肝细胞特异性消融在斑马鱼研究胆驱动肝再生
Summary
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.
Abstract
肝脏有很大的再生能力。 hepatocyte-或胆道驱动肝细胞再生：肝细胞，肝实质细胞，可以以两种方式之一再生。在肝细胞驱动的肝脏再生，再生肝细胞是从先前存在的肝细胞衍生的，而在胆汁驱动再生，再生肝细胞是从胆管上皮细胞（BEC中）而得。对于肝细胞驱动的肝脏再生，有优秀的啮齿动物模型已经显著有助于肝脏再生的目前了解。但是，没有这样的啮齿动物模型存在胆道驱动肝再生。我们最近报告了斑马鱼肝损伤模型中的BEC广泛引起肝细胞严重时肝细胞的损失。在此模型中，肝细胞是专门由药理学手段消融。在这里，我们将详细介绍的方法来烧蚀肝细胞并分析BEC驱动肝再生过程。此肝细胞特异性消融模型可以进一步用于发现的胆驱动肝脏再生潜在的分子和细胞机制。此外，这些方法可应用于化学筛选，以确定小分子增强或抑制肝再生。
Introduction
肝脏是一个高度再生的器官。在肝再生，再生肝细胞，肝实质细胞，衍生自预先存在的肝细胞（肝细胞驱动的肝脏再生）或的BEC（胆驱动肝再生）1,2。肝损伤引起，通常预先存在肝细胞的增殖;然而，当肝细胞增殖受到损害，建筑物能源效益守则可以促进肝细胞2-4。肝脏再生的这两种模式是临床显著。在手术切除（因为肝肿瘤或活肝供体例如 ）人类肝脏的一部分的，在剩余的肝的肝细胞增殖来恢复丢失的肝脏重量。与此相反，在患有严重肝病，肝细胞增殖受到很大损害，使得的BEC或肝脏祖细胞（LPCS）似乎有助于再生肝细胞5,6。啮齿动物的2/3部分肝切除模型，其中肝细胞增殖，以恢复丢失的肝脏肿块，已经显著促进了肝细胞驱动的肝脏再生7,8当前的理解。然而，存在其中再生肝细胞主要来源于BEC中没有有效的啮齿动物模型。尽管一些啮齿类动物肝脏毒素模型导致胆道驱动肝细胞再生2-4的鉴定，在小鼠近期谱系追踪研究表明建筑物能源效益守则，以在这些模型中9,10再生肝细胞最小的贡献。一些啮齿类肝损伤模型，其中包括部分肝切除11-13和对乙酰氨基酚引起的肝损害14,15，已经被应用到斑马鱼和导致牵连在肝再生新基因或通路的鉴定。然而，肝细胞驱动的，而不是BEC驱动，肝再生发生在这些斑马鱼肝损伤模型。因此，一种新的肝损伤模型中建筑物能源效益守则广泛有助于regenerat荷兰国际集团肝细胞都需要更好地了解BEC驱动的肝脏再生。
肝细胞消融模型的总体目标描述此处被（1），以产生一个肝损伤模型，其中的BEC广泛向再生肝细胞，和（2）阐明底层BEC驱动肝脏再生的分子和细胞机制。我们假设，损伤的严重程度决定了肝再生的模式;因此，我们预测，胆驱动肝细胞再生会引发严重时肝细胞损伤。为了检验这一假设，我们开发了斑马鱼肝损伤模型通过产生一个转基因品系，Tg为（fabp10a：CFP-NTR）S931，即高表达细菌硝基还原酶（NTR）融合青色荧光蛋白（CFP）的肝细胞特异性fabp10a下子。因为NTR转换无毒前药，甲硝唑（甲硝唑），成细胞毒性药物，它烧蚀只有预期NTR-表达细胞16-18，在这种情况下，肝细胞。通过操纵甲硝唑治疗的持续时间，肝细胞烧蚀的程度可被控制。利用该模型，我们最近报道说，在严重的肝细胞损失，BEC中广泛地再生肝细胞19，其进一步通过另外两个独立的研究20,21证实产生。因此，与上述的啮齿动物和斑马鱼肝损伤模型，我们的肝切除模型是用于研究BEC驱动肝再生更有利。
本协议描述了使用斑马鱼肝切除模型进行肝再生的实验过程。这个模型将是适当用于确定底层胆道驱动肝脏再生的机制和化学筛选，以确定小分子，可以抑制或增强肝再生。
Protocol
Representative Results
Discussion
严重的，但不是轻微，肝细胞消融引起胆道驱动的肝脏再生。 CFP-NTR基因：因此，以下甲硝唑治疗和清洗，以检查甲硝唑处理的幼虫，其可以通过固有的CFP荧光从fabp10a进行评估的肝脏大小是重要的。由于甲硝唑处理幼虫将显示非烧蚀（0-5％）或部分烧蚀（10-20％）的肝脏的一小部分，它是必不可少的梳理和只分析幼虫具有一个非常小的肝脏，这表明严重肝切除。
<p class="jove_…Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Drs. Hukriede and Tsang for discussions. M.K. was supported by an NIH training grant (T32EB001026). This work was supported in part by grants from the American Liver Foundation, the March of Dimes Foundation (5-FY12-39), and the NIH (DK101426) to D.S.
Materials
| 3-amino benzoic acid ethyl ester (Tricaine) powder | Sigma-Aldrich | A5040 | Make 0.4% (15 mM) tricaine stock : Bring 0.4 g tricaine to 100 mL with egg water. Adjust to pH 7. |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D8418 | |
| Metronidazole | Sigma-Aldrich | M1547 | |
| Triton X-100 | Sigma-Aldrich | T8532 | |
| Formaldehyde | Sigma-Aldrich | 252549 | |
| Calcium sulfate dihydrate | Sigma-Aldrich | C3771 | |
| N-Phenylthiourea (PTU) | Sigma-Aldrich | P7629 | |
| Mounting media (Vectashield) | Vector Laboratories | H-1000 | |
| 100 x 15 mm petri dish | Denville Scientific Inc. | M5300 | |
| clear nail polish | Fisher Scientific | 50949071 | |
| fine forceps | Fine Science Tools | 11251-20 | Dumont #5 |
| glass slide | Fisher Scientific | 12-544-1 | |
| glass coverslip | Fisher Scientific | 12-540-A and 12-542-A | 18 x 18 mm |
| zn-5 (mouse anti-Alcam) | ZIRC | zn-5 | 1:10 dilution |
| goat anti-Hnf4a | Santa Cruz | sc-6556 (C-19) | 1:50 dilution |
| rat anti-RFP | Allele Biotechnology | ACT-CM-MRRFP10 | 1:300 dilution |
| AlexaFluor 647 AffiniPure Donkey Anti-goat IgG (H+L) | Jackson laboratories | 705-605-147 | 1:500 dilution |
| AlexaFluor 647 AffiniPure Goat Anti-mouse IgG (H+L) | Invitrogen | A21240 | 1:500 dilution |
| Cy3 donkey anti-rat IgG | Jackson laboratories | 712-165-150 | 1:500 dilution |
| dissecting microscope | Leica Microsystems | S6F | |
| epifluorescence microscope | Leica Microsystems | M205FA | |
| confocal microscope | Carl Zeiss | LSM700 | |
| egg water | 0.3 g/L of sea salts ‘Instant Ocean’ and 0.5 mM CaSO4 in distilled water. | ||
| 10X PBS | 25.6 g Na2HPO4·7H2O, 80 g NaCl, 2 g KCl, 2 g KH2PO4. Bring to 1 L with distilled water. Adjust pH 7. Autoclave for 20 min at 121°C. | ||
| 1X PBSDT | 0.2% Triton X-100 and 0.2% DMSO in 1 X PBS. | ||
| PEM buffer | 30.2 g PIPES, 761 mg EGTA, 1 mM MgSO4. Bring to 1 L with distilled water. Adjust pH 7. | ||
| Blocking solution | Mix 1 ml of heat-inactivated horse serum with 9 ml of 1X PBSDT. |
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