Name: development of an ethanol-induced fibrotic liver model in zebrafish to study progenitor cell-mediated hepatocyte regeneration
Uploaded: 2016-05-13T14:00:00.000+00:00
Duration: 10 min 42 s
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

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。

斑马鱼中提出和使用符合国家卫生研究院和科技机构动物护理和使用委员会乔治亚理工学院获得批准的标准，一个标准协议孕育。 1.溶液的制备<ol><li>准备20升蛋水（与&#39;胚胎中期"交替使用），以维持胚胎/幼虫斑马鱼。溶解1.5克卡索4和6克250毫升蒸馏水中即时海洋海盐。倒入填充有20升蒸馏水和搅动一个大玻璃瓶。 </li><li>制备1升1-苯基-2-硫脲（PTU）原液（20倍）。在1升蒸馏水溶于水0.6克PTU粉末。将1ml每次20毫升胚胎培养基原液添加到0.003％作为工作溶液中的最终浓度。 注意:PTU可能会导致以下吸入或皮肤接触全身毒性。准备并根据适当的处理准则中使用。 </li><li>制备1升3- aminobenzoate甲磺酸酯（三卡因）原液（20倍）。溶解在蒸馏水4克三卡因粉末。通过加入1M的Tris缓冲液（pH 9）调节pH至7，并且使终体积到1L蒸馏水。分装于50ml并储存在-20°C。使用前解冻。 注意:三卡因可诱发感觉的损失。准备并根据适当的处理准则中使用。 </li><li>制备100ml幼虫甲硝唑（MTZ）的工作方案。通过在100毫升胚胎中期的0.1％二甲基亚砜溶解0.25克MTZ粉（DMSO）做出新的15毫米MTZ解决方案。通过剧烈摇晃完全溶解MTZ粉末。让新鲜MTZ解决方案，并用铝箔，以防止MTZ的光催化降解。 注意:MTZ可能引起刺激。准备并根据适当的处理准则中使用。 </li><li>制备100ml幼虫乙醇/甲硝唑（乙醇/ MTZ）的工作方案。让新鲜MTZ解决方案，并用铝箔，以防止光德MTZ的层次。刚好在使用之前添加1.5毫升100％乙醇入100ml MTZ溶液至1.5％的最终浓度。 </li><li>制成500毫升之成人MTZ工作溶液。通过在500毫升系统溶于水0.83克MTZ粉在0.1％DMSO做出新的10毫米MTZ解决方案。通过剧烈摇晃完全溶解MTZ粉末。让新鲜MTZ解决方案，并用铝箔，以防止MTZ的光催化降解。 </li><li>准备1升PEM缓冲。溶解30.2克PIPES，0.74克EGTA，室温下，用MgSO 4在蒸馏水中。调节pH至7，用NaOH。带来终体积为1升，用蒸馏水。 </li></ol> 2.幼虫斑马鱼的制备<ol><li> [ 的Tg（fabp10a:CFP-NTR）的设置配合GT1; TG（TP1:mCherry）jh11] 15,17与TG（HAND2:EGFP）成年斑马鱼PD24交配坦克13成年斑马鱼。使用分频器进行定时交配。 </li><li>取出Ðivider第二天早上，让鱼不中断交配。收获的胚胎后2小时由紧张系统的水和胚胎转移到百毫米培养皿胚胎中期。 </li><li>保持每培养皿不超过100的胚胎。在立体显微镜，删除使用玻璃吸管未受精的卵子。在28℃生长的胚胎和10小时后，受精后（HPF）添加苯基硫脲（PTU）至0.003％的终浓度，以抑制颜料的发展。 </li></ol> 3.乙醇，甲硝唑治疗与肝再生的斑马鱼幼体<ol><li>加入乙醇到胚胎培养基中的1.5％的终浓度制备新鲜的EtOH溶液。不要添加PTU。 </li><li>对待每个培养皿20毫升乙醇溶液56〜80 HPF胚胎。盖培养皿用保鲜膜，以防止乙醇蒸发。 </li><li>理清[ 的Tg（fabp10a:CFP-NTR）GT1; TG（TP1:mCherry）jh11 </SUP&gt;; TG（HAND2:EGFP）PD24]幼虫具有类似肝脏大小，由CFP表达式决定，在80 HPF。排序乙醇处理幼虫时，不要使用三卡因，因为这将导致高死亡率与随后乙醇/ MTZ治疗。保持排序的幼虫以每培养皿30的胚胎的密度。 </li><li>在0.1％DMSO准备新鲜的15毫米MTZ胚胎网上平台。添加乙醇适量到MTZ溶液，使1.5％的最终浓度。在28℃培养，每培养皿20ml的EtOH / MTZ溶液中24小时的幼虫。盖培养皿用保鲜膜来维持乙醇浓度，用铝箔避光。 </li><li>在治疗结束后，通过与新鲜胚胎中期幼虫转移到新的培养皿中除去乙醇/ MTZ解决方案。与胚胎中期幼虫洗3次，并保持在20毫升胚胎中期无PTU。 </li><li>要理清与肝细胞几乎完全消融的幼虫，观察荧光与80X的放大倍数在CFP滤波器的落射荧光显微镜下。成功消融导致在肝脏最小CFP荧光和显著降低肝脏体积。 </li><li>为了避免乙醇/ MTZ处理幼虫死亡，不使用三卡因排序。修正了免疫幼虫（参见第5节），或在28℃下保持有序的幼虫在培养皿中作进一步分析。 </li></ol> 4.化学屏幕在乙醇/ MTZ处理斑马鱼幼体<ol><li>带来含有10mM在DMSO化学股96孔板（参考材料清单用于与商业化学文库详情）从-80℃冷冻机中。用铝箔盖，防止化学品的光催化降解。用温和的摇摆解冻储备液在室温。 </li><li>通过稀释的10mM储备溶液与胚胎培养基中至50μM的（在96孔板中的终浓度制备化学溶液:初始小号creen;在1.5ml管:重新测试）。控制幼虫在胚胎中期同等浓度的DMSO处理。 </li><li>近乎完整的肝切除，这与1.5％乙醇/ 15毫米MTZ处理和分类如前所述，要么96孔（初始屏幕）或24孔转移幼虫（复检）板胚培养基的密度预充每孔5幼虫。使用重复的井每一种化学品。 </li><li>除去胚胎培养基和添加或250微升（96孔板）或500微升（24孔平板）的化学溶液每个孔中。盖板用铝箔和治疗幼虫在28℃50小时。每天检查化学浸泡幼虫并从单井任何死幼虫。 </li><li>在化学处理的结束时，在80X放大倍数观察与CFP滤波器的落射荧光显微镜下的CFP表达肝细胞的数目和/或强度。现场照片显示幼虫增强或降低D号码和/或用数码相机对记录CFP表达肝细胞的强度。 </li><li>在第5节保留通过甲醛固定的共焦成像幼虫。 </li><li>要重新测试便于在初始屏幕肝细胞再生的化学药品，检查它们的效力在更高和更低的浓度，以找到在4.1-4.5（&#39;复检&#39;）中描述的方法是最有效的浓度。 </li></ol> 5.斑马鱼幼体固定，免疫染色和共焦成像<ol><li>通过加入三卡因至0.02％的最终浓度麻醉幼虫。传送10-15幼虫到1.5ml管中，用磷酸缓冲盐水（PBS）洗一次。删除PBS和加入1 ml新鲜配制的2％甲醛的PEM缓冲修复O / N在4℃。 小心:甲醛会导致刺激和已知为人类致癌物。处理在化学通风橱。 </li><li>正确丢弃，然后固定解决方案WASH 3次PBS在室温。固定幼虫可以保持在PBS中在4℃下长达数天。在进行下一步骤及时提供了更好的免疫染色的结果。 </li><li>小心地取出蛋黄和皮肤覆盖使用立体显微镜下＃55钳肝区。 </li><li>通透并且在块缓冲块幼虫（4％牛血清白蛋白和0.3％的Triton X-100的PBS）O / N在4℃。 </li><li>用兔抗胶原蛋白I抗体孵育以1:1的稀释度:100在块缓冲器O / N在4℃。洗5次为用洗涤缓冲液（0.3％的Triton X-100的PBS），每次15分钟。 </li><li>在缓冲块Ø200 / N在4°C:用AlexaFluor 647孵育共轭驴抗兔抗体以1稀释。洗5次与洗涤液各15分钟。然后用PBS洗一次。 </li><li>轻轻幼虫转移到利用玻璃吸管，并与左外侧面朝上定向固定幼虫载玻片上。删除多余的PBS使用的Kimwipes。添加安装媒体一滴和密封玻璃盖用指甲油。在进行共焦成像，立即大大建议。 </li><li>使用配有40X / 1.3油透镜捕捉图像共焦系统。在20-50％使用的激光强度。在1微米的间隔拍摄ž堆栈的图像。 </li></ol> 6.准备成年斑马鱼的<ol><li> [ 的Tg（fabp10a:CFP-NTR）的设置配合GT1; TG（TP1:mCherry）jh11] 15,17成年斑马鱼交配坦克。嘉实由紧张系统的水和胚胎转移到100毫米培养皿胚胎中期第二天早上胚胎。 </li><li>保持每培养皿不超过100的胚胎。在立体显微镜，删除使用玻璃吸管未受精的卵子。在28℃生长的胚胎和后10 HPF添加PTU至0.003％的终浓度，以抑制颜料的发展。 </li><li>在4 DPF，使用三卡因麻醉幼虫和梳理[ 的Tg（fabp10a:CFP-NTR）GT1; TG（TP1:mCherry）jh11]幼虫。通过改变新鲜胚胎培养基几次洗出三卡因。允许，直到他们达到每分钟18次120-180正常的心脏率的幼虫在28°C恢复。 </li><li>提高排序的幼虫基于标准程序19 6-12个月大。 </li></ol> 7.乙醇，甲硝唑治疗和肝再生的成年斑马鱼<ol><li>传输6-12个月[ 的Tg（fabp10a:CFP-NTR）GT1; TG（TP1:mCherry）jh11]成年斑马鱼使用网交配坦克。保持鱼以每罐不超过10鱼的密度。 </li><li>通过在系统加水EtOH中的1％的终浓度制备新鲜的EtOH溶液。治疗成人鱼在每个水箱500毫升乙醇溶液中，并保持它们在28℃的孵化器。 </li><li>转成鱼新鲜的乙醇溶液每天丢弃死鱼道具erly作为一个生物危害。连续处理动物MTZ治疗前72小时。 </li><li>准备系统新鲜水10毫米MTZ解决方案在0.1％DMSO。预温在28℃培养箱的MTZ解决方案。治疗与1升油箱交叉500毫升MTZ解决8小时的鱼在28℃下，用铝箔避光。 </li><li>在治疗期间每小时观察并立即删除死鱼。正确丢弃死鱼作为一个生物危害。在MTZ治疗结束后，由动物转移到新鲜制水两次洗出MTZ。 </li><li>让动物在孵化器在28℃，恢复和维护。饲料，日用鱼转移到新鲜制水，直至用于分析的时间点。 </li></ol> 8.成年斑马鱼肝固定，免疫染色和共焦成像<ol><li>麻醉成年鱼用0.02％三卡因和在冰水安乐死15分钟。帕特在纸巾上的干鱼，并把它放在解剖垫。 </li><li>通过除去皮肤和肌肉如先前露出胃肠器官描述20。用剪刀从臀鳍切割沿腹部皮肤和下层肌肉厣，然后沿鱼的侧后方回臀鳍。 </li><li>把鱼在15ml锥形管中，并用PBS洗一次。删除PBS和添加4毫升新制备的2％甲醛的PEM缓冲修复O / N在4℃。 </li><li>丢弃正确定影溶液，并在室温用PBS洗3次。固定的样品可以保持在PBS中在4℃下数天。在进行下一步骤立即给出更好的免疫染色的结果。 </li><li>通过切割食道解剖与来自鱼的体腔肝整个肠。嵌入用4％的低熔琼脂糖在切片模具胃肠器官。 </li><li>使用vibratome在50微米的间隔在冰冷PBS切割样品成横截面，从前端开始。转移秒系统蒸发散到用PBS 6孔板用＃1漆刷。 </li><li>通透及缓冲块O /块切片ñ4℃。 </li><li>用兔抗胶原蛋白I抗体孵育以1:1的稀释度:100在块缓冲器O / N在4℃。洗5次，每次用洗涤缓冲液15分钟。 </li><li>在缓冲块Ø200 / N在4°C:用AlexaFluor 647孵育共轭驴抗兔抗体以1稀释。洗5次，15分钟，每用洗涤缓冲液并用PBS洗涤一次。 </li><li>轻轻的部分传送到使用＃1油漆刷载玻片上。删除多余的PBS使用的Kimwipes，加安装媒体的下降，封盖玻璃指甲油。在进行共焦成像立即强烈建议。 </li><li>使用配有40X / 1.3油透镜捕捉图像共焦系统。在20-50％使用的激光强度。在1微米的间隔拍摄ž堆栈的图像。 </li></ol>

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R., Saxena, M. T., Mumm, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+zebrafish+chemical+screening+technologies.">Advances in zebrafish chemical screening technologies.</a> Future Med Chem. 4 (14), 1811-1822 (2012).</li><li>Chen, C. H., Durand, E., Wang, J., Zon, L. I., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=zebraflash+transgenic+lines+for+in+vivo+bioluminescence+imaging+of+stem+cells+and+regeneration+in+adult+zebrafish.">zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish.</a> Development. 140 (24), 4988-4997 (2013).</li><li>Westhoff, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+automated+imaging+pipeline+for+the+analysis+of+the+zebrafish+larval+kidney.">Development of an automated imaging pipeline for the analysis of the zebrafish larval kidney.</a> PLoS One. 8 (12), e82137 (2013).</li><li>Perlman, Z. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidimensional+drug+profiling+by+automated+microscopy.">Multidimensional drug profiling by automated microscopy.</a> Science. 306 (5699), 1194-1198 (2004).</li><li>Chu, J., Sadler, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+school+in+liver+development:+lessons+from+zebrafish.">New school in liver development: lessons from zebrafish.</a> Hepatology. 50 (5), 1656-1663 (2009).</li><li>Choi, T. Y., Ninov, N., Stainier, D. Y., Shin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+conversion+of+hepatic+biliary+epithelial+cells+to+hepatocytes+after+near+total+loss+of+hepatocytes+in+zebrafish.">Extensive conversion of hepatic biliary epithelial cells to hepatocytes after near total loss of hepatocytes in zebrafish.</a> Gastroenterology. 146 (3), 776-788 (2014).</li><li>He, J., Lu, H., Zou, Q., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+liver+after+extreme+hepatocyte+loss+occurs+mainly+via+biliary+transdifferentiation+in+zebrafish.">Regeneration of liver after extreme hepatocyte loss occurs mainly via biliary transdifferentiation in zebrafish.</a> Gastroenterology. 146 (3), 789-800 (2014).</li><li>Yao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+structure,+enzyme+histochemistry,+and+immunohistochemistry+of+liver+in+zebrafish.">Fine structure, enzyme histochemistry, and immunohistochemistry of liver in zebrafish.</a> Anat Rec (Hoboken). 295 (4), 567-576 (2012).</li><li>Yovchev, M. 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>

The authors declare that they have no competing financial interests.

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?...

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。

<table><tbody><tr><td>Calcium sulfate hemihydrate (CaSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Acros</td><td>AC385355000</td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</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)-&lt;br /&gt;&lt;em&gt;N,N,N&amp;prime;,N&amp;prime;&lt;/em&gt;-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&amp;nbsp;</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>10&amp;nbsp;mM stock in DMSO</td></tr><tr><td>ActiProbe-1K Library</td><td>Timtec</td><td>ActiProbe-1K</td><td>10&amp;nbsp;mM stock in DMSO</td></tr><tr><td>SB 415286</td><td>Selleck Chemicals</td><td>S2729</td><td>Dissolve with DMSO to 10&amp;nbsp;mM</td></tr><tr><td>CHIR-99021</td><td>Selleck Chemicals</td><td>S2924</td><td>Dissolve with DMSO to 10&amp;nbsp;mM</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>Epifluoresence 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.

图1示出的幼虫斑马鱼的乙醇诱导的肝纤维化模型的发展。要优化斑马鱼暴露乙醇的协议，我们首先评估乙醇的毒性。 2.5天-受精后（dpf）的幼虫暴露在乙醇浓度为1％，1.5％或2％为24小时，随后由并发24小时的EtOH / MTZ治疗。暴露于2％的乙醇造成的死亡率高，而几乎所有的幼虫暴露于1％的乙醇或更少表现出与细胞外基质蛋白胶原蛋白的沉积罕见纤维化很小的变?...

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

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

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 ...

development-an-ethanol-induced-fibrotic-liver-model-zebrafish-to

Research

JoVE Journal

Developmental Biology

9.1K 次观看.  Georgia Institute of Technology. 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. 

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

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

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

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

科研

发育生物学

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

医学

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

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 morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

斑马鱼急性酒精性肝损伤的组织学分析

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 ...

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

癌症研究

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

使用头太空气色谱对斑马鱼胚胎中的乙醇水平进行定量

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social defects.

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, ...

行为学

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

斑马鱼 Tol2 系统:一种模块化且灵活的基于网关的转基因方法

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to ...

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

高脂饮食范式斑马鱼幼体:饲养，实时成像，和食物摄入的量化

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

生物学

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

成人斑马鱼的部分肝切除术

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...

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

摘要

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此视频中的章节

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

斑马鱼急性酒精性肝损伤的组织学分析

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

使用头太空气色谱对斑马鱼胚胎中的乙醇水平进行定量

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

斑马鱼 Tol2 系统:一种模块化且灵活的基于网关的转基因方法

高脂饮食范式斑马鱼幼体:饲养，实时成像，和食物摄入的量化

成人斑马鱼的部分肝切除术

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

摘要

探索更多视频

此视频中的章节

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

斑马鱼急性酒精性肝损伤的组织学分析

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

使用头太空气色谱对斑马鱼胚胎中的乙醇水平进行定量

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

斑马鱼 Tol2 系统:一种模块化且灵活的基于网关的转基因方法

高脂饮食范式斑马鱼幼体:饲养，实时成像，和食物摄入的量化

成人斑马鱼的部分肝切除术

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