Name: partial lobular hepatectomy: a surgical model for morphologic liver regeneration
Uploaded: 2018-05-31T13:30:00
Description: Watch this Scientific Journal Video about Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration at JoVE.com

感谢朱棣文的 & 和组织学的表现;还有麦卡蒂的讨论。研究得到了弗吉尼亚和交流的癌症研究基金会的资助;国家心脏、肺和血液研究所 (R01HL058770 和 U01HL099999);加州再生医学研究所 (RC1 00354) 资助 i.w.y. R. 得到了人类前沿科学项目职业发展奖 (CDA00017)、德国研究基金会 (RI 2787/1)、Siebel 干细胞研究所和托马斯和史黛西 Siebel 基金会 (1119368-104-GHBJI)。J.M.T. 得到了美国国立卫生研究院 (T32GM007365)、国家研究服务奖 (1F30DK108561) 和保罗和黛西. 索罗斯为新美国人提供的奖学金的支持。

所有动物实验都严格按照国际实验动物保育协会 (AAALAC) 和斯坦福大学实验室动物管理小组所规定的指导方针进行。护理 (亚太), (议定书号 #10266) 和在美国, 或《欧洲动物福利法》, 指令 2010/63/欧盟。该议定书获得了德国巴伐利亚政府动物实验伦理学委员会的批准, 并获得了许可号: 55.2-1-54-2532-150-2015。
1. 动物制剂
<ol><li>在加热垫上准备一个有适当床上用品的空笼子。</li>
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<ol>
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急性肝损伤传统上是使用化学 (扑热息痛, 乙醇, 四氯化碳), 或外科模型 (70% 部分肝切除)。70% 部分肝切除后再生反应的特点是涉及全球肝细胞肥大和多轮细胞分裂4,5。然而, 为了停止出血, 这个模型是有限的, 因为主要的血管必须结扎离开一个异常的环境, 以再生。因此, 许多研究都采用了其他不那么侵入性的急性损伤模型, 通过化学损伤, 使总的结构发生?...

再生器官的能力, 或恢复形式和功能, 一直被认为是在进化时期失去了大部分。在急性化学或物理损伤后, 成年哺乳动物肝脏的再生潜能涉及动员所有剩余的肝细胞, 导致肥大和几轮细胞分裂, 导致功能性但建筑上不同的器官1,2,3,4,5。最近, 研究已经开始描述新生儿哺乳动物器官在生命的第一周内对损伤的再生反应6,7,8。这些研究表明, 当在新生儿发育期间受伤时, 某些哺乳动物器官以形态学再生代替补偿生长或纤维化7,8。
最近的研究表明, 全球结构和功能的再生发生在早期新生儿期6,7,8。建立的肝损伤协议涉及化学伤害或酒精的管理9,10,11, 扑热息痛12,13,14,15, 四氯化碳16,17,18,19, 70% 部分肝切除4,20,21, 或删除左侧和中位裂片。化学管理导致肝细胞死亡, 但经常留下微和宏观结构完好。由于整体的肝脏结构没有被抹杀, 所以在这种背景下不能轻易地研究形态学再生。70% 部分肝切除术包括缝合结扎的主要血管, 这是必要的止血, 但留下一个非生理环境与永久性中断的血管。此外, 该方法仅用于成年啮齿动物, 其在新生儿中的应用在技术上极其困难。考虑到这一点, 我们开发了一种方法, 其中20-30% 的左叶顶点被移除在新生的 P0 鼠标 (图 1A-1B) 中。该方法手术保守, 微创, 不具技术挑战性, 导致形态学毛损, 不结扎血管, 留出再生空间。由此得出的分步协议, 允许任何研究员对新生小鼠进行部分小叶切除, 以便研究哺乳动物在产后早期的新生。这种方法在再生医学和干细胞生物学的比较研究中也有明显的应用, 因为它可以在生命后期的肝脏中使用。
最常见的急性肝损伤研究是化学性损伤, 成人肝脏截肢, 或70% 部分肝切除。化学损伤通常包括静脉注射、腹腔或口服乙酰氨基酚、四氯化碳或乙醇, 是一种相对容易和无创性的损伤模型。如前所述, 化学损伤导致肝细胞死亡, 但往往使基质和软组织结构完好, 使人们难以对形态学再生提出要求。化学损伤往往集中在肝脏血管, 使其成为一个有用的技术来研究现场和细胞特定的伤害, 但也使它难以在整个器官层面上审问其他人群, 可能是进一步从船只, 并可能有助于再生。尽管有这些限制, 化学损伤仍然是一个有用和高度生理相关的伤害模型。
成人70% 部分肝切除术包括切除肝血管结扎后的左侧和正中裂片。对肝切除的反应有很好的特点: 截肢后的肝14天后70% 部分肝切除与原始未受损的小叶形成严重不同的结构, 作为剩余右、尾状裂片的肝细胞经历肥厚和几轮细胞分裂4,5。这弥补了丢失的质量和功能, 但未能再生两个截切的裂片, 因此不取代毛形态。因此, 对70% 例部分肝切除术的损伤反应有助于研究有限再生的代偿生长机制。
在这里, 我们充分描述了一个新生儿部分小叶切除的协议。该程序涉及适当的动物选择和准备, 外科领域的准备, 手术和恢复。对议定书的不同应用可能需要对这些步骤进行优化和调整。
我们对野生型 C57BL/6J 幼崽 (jax-rpc 000664) 的这一协议进行了广泛的执行和优化, 但是, 为了研究不同的细胞种群和再生机制, 我们还使用了各种转基因动物, 包括饲养各种品种的小鼠和CreERT2 转基因和/或敲门 (Axin2CreERT2 jax-rpc 018867,和Sox9CreERT2 jax-rpc 018829) 与荧光记者, 如彩虹和 mTmG 系统 (R26VT2/GK3,R26mT/mG)22,23. 我们发现没有必要改变这种方法的不同的老鼠菌株, 因为没有差异的生存结局或再生潜能被观察到。
除了使用不同的动物菌株, 我们还对小分子治疗的新生小鼠进行了部分小叶 hepatectomies, 如 4-羟基三苯氧胺和 5-乙炔--2′-脱氧尿苷 (教育局)。以二甲基亚砜和乙醇为溶剂, 发现玉米油是引起发病率的重要原因。否则, 我们发现, 腹腔内对小分子的管理不影响生存或再生结果。我们预测, 这个协议将被修改, 以用于其他小分子, 以审问的各个方面的再生。
新生小鼠手术在技术上是有挑战性的, 可能需要专门的动物处理和显微解剖专家。畜牧业专门知识是必要的, 以避免在手术后和在立即恢复期间的孕妇同类相食。

<table>
	<tbody>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mother with litter of day 0 neonatal pups (any strain)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surrogate mother and surrogate litter (optional)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Standard Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Serum (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Providine-iodine or equivalent antiseptic solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Surgical Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Zeiss</td>
			<td>ZEMSDV4L MFR # 435421-9901-000</td>
			<td></td>
		</tr>
		<tr>
			<td>3mm straight spring micro scissors</td>
			<td>Vannas</td>
			<td>72932-01</td>
			<td></td>
		</tr>
		<tr>
			<td>5SF Forceps</td>
			<td>Dumont</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Kelly forceps</td>
			<td>Grainger</td>
			<td>17-050G</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Sunbeam</td>
			<td>000771-810-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott Labs</td>
			<td>0044-5260-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent Anesthesia System</td>
			<td>Kent Scientific</td>
			<td>1205S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze, 10.16 x 10.16cm</td>
			<td>Fisher Scientific</td>
			<td>13-761-52</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Standard Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 monocryl sutures</td>
			<td>Ethicon</td>
			<td>MCP489G</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher Scientific</td>
			<td>&#160;S35839</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Aid, Plain, 110V</td>
			<td>Drummond</td>
			<td>4-000-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Mettler Toledo NewClassic ME Analytical Balances</td>
			<td>Fisher Scientific</td>
			<td>01-912-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Cost Induction Chamber</td>
			<td>Kent Scientific</td>
			<td>SOMNO-0730</td>
			<td></td>
		</tr>
	</tbody>
</table>

partial lobular hepatectomy: a surgical model for morphologic liver regeneration

急性组织损伤后的形态学器官再生在下脊椎动物中很常见, 但在哺乳类动物产后生活中很少观察到。70% 部分肝切除后成人肝再生导致肝细胞肥大, 其余裂片复制, 代谢活性恢复, 但损伤叶的形态学和结构永久性丧失。在这里, 我们详细介绍了新生儿的一种新的外科方法, 它留下了一个有利于再生的生理环境。该模型涉及截肢的左叶顶端和随后的保守管理方案, 并缺乏必要的结扎的主要肝血管或化学伤, 留下一个生理环境, 再生可能发生。我们将这项议定书扩展到对幼年 (P7-14) 小鼠的截肢, 在此期间, 受伤的肝脏从器官再生过渡到代偿性生长。提出的, 简短的30分钟协议提供了一个框架, 以研究再生机制, 其年龄相关的下降, 哺乳动物, 和鉴定的肝茎或祖细胞。

图 1A详细介绍新生儿部分小叶切除术的一般时间线 (图 1B中的示意图), 以及预期等待的时间长度, 直到观察到再生。术后7-14 天可以观察到左叶的细微再生。术后56天, 常观察到全再生。小鼠在手术后不应显示生理异常的迹象。
接受部分小叶 hepatectomies 的小鼠可恢复2、7、14、35和56天?...

Watch this Scientific Journal Video about Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration at JoVE.com

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

部分小叶切除术: 形态学肝再生的外科模型

本文提出了新生儿 (0 天) 小鼠左肝叶部分切除的新方法。这一新的协议适用于研究急性肝损伤和损伤反应在新生儿的设置。

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

Research

JoVE Journal

Developmental Biology

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

肾再生的成年斑马鱼庆大霉素性损伤

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

科研

发育生物学

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

从诱导多能干得出视网膜色素上皮（RPE）（IPS），由胚体大小不同的细胞

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

Hepatocyte-specific Ablation in Zebrafish to Study 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 ...

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

心肌细胞DNA的合成和倍性的同时评价：一个方法来帮助心肌细胞再生和营业额的量化

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

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

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

手术消融分析在研究涡虫再生眼

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

真涡虫的化学截肢和咽部再生Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

诱导多潜能干细胞源性肝细胞的家族性高胆固醇血症人肝嵌合小鼠模型

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

Methods for the Study of Regeneration in Stentor

Methods for the Study of Regeneration in Stentor

Stentor再生的研究方法

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

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...