这项工作得到了澳大利亚国家卫生和医学研究理事会 (APP1042370、APP1061332、APP1087125) 澳洲的资助。作者希望感谢科科卫生创新研究所的工作人员提供技术援助。

1. 动物实验 在本调查中描述的所有动物研究都是由科大学动物伦理委员会 (批准编号: AEC_2014_28) 在开始之前批准的根据《澳大利亚动物保育和使用守则》进行实验和执行. 
<ol> <li> 动物 <ol> <li> 使用六周的老雄性 C57BL/6J 小鼠进行实验. </li>
	</ol> </li> <li> 实验设计 <ol> 到达动物设施后, 允许小鼠在任何实验开始前适应四天. <li>
		<li> 在小麦谷壳床上用品上的小鼠, 它已经耗尽了可见的谷物和茎秆, 并在单独通风的笼子里保持12小时的昼夜循环。改变床上用品在天三和七, 然后每周之后. </li>
		<li> 为鼠标提供 ad 随意 获得胆碱缺乏饮食和饮用水补充0.15% 的 dl-氨基。在4和 #176 中保持氨基补充水, 每隔一天更换一次饮用水, 以确保新鲜度并鼓励饮用。每隔一周, 每周一次彻底改变, 就会出现胆碱缺乏的食物. </li>
		<li> 在休息和处理过程中观察小鼠。监测动物健康的标准标志, 包括整体外貌、姿势、社会互动、仪容、皮毛状况和体重. </li>
		<li> 在实验的前两周每天对小鼠进行称重, 以确保任何有超过20% 体重减少的动物被安乐死, 以限制不必要的痛苦。这是典型的情况下, 在不到5% 的动物。两周后, 称重的频率可以减少到每周三次 ( 例如 , 星期一, 星期三, 星期五). </li>
	</ol> </li> <li> 肝脏灌注和隔离 <ol> 麻醉小鼠在指定的时间点 (在这项研究一, 两个, 和六周的多药) 与氯胺酮 (100 毫克/千克) 和嗪 (10 毫克/千克) 腹腔注射。测试踏板的撤退反射, 以确保足够的麻醉. </li>
		<li> 用70% 乙醇润湿毛皮, 在腹壁上垂直中线切开, 使用梅奥剪刀。肋骨笼可以被移除, 以便更容易进入心脏. </li>
		<li> 使用 25 G x 1/2 和 #34, 通过慢心穿刺收集血液; 常规壁针, 以避免心脏塌陷。允许血液在室温下在离心管内凝结, 在 2000 x g 离心10分钟后获得血清样品。后来, 通过标准的步骤测量血清丙氨酸转氨酶水平 12 . </li>
		<li> 将胃和小肠移到一侧, 并暴露门静脉。使用梅奥剪刀切割心脏, 允许液体退出和灌注肝脏与无菌磷酸盐缓冲盐水 (pH = 7.4) 通过插管门静脉使用 27 G x 1/2 和 #34; 规则壁针。一个均匀脱皮肝脏表明成功灌注所有肝裂片. </li>
		<li> 小心地将肝脏分离, 然后用镊子和梅奥剪刀把它放在培养皿中。除去多余的、肝的组织和胆囊. </li>
	</ol> </li> <li> 肝脏保存 <ol> 在总的肝脏重量被记录了之后, 切开一个小裂片成几平方毫米的几小块, 转移他们到无菌管, 并且在液氮结冰为以后 RNA和蛋白质提取, 按照标准的协议。在器官收集后尽快将这些组织块冷冻起来, 以避免任何组织退化. </li>
		<li> 根据标准协议, 在10% 中性缓冲福尔马林中收集一瓣以供以后处理和石蜡嵌入. </li>
		<li> 将一个叶放入一个充满最佳切削温度的模具中, cryomatrix 嵌入树脂和 #160; 在液氮中对齐冷. </li>
	</ol>
	</li>
</ol>

<ol>
	<li>Taub, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration:+from+myth+to+mechanism.">Liver regeneration: from myth to mechanism.</a> Nat Rev Mol Cell Biol. 5, 836-847 (2004).</li><li>Kohn-Gaone, J., Gogoi-Tiwari, J., Ramm, G. A., Olynyk, J. K., Tirnitz-Parker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+liver+progenitor+cells+during+liver+regeneration,+fibrogenesis,+and+carcinogenesis.">The role of liver progenitor cells during liver regeneration, fibrogenesis, and carcinogenesis.</a> Am J Physiol Gastrointest Liver Physiol. 310, G143-G154 (2016).</li><li>Lu, W. 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=Hepatic+progenitor+cells+of+biliary+origin+with+liver+repopulation+capacity.">Hepatic progenitor cells of biliary origin with liver repopulation capacity.</a> Nat Cell Biol. 17, 971-983 (2015).</li><li>Prakoso, 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=Analysis+of+the+intrahepatic+ductular+reaction+and+progenitor+cell+responses+in+hepatitis+C+virus+recurrence+after+liver+transplantation.">Analysis of the intrahepatic ductular reaction and progenitor cell responses in hepatitis C virus recurrence after liver transplantation.</a> Liver Transpl. 20, 1508-1519 (2014).</li><li>Tirnitz-Parker, J. E., Tonkin, J. N., Knight, B., Olynyk, J. K., Yeoh, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+immortalisation+of+hepatic+oval+cells+from+adult+mice+fed+a+choline-deficient,+ethionine-supplemented+diet.">Isolation, culture and immortalisation of hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented diet.</a> Int J Biochem Cell Biol. 39, 2226-2239 (2007).</li><li>Van Hul, N. K., Abarca-Quinones, J., Sempoux, C., Horsmans, Y., Leclercq, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+between+liver+progenitor+cell+expansion+and+extracellular+matrix+deposition+in+a+CDE-induced+murine+model+of+chronic+liver+injury.">Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury.</a> Hepatology. 49, 1625-1635 (2009).</li><li>Clouston, A. D., 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=Fibrosis+correlates+with+a+ductular+reaction+in+hepatitis+C:+roles+of+impaired+replication,+progenitor+cells+and+steatosis.">Fibrosis correlates with a ductular reaction in hepatitis C: roles of impaired replication, progenitor cells and steatosis.</a> Hepatology. 41, 809-818 (2005).</li><li>Forbes, S. J., Newsome, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration+-+mechanisms+and+models+to+clinical+application.">Liver regeneration - mechanisms and models to clinical application.</a> Nat Rev Gastroenterol Hepatol. 13, 473-485 (2016).</li><li>Akhurst, B., 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=A+modified+choline-deficient,+ethionine-supplemented+diet+protocol+effectively+induces+oval+cells+in+mouse+liver.">A modified choline-deficient, ethionine-supplemented diet protocol effectively induces oval cells in mouse liver.</a> Hepatology. 34, 519-522 (2001).</li><li>Shinozuka, H., Lombardi, B., Sell, S., Iammarino, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+histological+and+functional+alterations+of+ethionine+liver+carcinogenesis+in+rats+fed+a+choline-deficient+diet.">Early histological and functional alterations of ethionine liver carcinogenesis in rats fed a choline-deficient diet.</a> Cancer Res. 38, 1092-1098 (1978).</li><li>Knight, B., Tirnitz-Parker, J. E., Olynyk, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-kit+inhibition+by+imatinib+mesylate+attenuates+progenitor+cell+expansion+and+inhibits+liver+tumor+formation+in+mice.">C-kit inhibition by imatinib mesylate attenuates progenitor cell expansion and inhibits liver tumor formation in mice.</a> Gastroenterology. 135, 969-979 (2008).</li><li>Kohn-Gaone, J., 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=Divergent+Inflammatory,+Fibrogenic,+and+Liver+Progenitor+Cell+Dynamics+in+Two+Common+Mouse+Models+of+Chronic+Liver+Injury.">Divergent Inflammatory, Fibrogenic, and Liver Progenitor Cell Dynamics in Two Common Mouse Models of Chronic Liver Injury.</a> Am J Pathol. 186, 1762-1774 (2016).</li><li>Knight, B., 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=Attenuated+liver+progenitor+(oval)+cell+and+fibrogenic+responses+to+the+choline+deficient,+ethionine+supplemented+diet+in+the+BALB/c+inbred+strain+of+mice.">Attenuated liver progenitor (oval) cell and fibrogenic responses to the choline deficient, ethionine supplemented diet in the BALB/c inbred strain of mice.</a> J Hepatol. 46, 134-141 (2007).</li><li>Dwyer, B. J., Olynyk, J. K., Ramm, G. A., Tirnitz-Parker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TWEAK+and+LTbeta+Signaling+during+Chronic+Liver+Disease.">TWEAK and LTbeta Signaling during Chronic Liver Disease.</a> Front Immunol. 5, 39 (2014).</li><li>Karin, M., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reparative+inflammation+takes+charge+of+tissue+regeneration.">Reparative inflammation takes charge of tissue regeneration.</a> Nature. 529, 307-315 (2016).</li><li>Ruddell, R. G., 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=Lymphotoxin-beta+receptor+signaling+regulates+hepatic+stellate+cell+function+and+wound+healing+in+a+murine+model+of+chronic+liver+injury.">Lymphotoxin-beta receptor signaling regulates hepatic stellate cell function and wound healing in a murine model of chronic liver injury.</a> Hepatology. 49, 227-239 (2009).</li><li>Tirnitz-Parker, J. 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=Tumor+necrosis+factor-like+weak+inducer+of+apoptosis+is+a+mitogen+for+liver+progenitor+cells.">Tumor necrosis factor-like weak inducer of apoptosis is a mitogen for liver progenitor cells.</a> Hepatology. 52, 291-302 (2010).</li><li>Viebahn, C. S., Yeoh, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+fires+prometheus?+The+link+between+inflammation+and+regeneration+following+chronic+liver+injury.">What fires prometheus? The link between inflammation and regeneration following chronic liver injury.</a> Int J Biochem Cell Biol. 40, 855-873 (2008).</li><li>Hayner, N. T., Braun, L., Yaswen, P., Brooks, M., Fausto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isozyme+profiles+of+oval+cells,+parenchymal+cells,+and+biliary+cells+isolated+by+centrifugal+elutriation+from+normal+and+preneoplastic+livers.">Isozyme profiles of oval cells, parenchymal cells, and biliary cells isolated by centrifugal elutriation from normal and preneoplastic livers.</a> Cancer Res. 44, 332-338 (1984).</li><li>Tee, L. B., Smith, P. G., Yeoh, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+alpha,+mu+and+pi+class+glutathione+S-transferases+in+oval+and+ductal+cells+in+liver+of+rats+placed+on+a+choline-deficient,+ethionine-supplemented+diet.">Expression of alpha, mu and pi class glutathione S-transferases in oval and ductal cells in liver of rats placed on a choline-deficient, ethionine-supplemented diet.</a> Carcinogenesis. 13, 1879-1885 (1992).</li><li>Chayanupatkul, M., 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=Hepatocellular+carcinoma+in+the+absence+of+cirrhosis+in+patients+with+chronic+hepatitis+B+virus+infection.">Hepatocellular carcinoma in the absence of cirrhosis in patients with chronic hepatitis B virus infection.</a> J Hepatol. 66, 355-362 (2017).</li><li>Mittal, S., 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=Hepatocellular+Carcinoma+in+the+Absence+of+Cirrhosis+in+United+States+Veterans+is+Associated+With+Nonalcoholic+Fatty+Liver+Disease.">Hepatocellular Carcinoma in the Absence of Cirrhosis in United States Veterans is Associated With Nonalcoholic Fatty Liver Disease.</a> Clin Gastroenterol Hepatol. 14, 124-131 (2016).</li></ol>

作者没有什么可透露的。

慢性肝病通常是一种无声的疾病, 大多数患者无症状, 它是一个主要的贡献, 发病率和死亡率在世界范围内。慢性酒精中毒和丙肝病毒感染是主要原因。慢性肝损伤的特点是肝脏炎症、纤维化和严重的肝硬化、肝癌和最终肝功能衰竭。目前尚无治疗方法, 虽然已取得重大进展, 了解肝脏疾病的机制, 但仍然迫切需要新的治疗途径。
时间饮食是一个简单的, 容易管理的模型, 诱导和?...

肝脏是身体的最大的腺新陈代谢的器官并且有许多复杂作用。肝脏的关键作用包括消化、新陈代谢、解毒、储存必要的营养素、生产血浆蛋白组分、以及通过驻留巨噬细胞或枯否体细胞介导的免疫。肝脏有很大的能力再生, 即使高达70-90% 的总质量损失。在急性肝损伤的情况下, 如看到部分肝切除或扑热息痛中毒, 其余健康肝细胞增殖, 以修复在高度协调的过程中的损害1。然而, 当肝细胞因长期病毒感染、酒精或非酒精性脂肪肝而长期受伤时, 炎症微环境引发肝纤维化的活化, 并肝祖细胞增殖 (LPCs), 有可能分化为胆管或肝细胞2,3,4,5。确切的起源, 分化命运的 LPCs, 他们对肝脏再生的贡献, 和肝癌一直是激烈辩论的话题, 很可能取决于伤害的严重性和上下文2。早期再生相关事件的顺序也有争议的讨论, 一些研究人员指出, 肝星状细胞活化和基质重塑对于产生 LPC 偏爱的小生境6是必不可少的, 而其他报告, 要触发纤维7, 需要 LPC 扩展和 so-called 胆管反应。有许多动物模型研究损伤和再生的具体方面, 试图了解所有的潜在因素, 调节疾病进展, 并最终制定新的治疗策略的病人8。
本发明的胆碱缺乏和氨基补充的饮食模型最初是为大鼠使用的, 后来改性用于小鼠的慢性肝损伤诱导,9,10。膳食缺乏的胆碱导致受损的组装和分泌非常低密度脂蛋白。结合 hepatocarcinogen dl-氨基, 这种方案导致过量的肝脏脂肪负荷, 连续炎症, periportal 纤维化, LPC 反应和长期肝癌的发展11,12.然而, 重要的是, 不同的小鼠菌株表现出鲜明的炎症, 化和 LPC 反应动力学13。本协议描述了慢性肝损伤诱导的 C57BL/6J 小鼠, 最常用的近交系小鼠品系。
在慢性肝病的研究中, 典型的分析包括苏木精和红素的组织学评估以及天狼星红色染色, 以可视化胶原沉积、免疫组织化学或荧光检测肝细胞的数量,组通过复杂生长因子和细胞因子网络协调诱导细胞变化的肝脏微环境分析14,15,16,17,18。

<table border="0" cellpadding="0" cellspacing="0" width="982">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Six-week-old male C57BL/6J mice&#160;</td>
			<td style="width:367px;">Animal Resource Centre of Western Australia, Murdoch, WA,&#160; Australia</td>
			<td style="width:121px;">N/A</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 Kg Steam Cut Wheaten Chaff&#160;</td>
			<td>Specialty Feeds, Glen Forrest, WA, Australia&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water for irrigation 1000ml bottle (Baxter)&#160;</td>
			<td>Surgical House, Perth, WA, Australia&#160;</td>
			<td>AHF7114A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Choline- deficient diet, modified (pellets)&#160;</td>
			<td colspan="2">MP Biomedicals Australasia Pty Limited, WA, Australia 02960210</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DL-Ethionine&#160;</td>
			<td>Sigma-Aldrich, Castle Hill, NSW,&#160; Australia&#160;</td>
			<td>E5139-25G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamil injection&#160;</td>
			<td>Troy Laboratories Pty Limited, Glendenning, NSW, Australia&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ilum Xylazil-20 injection&#160;</td>
			<td>Troy Laboratories Pty Limited, Glendenning, NSW, Australia&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">27G x 1/2&#34;, Regular Wall Needle&#160;</td>
			<td>Terumo Australia Pty Limited, NSW, Australia&#160;</td>
			<td>NN-2713R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringes Terumo 1ml and 10ml&#160;</td>
			<td>Terumo Australia Pty Limited, Macquarie Park, NSW, Australia&#160;</td>
			<td>1018242, 1018037</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue-Tek OCT compound&#160;</td>
			<td>VWR International Pty Limited, Tingalpa, QLD, Australia&#160;</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neutral buffered formalin&#160;</td>
			<td>Amber Scientific, Midvale, WA, Australia&#160;</td>
			<td>NBF-2.5L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol absolute anaLaR normalpur&#160;</td>
			<td>VWR International Pty Limited, Tingalpa, QLD, Australia&#160;</td>
			<td>20821.33</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Superfrost Plus slides&#160;</td>
			<td>Grale Scientific Pty Limited, Ringwood, VIC, Australia&#160;</td>
			<td>SF41296SP</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dako Protein Block, serum-free&#160;</td>
			<td>Dako Australia Pty Limited, North Sydney, NSW, Australia&#160;</td>
			<td>&#160;X090930-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dako antibody diluent&#160;</td>
			<td>Dako Australia Pty Limited, North Sydney, NSW, Australia&#160;&#160;</td>
			<td>s0809</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rat anti-CD45&#160;</td>
			<td>eBioscience, San Diego, California, USA&#160;</td>
			<td>m0701&#160;</td>
			<td>1/200 dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rabbit anti-panCK&#160;</td>
			<td>Dako Australia Pty Limited, North Sydney, NSW, Australia&#160;&#160;</td>
			<td>Z0622</td>
			<td>1/300 dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rabbit (Alexa Fluor 488)</td>
			<td>Life Technologies Australia Pty Limited, Mulgrave, VIC, Australia</td>
			<td>A-11008</td>
			<td>1/500 dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rat IgG (Alexa Fluor 594)&#160;</td>
			<td>Life Technologies Australia Pty Limited, Mulgrave, VIC, Australia</td>
			<td>&#160;A-11007</td>
			<td>1/500 dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ProLong Gold Antifade Reagent with DAPI&#160;</td>
			<td>Life Technologies Australia Pty Limited, Mulgrave, VIC, Australia&#160;</td>
			<td>P36935&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Picrosirius Red Stain Kit</td>
			<td>Polysciences Inc., Warrington, PA, USA&#160;</td>
			<td>ab150681&#8195;</td>
			<td></td>
		</tr>
	</tbody>
</table>

the murine choline-deficient, ethionine-supplemented (cde) diet model of chronic liver injury

慢性肝病, 如病毒性肝炎、酒精性肝病或非酒精脂肪肝, 其特征是持续性炎症、肝实质的渐进破坏和再生, 肝祖细胞增殖和纤维化。每个慢性肝病的终末期是肝硬化, 是肝癌发展的主要危险因素。为了研究控制疾病的起始、建立和进展的过程, 实验室中使用了几种动物模型。在这里, 我们描述了六周的时间过程中的胆碱缺乏和氨基补充 (多) 小鼠模型, 其中包括喂养六周的老雄性 C57BL/6J 小鼠与胆碱缺乏的 chow 和 0.15% DL 氨基补充饮用水。对动物健康的监测和典型的体重损失曲线进行了解释。该协议显示了经心脏穿刺治疗的肝脏和血液采集的总检查结果, 随后进行血清分析。其次, 肝脏灌注技术和收集不同的肝叶的标准评价显示, 包括肝组织学评估的苏木精和曙红或天狼星红色染色, 荧光检测肝细胞数量以及对肝脏微环境的转录分析。这种小鼠模型适于研究慢性肝病诱发的炎症、化和肝祖细胞动力学, 并可用于测试可能调节这些过程的潜在治疗剂。

在六周的时间过程中, 多因素诱发慢性肝损伤, 参数评估的天数 7 (归纳阶段), 14 和 21 (建立阶段) 和 42 (维护阶段)。与对照组小鼠相比, 经治疗的小鼠在初始适应阶段损失了20% 的初始体重, 并倾向于在建立和维护阶段重拾重量(图 1)。体重与血清丙氨酸转氨酶水平呈反比关系, 这是肝细胞损伤的指标(图 2)。...

Watch this Scientific Journal Video about The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury at JoVE.com

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

在这里, 我们描述了一个常见的方法来诱导慢性肝损伤小鼠通过喂养的胆碱缺乏和氨基补充 (多) 饮食。我们展示健康监测, 肝脏灌注, 隔离和保存。六周的时间过程可以告知肝损伤、病理、纤维化、炎症和肝祖细胞反应。

慢性肝损伤小鼠胆碱缺乏、氨基补充的饮食模型

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

the-murine-choline-deficient-ethionine-supplemented-cde-diet-model

Research

JoVE Journal

Biology

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

11.6K 次观看.  Curtin University. 在这里, 我们描述了一个常见的方法来诱导慢性肝损伤小鼠通过喂养的胆碱缺乏和氨基补充 (多) 饮食。我们展示健康监测, 肝脏灌注, 隔离和保存。六周的时间过程可以告知肝损伤、病理、纤维化、炎症和肝祖细胞反应。

视频: The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

激光诱导SD大鼠慢性高眼压高压模型

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular ...

科研

生物学

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

肢体缺血小鼠模型

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

小鼠肾移植程序

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Chronic Salmonella Infected Mouse Model

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Chronic Salmonella Infected Mouse Model

Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.

慢性沙门氏菌感染小鼠模型

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

大鼠门户成纤维细胞的分离<em&gt;原位</em&gt;肝脏灌注

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

小鼠主动脉夹层损伤:高效<em&gt;体内</em&gt;平滑肌细胞增殖和内皮功能模型

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

A Murine Model of Vertical Sleeve Gastrectomy

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. Bariatric surgery is currently the most effective long-term treatment for obesity. In addition to weight loss, bariatric surgery produces additional health benefits such as remission of type 2 diabetes, remission of hypertension, and decreased risk of developing certain types of cancer. The mechanisms beyond weight loss for these benefits remain incompletely defined. Therefore, animal models of bariatric surgery are being developed and validated to identify the mechanisms leading to these benefits, with the goal of improving understanding of gastrointestinal physiology and identifying new therapeutic targets. VSG has become the most commonly performed bariatric procedure in the clinic in the United States because it is highly effective at producing weight loss and metabolic improvement, and is simpler to perform than other bariatric procedures. Therefore, we have developed and validated a murine model of VSG. This murine VSG model recapitulates many of the effects of VSG seen in humans, including improved glucose and blood pressure regulation. The method is based on isolation of the stomach, ligation of gastric vessels, and removal of 70% of the stomach by transecting along the greater curvature of the stomach. We have successfully applied this surgical protocol to various genetically modified mouse lines to define the mechanistic contributors to the benefits of VSG. Furthermore, this murine VSG model has been combined with other surgical techniques, to achieve deeper mechanistic insight. Therefore, this is a simple and versatile model for studying gastrointestinal physiology and the health benefits of bariatric surgery.

The following describes the performance of vertical sleeve gastrectomy in mice. This is a type of weight-loss surgery that involves removal of approximately 70% of the stomach.

小鼠垂直套胃切除术的动物模型

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

小鼠部分胆管结扎术: 局部梗阻性胆汁淤积症的对照模型

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...