Name: development of an ethanol-induced fibrotic liver model in zebrafish to study progenitor cell-mediated hepatocyte regeneration
Uploaded: 2016-05-13T14:00:00
Description: Watch this Scientific Journal Video about Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration at JoVE.com

이 작품은 우리는 원고의 중요한 읽기 알렘 Giorgis 감사 CHS에 GTEC (2731336 및 1411318)는 NIH (K01DK081351)와 NSF (1354837)에서 교부금에 의해 부분적으로 지원되었다.

Zebrafish의 제기 및 기술 기관 동물 관리 및 사용위원회의 조지아 연구소의 승인을 국민 건강의 연구소와의 기준을 충족하는 표준 프로토콜을 사용하여 사육 하였다. 솔루션 1. 준비 <ol><li> 배아 / 애벌레 제브라 피쉬를 유지하기 위해 (상호 교환 &#39;배아 매체&#39;와 함께 사용) 20 L 계란 물을 준비합니다. 증류수 250 ml의 1.5 g CASO 4, 6 g 즉시 바다 바다 소금을 녹여. 20 L 증류수와 선동으?...

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I., Xue, Y., Shafritz, D. A., Locker, J., Oertel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repopulation+of+the+fibrotic/cirrhotic+rat+liver+by+transplanted+hepatic+stem/progenitor+cells+and+mature+hepatocytes.">Repopulation of the fibrotic/cirrhotic rat liver by transplanted hepatic stem/progenitor cells and mature hepatocytes.</a> Hepatology. 59 (1), 284-295 (2014).</li></ol>

우리는 심지어 내가 콜라겐 섬유의 유형을 포함하여 ECM 단백질의 상당한 양의 존재의의 HPC가 간세포로 재생하기 위해 자신의 역량을 유지하는 것이 시사의 EtOH / MTZ 처리 복구 간에서 HPC 매개 간세포의 재생을 관찰했다. 의 EtOH 만 처리는 HPC 활성화 (15)를 유도하지 않은 반면, MTZ 전용 치료는 크게 ECM 단백질의 침착을 증가하지 않았다. 조합의 EtOH / MTZ 처리를 이용하여, 우리는 섬유 성 간에...

간 실질 주요 세포 타입 간세포 하나의 놀라운 재생 능력에도 불구하고, 만성 간부전 간장 전구 세포 (HPC) 의존성이 재생 선도,이 능력을 손상시킨다. 만성 간 손상은 주로 알코올 남용, 만성 C 형 간염 바이러스 (HCV) 감염 (3) 및 비 - 알코올성 지방 간 질환 (NAFLD) (4)로부터 유도된다. 이 세포 외 기질 (ECM) 단백질의 축적과 연관되어 유지 간 섬유증으로 이끈다. 지속 ECM 축적 이후 높은 이환율과 사망률과 간경변의 결과로, 섬유 반흔 조직 (5)을 형성하여 그대로 간 구조를 왜곡. 많은 시도가 profibrogenic 사이토 카인 활성을 억제 근섬유 6에 초점을 주로 섬유 성 반응을 완화하기 위해 이루어졌다. 후자는 주로 H (간 성상 세포에서 유래희주), 간 흉터 형성 (4)에 대한 책임 원칙 간 비 실질 세포. 그럼에도 불구하고, 지속적인 폐의 섬유 성 모욕의 존재 간세포를 재생하는 HPC의 등 내인성 세포 소스를 자극 재생 치료법은 추가 조사를 기다리고 있습니다. 간 섬유화 많은 실험 모델은 포유류에 기재되어있다. 사염화탄소 (사염화탄소)의 반복적 인 주입 널리 뮤린 및 래트 모델 7 간 섬유화를 유도하는 데 사용되어왔다. 높은 지방 (HF)식이 요법과 함께 사용하면 알코올 profibrogenic 유전자 발현 및 간 섬유화 (8)의 실질적인 상향 조절되었다. 지방증 (지질 축적) 급성 알코올 노출의 결과이지만, 더 심각한 간 손상에 취약 9 간을 만든다. 제브라 피쉬, 다니오 레 리오는 재생 연구를위한 귀중한 척추 동물 모델 시스템으로 떠오르고있다. 그래도이러한 도롱뇽과 axolotls 같은 다른 낮은 척추 동물은 제브라 피쉬는 잠재적 인 재생을 조작하는 데 필요한 유전자 조작 및 시각화 전략에 관해서 다른 모델 시스템에 비해 장점이 있습니다 (10) 요인, 재생에 놀라운 능력을 가지고있다. 제브라 피쉬는 단순히 물에 에탄올 (EtOH로)를 추가하여 알코올성 간 질환 (ALD)을 연구하는 매력적인 척추 동물 모델을 나타냅니다. 애벌레와 성인 제브라 피쉬에 급성 EtOH로 노출 간 지방증 11-13 발생했습니다. 성인 지브라 피쉬가 확장을 EtOH 노출을 수신하면, 콜라겐 침착은 섬유증 관련 유전자 (14)의 상향 조절을 관찰 하였다. 그러나, 필요는 폐의 섬유 자극 등의 EtOH에 응답 간 재생을 연구 모델의 개발에 대한 필요성이 존재한다. 최근에는 제브라 15에서 EtOH로 인한 간 섬유화 모델을 개발 하였다. 우리는 애벌레 및 adul에 EtOH로 처리와 간세포 특이 유전자 제거 시스템을 결합t의 제브라 피쉬. 우리가 두 개의 형질 전환 라인을 생성, Tg는 (fabp10a : CFP-NTR) GT1, Tg 및 (fabp10a : mCherry-NTR) GT2, 대장균 nitroreductase (NTR)의 제어하에 각각 시안 mCherry 형광 단백질에 융합 된 단백질 10A, 간 기본 (fabp10a) 프로모터 바인딩 간세포 특이 지방산. 이 시스템에서, NTR은 간세포의 명시 적 죽음을 유도하는 DNA 간 가닥 가교제 (16)로 독성 전구 약물 메트로니다졸 (MTZ)을 변환합니다. 이 모델을 사용하여, 우리는 노치 신호 전달에 반응하는 간 세포의 집단은 간세포의 근처에 부재와 ECM의 초과 간세포로 변환하는 것이 보였다. 우리는 HPC의 이러한 세포를 지정. 또한, 화학 화면을 통해, 우리는 섬유 성 간에서 간세포의 재생을 증가 작은 분자 Wnt 신호의 활성화 및 노치 신호 전달의 억제를 확인 하였다. Therefo다시는 제브라 피쉬에서 우리의 섬유 성 간 모델은 셀 문화 - 또는 포유류 기반 검사 시스템에 비해 뛰어난 화학 검사 시스템을 나타냅니다. 그것은 상당한 비용과 시간을 절약 혜택을 생체 시스템입니다. 여기에서 우리는 EtOH로 유도 된 섬유 성 간 모델을 확립하고 제브라 피쉬에서이 모델을 사용하여 화학 화면을 수행하기위한 세부 절차에 대해 설명합니다. 또한, 시간 코스 분석은 섬유 성 간에서 발생하는 방법 간세포 재생 조사를 실시 하였다. 이 프로토콜은 메커니즘과 섬유 성 간에서 간세포의 재생을 향상시키는 전략을 연구하는 귀중한 도구를 제공합니다.

<table>
	<tbody>
		<tr>
			<td>Calcium sulfate hemihydrate (CaSO4)</td>
			<td>Acros</td>
			<td>AC385355000</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>EMD</td>
			<td>MX0075</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Piperazinediethanesulfonic acid (PIPES)</td>
			<td>Sigma-Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>200 proof</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>F79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Metronidazole (MTZ)</td>
			<td>Sigma-Aldrich</td>
			<td>M3761</td>
			<td></td>
		</tr>
		<tr>
			<td>1-phenyl-2-thiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>3-amino benzoic acid ethyl ester (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) tablet</td>
			<td>Amresco</td>
			<td>E404</td>
			<td>Dissolve one tablet with 100 ml distilled water</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Fisher Scientific</td>
			<td>BP1600</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-melting agarose&#160;</td>
			<td>Amresco</td>
			<td>BP165</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem Cell Signaling Compound Library</td>
			<td>Selleck Chemicals</td>
			<td>L2100</td>
			<td>10mM stock in DMSO</td>
		</tr>
		<tr>
			<td>ActiProbe-1K Library</td>
			<td>Timtec</td>
			<td>ActiProbe-1K</td>
			<td>10mM stock in DMSO</td>
		</tr>
		<tr>
			<td>SB 415286</td>
			<td>Selleck Chemicals</td>
			<td>S2729</td>
			<td>Dissolve with DMSO to 10mM</td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>Selleck Chemicals</td>
			<td>S2924</td>
			<td>Dissolve with DMSO to 10mM</td>
		</tr>
		<tr>
			<td>Anti-Collagen I antibody</td>
			<td>Abcam</td>
			<td>ab23730</td>
			<td>Use at 1:100 for immunostaining, reacts with fish</td>
		</tr>
		<tr>
			<td>AlexaFluor 647 Donkey anti-rabbit IgG (H+L)</td>
			<td>Molecular Probes</td>
			<td>A31573</td>
			<td>Use at 1:200 for immunostaining</td>
		</tr>
		<tr>
			<td>Mounting media (Vectorshield)</td>
			<td>Vector Laboratories</td>
			<td>H-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>VWR</td>
			<td>10062-896</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td>Dumont #55</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>75x25 mm</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>48366-045</td>
			<td>18 mm</td>
		</tr>
		<tr>
			<td>Plastic wrap</td>
			<td>Fisher Scientific</td>
			<td>22305654</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher Scientific</td>
			<td>1213100</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrotome</td>
			<td>Leica</td>
			<td>VT1000 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica</td>
			<td>M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluoresent microscope</td>
			<td>Leica</td>
			<td>M205 FA</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocol microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an ethanol-induced fibrotic liver model in zebrafish to study progenitor cell-mediated hepatocyte regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

도 1은 유충 지브라 피쉬의 EtOH로 인한 간 섬유화 모델의 개발을 도시한다. 의 EtOH에 제브라 피쉬 애벌레를 노출 프로토콜을 최적화하기 위해, 우리는 먼저 EtOH로 독성을 평가 하였다. 2.5 일 후 - 수정 (DPF) 유충은 동시 24 시간을 EtOH / MTZ 처리 한 다음 24 시간 동안 EtOH 중 농도 1 %, 1.5 %, 2 %를 노출시켰다. 거의 모든 애벌레 콜라겐 등 세포 외 기질 단백질의 희귀 증...

Watch this Scientific Journal Video about Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration at JoVE.com

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Zebrafish의에서 에탄올에 의한 섬유 성 간 모델의 개발은 전구 세포 매개 간세포의 재생을 연구하는

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Research

JoVE Journal

Developmental Biology

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

코스닥 시스템의 사용은 분자 메커니즘 Zebrafish의 재생의 연구를

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

연구

발생학

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

담즙 중심의 간 재생을 연구하는 Zebrafish의에서 간세포 특이 절제

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

마우스 자궁 내막 상피와 기질 세포의 분리에 대한<em&gt; 체외</em&gt; 탈락

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Zebrafish에서의 급성 알코올성 간 손상의 조직 학적 분석

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

조사 Trypanosoma cruzi 운동 비보 를 동물 모델로 서 애벌레 Zebrafish의 설립

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

조 혈 줄기와 뿌리를 Quantitate를 생체 외에서 시험의 개발 세포 (HSPCs) Zebrafish 태아를 개발

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

부분 Lobular Hepatectomy: Morphologic 간 재생에 대 한 수술 모델

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

Methods for the Study of Regeneration in Stentor

Methods for the Study of Regeneration in Stentor

Stentor 에 중생의 연구 방법

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

혈관 발달에서 엔도글린의 역할을 연구하기 위해 iPSC-ECs의 제브라피시 배아 및 튜브 형성 분석에서 시투 혼성화에 대한 전체 마운트

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

클로스트리디오이드 디피실을 위한 애벌레 제브라피쉬 감염 모델 개발

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...