乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的肝细胞的再生
Summary
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.
Abstract
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.
Introduction
尽管肝细胞1，它是在肝脏的主要实质细胞类型的显着的再生能力，慢性肝衰竭损害这种能力，导致肝祖细胞（HPC）依赖性再生2。
慢性肝损伤主要由酗酒，慢性丙型肝炎病毒（HCV）感染3和非酒精性脂肪肝病（NAFLD）4而得。它导致持续的肝纤维化，这是与细胞外基质（ECM）蛋白的积累相关联。持续ECM积聚通过形成纤维疤痕组织5，随后形成具有高发病率和死亡率肝硬化扭曲完好肝架构。已经进行了许多尝试来通过注重抑制纤维化的细胞因子和活化的肌成纤维细胞6主要减轻纤维化反应。后者主要由肝星状细胞衍生（HSCS）负责肝瘢痕形成4原理肝非实质细胞。尽管如此，刺激内源性细胞来源，包括高性能计算机再生肝细胞纤维化的持续侮辱的存在再生疗法等待进一步的调查。
肝纤维化的许多实验模型已在哺乳动物中已经描述。四氯化碳（CCL 4）的重复注射已广泛用于诱导肝纤维化的小鼠和大鼠模型7。当与高脂肪（HF）饮食组合，酒精导致纤维化基因表达和肝纤维化8的相当上调。虽然脂肪变性（脂肪蓄积）急性酒精暴露的结果，它使肝脏容易受到更严重的肝损伤9。
斑马鱼， 斑马鱼已成为研究为再生一个宝贵的脊椎动物模型系统。虽然其他低等脊椎动物如蝾螈和蝾螈有一个显着的能力，再生，斑马鱼比其他模型系统的优势在问候操纵潜在的再生所需要的基因操作和可视化战略的因素10。斑马鱼也代表了由简单地将乙醇（EtOH中）至其水研究酒精性肝病（ALD）一个有吸引力的脊椎动物模型。急性乙醇暴露在幼虫和成年斑马鱼引起的脂肪肝11-13。当成年斑马鱼接收扩展EtOH中曝光，用的纤维化相关基因14上调观察胶原沉积。然而，需要一种用于开发模型来研究肝脏再生响应于EtOH中作为纤维化刺激。
最近，我们制定了斑马鱼的15乙醇诱导肝纤维化模型。我们幼虫和adul结合乙醇治疗肝细胞特异性基因消融系统Ť斑马鱼。我们生成了两个转基因株系，TG（fabp10a：CFP-NTR）GT1和Tg的（fabp10a：mCherry-NTR）GT2，其中大肠杆菌硝基还原酶（NTR）分别熔接到青色和mCherry荧光蛋白，的控制下的肝细胞特异性脂肪酸结合蛋白10a中，肝基本 （fabp10a）启动子。在这个系统中，NTR转换无毒的前药甲硝唑（MTZ）插入的DNA间链交联剂16，诱导肝细胞的明确的死亡。利用该模型，我们表明，肝细胞，这是响应于Notch信号群，换算成肝细胞，在不久的缺乏肝细胞，并在过量的ECM。我们指定这些细胞为高性能计算机。此外，通过化学的屏幕，我们确定了Wnt信号的小分子活化剂和Notch信号抑制剂增加在肝纤维化肝细胞的再生。 Therefo再次，我们在斑马鱼肝纤维化模型相比，细胞的文化 – 或基于哺乳动物筛选系统代表了一个极好的化学筛选系统。它是与显著成本和节省时间的益处在体内系统中。在这里，我们描述了建立乙醇诱导肝纤维化模型，并用于执行使用斑马鱼这种模式化学屏幕的详细过程。此外，进行了时间过程分析来调查再生如何肝细胞中的肝纤维化发生。该协议将提供一个宝贵的工具来研究机制，并在肝纤维化增强肝细胞的再生战略。
Protocol
Representative Results
Discussion
我们在乙醇/ MTZ处理回收肝观察HPC介导的肝细胞的再生，这表明即使是在ECM蛋白，包括纤维状I型胶原的显着量的存在下，在高性能计算机保留他们的能力来再生为肝细胞。该MTZ唯一治疗没有增加细胞外基质蛋白沉积显著，而乙醇只能处理没有诱导激活HPC 15。通过利用合并的EtOH / MTZ处理，我们能够调查HPC驱动再生的肝纤维化。由于肝细胞几乎完全消除引起HPC驱动的肝细胞的再生，它是由集?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由部分从GTEC（2731336和1411318），美国国立卫生研究院（K01DK081351）和美国国家科学基金会（1354837），以CHS补助感谢阿莱姆·乔治斯的手稿的批判性阅读的支持。
Materials
| Calcium sulfate hemihydrate (CaSO4) | Acros | AC385355000 | |
| Magnesium sulfate (MgSO4) | EMD | MX0075 | |
| 1,4-Piperazinediethanesulfonic acid (PIPES) | Sigma-Aldrich | P6757 | |
| Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) | Sigma-Aldrich | E3889 | |
| Ethanol | Sigma-Aldrich | E7023 | 200 proof |
| Formaldehyde | Fisher Scientific | F79-500 | |
| Metronidazole (MTZ) | Sigma-Aldrich | M3761 | |
| 1-phenyl-2-thiourea (PTU) | Sigma-Aldrich | P7629 | |
| 3-amino benzoic acid ethyl ester (Tricaine) | Sigma-Aldrich | A5040 | |
| Phosphate-buffered saline (PBS) tablet | Amresco | E404 | Dissolve one tablet with 100 ml distilled water |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D2438 | |
| Bovine serum albumin | Fisher Scientific | BP1600 | |
| Triton X-100 | Fisher Scientific | BP151 | |
| Low-melting agarose | Amresco | BP165 | |
| Stem Cell Signaling Compound Library | Selleck Chemicals | L2100 | 10mM stock in DMSO |
| ActiProbe-1K Library | Timtec | ActiProbe-1K | 10mM stock in DMSO |
| SB 415286 | Selleck Chemicals | S2729 | Dissolve with DMSO to 10mM |
| CHIR-99021 | Selleck Chemicals | S2924 | Dissolve with DMSO to 10mM |
| Anti-Collagen I antibody | Abcam | ab23730 | Use at 1:100 for immunostaining, reacts with fish |
| AlexaFluor 647 Donkey anti-rabbit IgG (H+L) | Molecular Probes | A31573 | Use at 1:200 for immunostaining |
| Mounting media (Vectorshield) | Vector Laboratories | H-1400 | |
| 100 mm petri dish | VWR | 25384-088 | |
| 24-well plate | VWR | 10062-896 | |
| Forceps | Fine Science Tools | 11255-20 | Dumont #55 |
| Glass slide | VWR | 48312-003 | 75×25 mm |
| Cover glass | VWR | 48366-045 | 18 mm |
| Plastic wrap | Fisher Scientific | 22305654 | |
| Aluminum foil | Fisher Scientific | 1213100 | |
| Kimwipes | Kimberly-Clark | 34155 | |
| Vibrotome | Leica | VT1000 S | |
| Stereo microscope | Leica | M80 | |
| Epifluoresent microscope | Leica | M205 FA | |
| Confocol microscope | Zeiss | LSM700 |
References
- Michalopoulos, G. K. Liver regeneration. J Cell Physiol. 213 (2), 286-300 (2007).
- Duncan, A. W., Dorrell, C., Grompe, M. Stem cells and liver regeneration. Gastroenterology. 137 (2), 466-481 (2009).
- Shepard, C. W., Finelli, L., Alter, M. J. Global epidemiology of hepatitis C virus infection. Lancet Infect Dis. 5 (9), 558-567 (2005).
- Hernandez-Gea, V., Friedman, S. L. Pathogenesis of liver fibrosis. Annu Rev Pathol. 6, 425-456 (2011).
- Bataller, R., Brenner, D. A. Liver fibrosis. J Clin Invest. 115 (2), 209-218 (2005).
- Kisseleva, T., Brenner, D. A. Anti-fibrogenic strategies and the regression of fibrosis. Best Pract Res Clin Gastroenterol. 25 (2), 305-317 (2011).
- Constandinou, C., Henderson, N., Iredale, J. P. Modeling liver fibrosis in rodents. Methods Mol Med. 117, 237-250 (2005).
- Gabele, E., et al. A new model of interactive effects of alcohol and high-fat diet on hepatic fibrosis. Alcohol Clin Exp Res. 35 (7), 1361-1367 (2011).
- Lieber, C. S. Alcoholic fatty liver: its pathogenesis and mechanism of progression to inflammation and fibrosis. Alcohol. 34 (1), 9-19 (2004).
- Poss, K. D. Advances in understanding tissue regenerative capacity and mechanisms in animals. Nat Rev Genet. 11 (10), 710-722 (2010).
- Jang, Z. H., et al. Metabolic profiling of an alcoholic fatty liver in zebrafish (Danio rerio). Mol Biosyst. 8 (7), 2001-2009 (2012).
- Passeri, M. J., Cinaroglu, A., Gao, C., Sadler, K. C. Hepatic steatosis in response to acute alcohol exposure in zebrafish requires sterol regulatory element binding protein activation. Hepatology. 49 (2), 443-452 (2009).
- Yin, C., Evason, K. J., Maher, J. J., Stainier, D. Y. The basic helix-loop-helix transcription factor, heart and neural crest derivatives expressed transcript 2, marks hepatic stellate cells in zebrafish: analysis of stellate cell entry into the developing liver. Hepatology. 56 (5), 1958-1970 (2012).
- Lin, J. N., et al. Development of an animal model for alcoholic liver disease in zebrafish. Zebrafish. 12 (4), 271-280 (2015).
- Huang, M., et al. Antagonistic interaction between Wnt and Notch activity modulates the regenerative capacity of a zebrafish fibrotic liver model. Hepatology. 60 (5), 1753-1766 (2014).
- Curado, S., Stainier, D. Y., Anderson, R. M. Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies. Nat Protoc. 3 (6), 948-954 (2008).
- Parsons, M. J., et al. Notch-responsive cells initiate the secondary transition in larval zebrafish pancreas. Mech Dev. 126 (10), 898-912 (2009).
- Baker, K., Warren, K. S., Yellen, G., Fishman, M. C. Defective ‘pacemaker’ current (Ih) in a zebrafish mutant with a slow heart rate. Proc Natl Acad Sci U S A. 94 (9), 4554-4559 (1997).
- Avdesh, A., et al. Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction. J Vis Exp. (69), e4196 (2012).
- Gupta, T., Mullins, M. C. Dissection of organs from the adult zebrafish. J Vis Exp. (37), (2010).
- Paku, S., Schnur, J., Nagy, P., Thorgeirsson, S. S. Origin and structural evolution of the early proliferating oval cells in rat liver. Am J Pathol. 158 (4), 1313-1323 (2001).
- Turner, R., et al. Human hepatic stem cell and maturational liver lineage biology. Hepatology. 53 (3), 1035-1045 (2011).
- Kodama, Y., Hijikata, M., Kageyama, R., Shimotohno, K., Chiba, T. The role of notch signaling in the development of intrahepatic bile ducts. Gastroenterology. 127 (6), 1775-1786 (2004).
- Ryback, R., Percarpio, B., Vitale, J. Equilibration and metabolism of ethanol in the goldfish. Nature. 222 (5198), 1068-1070 (1969).
- Mathias, J. R., Saxena, M. T., Mumm, J. S. Advances in zebrafish chemical screening technologies. Future Med Chem. 4 (14), 1811-1822 (2012).
- Chen, C. H., Durand, E., Wang, J., Zon, L. I., Poss, K. D. zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish. Development. 140 (24), 4988-4997 (2013).
- Westhoff, J. H., et al. Development of an automated imaging pipeline for the analysis of the zebrafish larval kidney. PLoS One. 8 (12), e82137 (2013).
- Perlman, Z. E., et al. Multidimensional drug profiling by automated microscopy. Science. 306 (5699), 1194-1198 (2004).
- Chu, J., Sadler, K. C. New school in liver development: lessons from zebrafish. Hepatology. 50 (5), 1656-1663 (2009).
- Choi, T. Y., Ninov, N., Stainier, D. Y., Shin, D. Extensive conversion of hepatic biliary epithelial cells to hepatocytes after near total loss of hepatocytes in zebrafish. Gastroenterology. 146 (3), 776-788 (2014).
- He, J., Lu, H., Zou, Q., Luo, L. Regeneration of liver after extreme hepatocyte loss occurs mainly via biliary transdifferentiation in zebrafish. Gastroenterology. 146 (3), 789-800 (2014).
- Yao, Y., et al. Fine structure, enzyme histochemistry, and immunohistochemistry of liver in zebrafish. Anat Rec (Hoboken). 295 (4), 567-576 (2012).
- Yovchev, M. I., Xue, Y., Shafritz, D. A., Locker, J., Oertel, M. Repopulation of the fibrotic/cirrhotic rat liver by transplanted hepatic stem/progenitor cells and mature hepatocytes. Hepatology. 59 (1), 284-295 (2014).
