after fertilization. After daily treatment, pancreatic beta cells were killed.
4. 상보적 DNA 합성 및 실시간 4. Complementary DNA synthesis and real-time 중합효소연쇄반응polymerase chain reaction 분석 analyze
인슐린 전사체의 상대적 발현량 비교분석을 위해 다이메틸 설폭사이드 또는 메트로니다졸이 6일 동안 처리된 형질전환 제브라피쉬 수정 후 9일 치어 단계에서 Trizol 시약(Roche)으로 RNA를 추출하였다. 추출된 RNA는 역전사 도구(reverse transcription kit, ImProm-ⅡTM reverse transcriptase, Promega)를 사용하여 상보적 DNA(complementary DNA, cDNA)로 합성되었다.For comparative analysis of the relative expression level of insulin transcripts, RNA was extracted with Trizol reagent (Roche) from the fry stage 9 days after fertilization of transgenic zebrafish treated with dimethyl sulfoxide or metronidazole for 6 days. The extracted RNA was extracted using a reverse transcription kit (ImProm-Ⅱ TM) . It was synthesized into complementary DNA (cDNA) using reverse transcriptase (Promega).
실시간 중합효소연쇄반응은 cDNA 2.5 μl, 0.2 μM 프라이머, 2X Fast Start Essential DNA Green Master Mix (Roche)를 혼합하여 LightCycler 96 기기(Roche)에서 95℃로 10분, 95℃로 10초, 60℃로 10초, 72℃로 10초의 40주기(cycles)로 증폭되었다. 인슐린 전사체의 발현량을 측정하기 위해 사용된 프라이머는 다음과 같다.Real-time polymerase chain reaction was performed by mixing 2.5 μl of cDNA, 0.2 μM primer, and 2 Amplification was performed with 40 cycles of 10 seconds at 72°C. The primers used to measure the expression level of insulin transcripts are as follows.
ins_qRT_F: ATGGCAGTGTGGCTTCAGGCins_qRT_F:ATGGCAGTGTGGCTTCAGGC
ins_qRT_R: CAGCCACCTCAGTTTCCTGGins_qRT_R: CAGCCACCTCAGTTTCCTGG
5. 혈당 측정 및 5. Blood sugar measurement and YOY.O. -- PRO1PRO1 염색법 process of dyeing
제브라피쉬 치어 단계에서 혈당을 측정하기 위해 다이메틸 설폭사이드 또는 메트로니다졸이 처리된 수정 후 9일 단계의 치어들을 분쇄하여 균질화한 후 원심분리(12000rpm, 1분)한다. 혈당측정기(On Call Extra, ACON Laboratories Inc., CA, USA)를 이용하여 원심분리된 상등액으로부터 혈당을 측정한다. 제브라피쉬 치어 섬모세포 염색을 위해 0.1% 다이메틸 설폭사이드가 포함된 배아 배지에 YO-PRO1(Invitrogen; 1 μM)을 용해시켜 염색약을 제조한 후, 제브라피쉬 치어에 28.5℃ 1시간 조건으로 배양시키고 배아 배지를 이용해 염색약을 씻어낸다.To measure blood sugar at the zebrafish fry stage, 9-day post-fertilization fry treated with dimethyl sulfoxide or metronidazole were pulverized, homogenized, and centrifuged (12000 rpm, 1 minute). Blood sugar is measured from the centrifuged supernatant using a blood glucose meter (On Call Extra, ACON Laboratories Inc., CA, USA). To stain zebrafish fry cilium cells, a dye was prepared by dissolving YO-PRO1 (Invitrogen; 1 μM) in embryo medium containing 0.1% dimethyl sulfoxide, and then incubated in zebrafish fry at 28.5°C for 1 hour. Wash off the dye using embryo medium.
6. 6. 제브라피쉬zebrafish 주류성 행동분석 Mainstream behavior analysis
주류성 행동분석 장치의 구조는 3cm×10cm×4cm(가로×세로×높이) 크기의 수조 4개가 연동펌프(Peristaltic pump, World precision instrument, Peri-StarTM pro)에 연결되어 있다. 주류성 행동분석은 연동펌프에서 만들어지는 84ml/min 유속조건에서 물의 흐름방향을 마주한 제브라피쉬 치어 몸의 각도를 ImageJ 프로그램을 이용하여 측정한다. The structure of the mainstream behavior analysis device consists of four tanks measuring 3cm x 10cm x 4cm (width x length x height) and a peristaltic pump (Peristaltic pump, World precision instrument, Peri-Star TM) . pro). In the mainstream behavior analysis, the angle of the zebrafish fry's body facing the direction of water flow is measured using the ImageJ program at a flow rate of 84 ml/min produced by a peristaltic pump.
7. 통계적 분석7. Statistical analysis
섬모세포 및 감각신경섬유의 정량적 분석을 위해서, 대조군 및 실험군 비교분석으로 Kruskal-Wallis one-way analysis of variance(ANOVA)를 수행하였다. 주류성 행동분석의 경우 Chi-Square를 수행하여 집단간 비교분석하였다. 모든 통계 데이터에서 통계적 유의성은 P 양측 검증 (two-tailed probability)이 0.05미만 일 때 유효성을 가지는 것으로 보았다. 모든 통계분석은 GraphPad Prism 7 소프트웨어를 사용하여 분석하였다.For quantitative analysis of ciliated cells and sensory nerve fibers, Kruskal-Wallis one-way analysis of variance (ANOVA) was performed to compare the control and experimental groups. In the case of mainstream behavior analysis, Chi-Square was performed to conduct comparative analysis between groups. In all statistical data, statistical significance was considered valid when P two-tailed probability was less than 0.05. All statistical analyzes were performed using GraphPad Prism 7 software.
<< 실시예Example > >
제브라피쉬 췌장 베타세포는 수정 후 3일 치어단계부터 인슐린 유전자를 발현하기 때문에 메트로니다졸을 발현단계에 맞추어 6일간 처리하여 수정 후 9일 치어단계에서 모든 분석을 진행하고자 하였고, 그 실험계획표를 도 1A에 나타내었다. Since zebrafish pancreatic beta cells express the insulin gene starting from the fry stage on the 3rd day after fertilization, we attempted to treat metronidazole for 6 days according to the expression stage and conduct all analyzes at the fry stage on the 9th day after fertilization. The experimental plan is shown in Figure 1A. indicated.
먼저, 메트로니다졸에 의한 췌장 베타세포의 사멸을 확인하기 위하여 췌장 베타세포 및 적색 형광단백질의 강도를 관찰하였다. 그 결과, 메트로니다졸 실험군에서 적색 형광단백질을 발현하는 췌장 베타세포의 감소를 확인하였다(도 1B 및 도 1C). 이로 인하여 췌장 베타세포에서 발현되는 인슐린 mRNA 전사체량도 유의미하게 감소되었고(도 1D), 혈당 수치도 2-3배 증가된 것을 관찰할 수 있었다(도 1E). 이를 통해 제조된 형질전환 제브라피쉬에서 고혈당 표현형이 나타남을 확인하였다.First, to confirm the death of pancreatic beta cells by metronidazole, the intensity of pancreatic beta cells and red fluorescent protein was observed. As a result, a decrease in pancreatic beta cells expressing red fluorescent protein was confirmed in the metronidazole experimental group (Figures 1B and 1C). As a result, the amount of insulin mRNA transcript expressed in pancreatic beta cells was significantly reduced (Figure 1D), and blood sugar levels were also observed to increase 2-3 times (Figure 1E). Through this, it was confirmed that the produced transgenic zebrafish exhibited a hyperglycemic phenotype.
다음으로, 고혈당으로 인한 말초신경손상을 확인하기 위하여 감각신경에서 발현되는 형광리포터 형질전환 제브라피쉬 Tg(nbt:dsred)를 Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry)와 교배하였고, 이로부터 얻은 치어의 말초 측면 신경(Periphreal lateral line nerve)과 섬모세포(hair cell)의 손상정도를 관찰하였다. 감각신경섬유의 손상정도는 섬모세포와 신경연접된 축삭가지의 개수를 정량하였고 섬모세포의 손상정도는 섬모세포의 개수를 정량하여 분석하였다. 그 결과, 메트로니다졸 실험군에서 감각신경섬유의 축삭가지 및 섬모세포의 개수가 대조군 대비 유의성 있게 감소하였다(도 2A-E). 이를 통해 형질전환 제브라피쉬에서 고혈당으로 인해 말초신경손상과 섬모세포손상을 확인하였다.Next, to confirm peripheral nerve damage caused by hyperglycemia, the fluorescent reporter transgenic zebrafish Tg(nbt:dsred) expressed in sensory nerves was crossed with Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry). The degree of damage to the peripheral lateral line nerve and hair cells of the fry obtained from was observed. The degree of damage to sensory nerve fibers was analyzed by quantifying the number of ciliated cells and axonal branches connected to the nerve, and the degree of damage to ciliated cells was analyzed by quantifying the number of ciliated cells. As a result, the number of axonal branches and ciliated cells of sensory nerve fibers in the metronidazole experimental group was significantly decreased compared to the control group (Figure 2A-E). Through this, peripheral nerve damage and ciliated cell damage were confirmed due to hyperglycemia in transgenic zebrafish.
다음으로, NTR/MTZ 시스템과 epNTR/MTZ 시스템의 당뇨모델에서 말초신경손상을 비교분석하기 위하여 감각신경에서 발현되는 형광리포터 형질전환 제브라피쉬 Tg(nbt:dsred)를 Tg(ins:NTR-mcherry), Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry)와 각각 교배하였고, 이로부터 얻은 치어의 말초 측면 신경의 손상정도를 관찰하였다. 그 결과, Tg(ins:NTR-mcherry) 동물모델은 MTZ 실험군에서 감각신경섬유의 축삭가지 개수가 대조군 대비 유의성 있게 차이가 나타나지 않았지만(도 3A-C, 도 3G), Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) 동물모델은 MTZ 실험군에서 대조군 대비 유의성 있게 감각신경섬유의 축삭가지 개수가 감소하였다(도 3E-F, 도 3G). Tg(ins:NTR-mcherry) 동물모델과 Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) 동물모델의 MTZ 실험군을 비교했을 때 췌장베타세포사멸로 인해 감각신경섬유의 축삭가지 개수가 Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) 동물모델에서 유의성 있게 감소하였다(도 3B, 도 3E, 도 3G). 이를 통해, Tg(ins:NTR-mcherry) 동물모델에서 췌장베타세포 사멸로 인한 말초신경손상이 나타나지 않았으며, Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) 동물모델에서 효과적으로 말초신경손상이 유발되었다.Next, to compare and analyze peripheral nerve damage in diabetes models of the NTR/MTZ system and the epNTR/MTZ system, the fluorescent reporter transgenic zebrafish Tg(nbt:dsred) expressed in sensory nerves was transformed into Tg(ins:NTR-mcherry). , Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry), respectively, and the degree of damage to the peripheral lateral nerves of the resulting fry was observed. As a result, in the Tg(ins:NTR-mcherry) animal model, there was no significant difference in the number of axonal branches of sensory nerve fibers in the MTZ experimental group compared to the control group (Figure 3A-C, Figure 3G), but Tg(ins:gal4vp16;5uas) :epNTR-p2a-mcherry) animal model, the number of axonal branches of sensory nerve fibers was significantly reduced in the MTZ experimental group compared to the control group (Figure 3E-F, Figure 3G). When comparing the MTZ experimental group of the Tg(ins:NTR-mcherry) animal model and the Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) animal model, the number of axonal branches of sensory nerve fibers decreased due to pancreatic beta cell death in Tg( ins:gal4vp16;5uas:epNTR-p2a-mcherry) was significantly decreased in the animal model (Figure 3B, Figure 3E, Figure 3G). Through this, peripheral nerve damage due to pancreatic beta cell death did not appear in the Tg(ins:NTR-mcherry) animal model, and peripheral nerve damage was effectively prevented in the Tg(ins:gal4vp16;5uas:epNTR-p2a-mcherry) animal model. It was triggered.
다음으로, 말초 측면 신경손상에 따른 제브라피쉬의 주류성 변화를 관찰하였다. 주류성이란 제브라피쉬가 물이 흐르는 방향으로 거슬러 이동하는 현상을 말한다. 말초 측면 신경은 물의 유동성 탐지를 위해 필요하기 때문에 말초 측면 신경이 손상되면 물의 흐름을 느끼지 못하여 주류성이 감소된다. 이에 따라, 고혈당으로 인한 주류성 손상을 확인하기 위하여 제브라피쉬 주류성 행동 분석실험을 수행하였다. 주류성 행동 분석은 물의 흐름방향을 마주한 제브라피쉬 치어몸의 각도가 0°-30°이면 주류성을 띄고 30°를 벗어나면 주류성이 없는 것으로 판단한다(도 4A). 그 결과, 메트로니다졸 실험군에서 대조군 대비 30°를 벗어난 치어의 수가 유의성 있게 증가하여 주류성 행동양상이 감소된 것을 관찰할 수 있었다(도 4B-C). 이를 통해, 고혈당으로 인한 말초신경손상으로 제브라피쉬 주류성이 감소됨을 확인하였다.Next, we observed changes in the mainstreaming of zebrafish due to peripheral lateral nerve damage. Mainstreaming refers to the phenomenon in which zebrafish move against the direction of water flow. Because the peripheral lateral nerve is necessary for detecting water fluidity, if the peripheral lateral nerve is damaged, water flow cannot be felt and fluidity is reduced. Accordingly, a zebrafish mainstreaming behavior analysis experiment was performed to confirm mainstreaming damage caused by hyperglycemia. In the mainstreaming behavior analysis, if the angle of the zebrafish fry body facing the direction of water flow is 0°-30°, it is judged to be mainstream, and if it is beyond 30°, it is judged to be mainstream (Figure 4A). As a result, in the metronidazole experimental group, the number of fry that deviated from 30° significantly increased compared to the control group, and it was observed that mainstream behavior was reduced (Figures 4B-C). Through this, it was confirmed that zebrafish mainstreaming was reduced due to peripheral nerve damage caused by hyperglycemia.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.
Claims (7)
상기 제조된 형질전환 제브라피쉬에 메트로니다졸(Metronidazole, MTZ)을 처리하는 단계를 포함하는 당뇨병성 말초신경병증 제브라피쉬 동물모델 제조방법.Preparing transgenic zebrafish expressing enhanced potency nitroreductase (epNTR) specifically for pancreatic beta cells; and
A method of producing a diabetic peripheral neuropathy zebrafish animal model comprising treating the prepared transgenic zebrafish with metronidazole (MTZ).
1) 인슐린 프로모터 유전자, gal4vp16 유전자 및 polyA 서열을 포함하는 ins:gal4vp16pA 플라스미드를 제조하는 단계;
2) 5uas 프로모터 유전자, epNTR 유전자 및 P2A 펩타이드가 결합된 적색 형광단백질(mCherry) 유전자를 포함하는 5uas:epNTR-p2a-mCherry 플라스미드를 제조하는 단계;
3) 상기 1) 단계 및 2) 단계에서 제조된 플라스미드를 유전자전이효소(Transposase)와 함께 제브라피쉬 수정란에 미세주입(microinjection)하는 단계; 및
4) 상기 미세주입된 수정란의 성체(F0)와 야생형 제브라피쉬를 교배하여, 적색 형광단백질을 발현하는 형질전환 제브라피쉬(F1)를 선별하는 단계를 포함하는 것을 특징으로 하는 당뇨병성 말초신경병증 제브라피쉬 동물모델 제조방법.The method of claim 1, wherein the step of preparing the transgenic zebrafish is
1) preparing an ins:gal4vp16pA plasmid containing the insulin promoter gene, gal4vp16 gene, and polyA sequence;
2) preparing a 5uas:epNTR-p2a-mCherry plasmid containing the 5uas promoter gene, the epNTR gene, and the red fluorescent protein (mCherry) gene combined with the P2A peptide;
3) microinjecting the plasmids prepared in steps 1) and 2) together with a gene transferase into a zebrafish fertilized egg; and
4) Crossbreeding the adult (F0) of the microinjected fertilized egg with wild-type zebrafish, and selecting transgenic zebrafish (F1) expressing red fluorescent protein. Diabetic peripheral neuropathy zebrafish. Fish animal model manufacturing method.
상기 약물 후보 물질을 처리한 동물 모델에서 고혈당으로 인해 말초신경손상 또는 섬모세포손상 수준을 측정하는 단계; 및
대조군과 비교하여 상기 동물 모델의 말초신경손상 또는 섬모세포손상 수준이 감소된 약물 후보 물질을 선별하는 단계를 포함하는 당뇨병성 말초신경병증 치료제 스크리닝 방법. Treating the drug candidate material to the diabetic peripheral neuropathy zebrafish animal model according to claim 5 or 6; and
Measuring the level of peripheral nerve damage or ciliated cell damage due to hyperglycemia in an animal model treated with the drug candidate; and
A screening method for the treatment of diabetic peripheral neuropathy, comprising the step of selecting a drug candidate with a reduced level of peripheral nerve damage or ciliated cell damage in the animal model compared to the control group.
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