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>
	<li>在用手套抚摸动物之前, 把母亲的被褥揉到手套上。</li>
	<li>把所有幼崽从母亲身上移开, 放入空笼子里。删除一些母亲的床上用品, 并把它放在空笼与幼崽。</li>
	<li>把母亲放在一个独立的、干净的、干燥的笼子里, 远离手术场。</li>
	<li>新的, 干净的无菌手套在外科领域准备之前。</li>
</ol>2. 手术现场准备
<ol><li>使用10毫升吸管, 添加10毫升磷酸盐缓冲盐水 (PBS) 到10厘米培养皿。</li>
	<li>吸管2毫升的 betadine 或等效的抗脓毒溶液在10厘米培养皿。</li>
	<li>在手术领域放置解剖范围。打开解剖范围灯, 并调整光的水平, 以外科医生的舒适。小狗可以放置在解剖范围灯下面的手术领域。足够的光线可以通过小狗腹面上没有阴影来证实。</li>
	<li>用鼻锥准备异氟醚麻醉室 (参见材料表)。该分庭应在没有尿液或粪便证据的情况下进行清洗。将鼻锥及相关导管置于手术领域的解剖范围内。将氧气和异氟醚的流动完全转移到鼻锥上。</li>
	<li>准备手术后恢复区与加热垫设置在大约37°c。手术后的恢复应包括4片纱布放在加热垫上。如果可能的话, 擦纱布垫在母亲的床上用品, 粪便, 和尿液, 以保存母亲的气味。 注意: 使用热灯是不明智的, 因为这使得控制温度变得困难。高温会导致新生鼠死亡。</li>
	<li>用高压釜准备和消毒所有外科器械。所需的工具包括: 显微解剖剪刀, 显微解剖钳, 纱布, 止血, 和任何6-0 单丝不可吸收缝合。手术器械可以 resterilzed 任何玻璃珠消毒器。</li>
</ol>3. 部分小叶切除术
<ol><li>麻醉把小狗放在鼻子锥上, 然后轻轻地把脚和手贴在原处。小狗应该在氧气中接收5% 异氟醚。<ol>
<li>允许幼犬坐5分钟或直到充分麻醉, 这可以通过脚趾捏测试验证。</li>
		<li>在切口前注射5毫克/千克卡洛芬。 注: 整个手术应不超过30分钟. 在全身麻醉下的新生儿中, 可能会发现较贫穷的结局30分钟以上。采取预防措施, 最大限度地减少手术的长度, 通过彻底的现场准备和润湿皮肤之前关闭, 以尽量减少缝合引起的皮肤撕裂。</li>
	</ol>
</li>
	<li>用 betadine 湿的小纱布轻轻擦拭后腹壁。允许 betadine 干燥1分钟。</li>
	<li>用微解剖剪刀和镊子将左中锁骨0.5 厘米切口立即放在肋骨笼下面。用镊子轻轻地将皮肤分开, 然后将第二个更深的切口插入腹膜腔 (参见图 1C, 左和中心)</li>
	<li>轻轻地施加腹部两侧的侧压, 使用背部, 钝端的微解剖剪刀和镊子, 以迫使左叶顶端的腹膜腔。左叶的左尖应该很容易被可视化 (图 1C, 右)。</li>
	<li>
从先端, 截肢, 并权衡要去除的组织数量。
	<ol>
<li>使用微解剖剪刀, 从左叶的顶端轻轻地切除所需的组织数量。</li>
		<li>将被截去的组织放进一个1.5 毫升的试管中, 用 PBS 填充。使用分析天平对被截断的组织进行称量 (请参阅材料表)。 注: 在天平上放一块纱布或纸, 并将其包装。然后将被截断的组织放在纱布或纸上, 并测量其质量。</li>
		<li>将2% 多聚甲醛的截面积固定在室温下1小时, 并将最佳切削温度 (OCT) 在干冰中放置, 用于冷冻切片分析。通过使用任何标准恒温器切割7-10 µm 切片来分析冰冻切片。</li>
	</ol>
</li>
	<li>使用卷纱布, 轻轻地将左叶替换成腹腔。把纱布留在洞里直到出血停止。</li>
	<li>取出纱布, 并用 PBS 浸泡的纱布将手术部位和周围区域弄湿。</li>
	<li>关闭手术部位与6-0 单丝, 不可吸收缝线与运行针。腹膜和皮肤应分别闭合。 注: 用 PBS 浸泡纱布轻轻润湿皮肤, 可减少缝合引起的泪。</li>
	<li>轻轻地, 但彻底清洁小狗用纱布浸泡与 PBS, 并确保没有血液或 betadine 遗骸。卷上浸泡的纱布垫, 轻轻擦洗伤口标志, 清除任何血液或 betadine。 注意: 这是特别重要的, 因为母亲可能蚕食幼崽, 如果他们被清理得不充分。</li>
	<li>从鼻锥中取出幼犬, 将其放在恢复区, 包括暴露在母亲粪便和尿液中的纱布垫。一旦幼崽恢复, 用母亲的被褥替换空笼子里的小狗。 注: 请勿使用散热灯。使用热灯会导致新生儿过热。</li>
	<li>对所需的幼崽数重复该过程。虽然所有的幼崽都可以使用 , 但一般是可取的 , 让一些未操作的幼崽与作的幼崽一起替换。</li>
	<li>在母亲的笼子里同时用母亲的被褥替换所有幼崽。</li>
</ol>4. 恢复和分析
<ol><li>
每天跟踪老鼠。
	<ol>
<li>检查伤口是否保持闭合, 并清除任何死幼崽, 如果存在。</li>
		<li>如果伤口重新打开, 准备手术部位和恢复区如前所述, 并重复步骤3.8 至3.12。</li>
		<li>注射5毫克/千克的 carpofen 皮下24和48小时后的程序。</li>
		<li>跟踪幼崽56天或更多。</li>
	</ol>
</li>
	<li>通过二氧化碳 (CO2) 暴露和宫颈脱位, 弄死动物在手术后所需的天数。<ol>
<li>在诱导/安乐死室放置老鼠, 并打开 CO2直到老鼠停止呼吸。</li>
		<li>通过颈椎脱位确保安乐死。使用前指和拇指向下推鼠标的背颈, 然后用另一只手向下拉下尾部。</li>
	</ol>
</li>
	<li>
删除整个肝脏en 集团和权衡它。
	<ol>
<li>仔细地分开每个裂片并且单独称重。 注: 仔细解剖膈肌和肝门血管, 取出肝脏。通过仔细解剖每一个裂片在其近端的附件, 分离出不同的叶。</li>
	</ol>
</li>
	<li>通过将被截离的左叶质量与整个肝脏的质量进行比较, 确定再生的程度。没有受伤的左叶大约是整个肝脏的30%。</li>
</ol>

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作者没有披露。

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。最近, Porrello et等和嫦娥et 等. 显示了明显不同的新生儿再生反应后, 急性损伤心脏, 数字提示, 和耳朵7,8。他们的结果平行地提出结论肝脏也经历一个明显的再生现象在新生儿生活中6。在主要器官中有多个相似的发现, 在产后发育的早期阶段再生是一个新兴领域, 可能对干细胞生物学产生影响。
新生儿部分小叶 hepatectomies 的早期死亡率往往来自于不充分的康复、重大出血或产妇疏忽。如前所述, 使用更高强度的热源, 如热灯来恢复, 可能导致手术后死亡。新生小鼠至少在生命的前两周依赖母亲。同时, 如果她感觉到异常 (如血液或其他化学物质的气味)24,25, 母亲将经常忽略和或蚕食她的年轻.因此, 非常重要的是, 在手术后彻底清洁新生儿, 并与母亲床上用品摩擦, 以掩饰任何进攻性气味。如果这些问题得到充分解决, 生存可以达到100%。如果母性的同类相食成为一个问题, 幼崽可以被安置在一个笼子里, 有一个代孕母亲与她的一些幼崽。如果是这种情况, 请在前面的步骤中使用代理母亲的床上用品。
切除20-30% 的新生儿左叶和随后的再生可能不是只有左叶固有。目前, 这种方法仅在左侧进行了测试, 因为暴露中位和更多的后右和尾状裂片将需要一个更大的剖腹手术, 从而导致更高的出血风险, 间接地, 产妇同类相食的风险更高的新生儿。然而, 新生儿再生机制在肝脏内是否存在异质性是一个重要的问题, 因此, 对该议定书进行手术调整, 以询问其他肝叶。
这些新生儿肝切除研究的结果显示了一个时间段 (P0-P7), 在此期间, 再生是可以发生的。对幼年小鼠 (P7、P10、P14) 也进行了类似的肝切除, 并不会导致完全再生, 显示有疤痕和纤维化, 标志着截肢发生的明确区域。虽然部分小叶切除术后幼年小鼠的损伤反应不是初步研究的重点, 但新生小鼠再生潜能的差异, 以及器官组织重建能力的丧失建筑学, 将是必不可少的理解什么机制茎或祖细胞新生儿再生发生。
我们以前已经证明, 新生儿再生肝脏不仅在建筑和结构上出现相同, 而且也与功能无法区分。免疫荧光染色的功能性肝酶, 如谷氨酰胺合成酶 (GS), carbamoylphosphate 合成酶 (CPS) 和细胞色素 p450 2E1 显示了类似的分布在再生地区相比未受伤的裂片。然而, 再生新生儿的次生再生潜能还未被测试。由于新生小鼠可以恢复56天, 在生理上与未受伤的对照组有区别, 在成人70% 部分肝切除术后, 可能会发生经典的再生反应。然而, 这种肝再生往往受到肝细胞衰竭的限制, 因此, hepatectomies 的部分小叶 hepatectomies 将是一个重要的研究。

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。在恢复后, 这些小鼠左裂片的苏木精和伊红 (H & E) 显示在图 2中。值得注意的是, 在56天后, 被截肢的左叶可能与对照, 未受伤的裂片看起来无法区分。对 P14 幼年小鼠进行的手术进行比较, 并允许恢复 7, 14, 56 天后手术 (图 S1)。
为描述新生儿再生, 45 只小鼠在0天内进行了部分小叶切除术, 其所有裂片的肿块被服用56天后手术。与其他未受伤的中位、右、尾状裂片 (图 3A) 和未受伤的对照组相比, 受伤左叶的质量发生了更大的变化, 接近于 P56 未受伤左叶的质量。这表明新生儿肝损伤后的再生是局部的左叶。对 P14 小鼠进行的手术进行比较, 结果表明左叶再生减少, 未受伤的裂片增加补偿 (图 3B), 表明14天后, 急性切除的损伤反应从叶的特定再生转向全球补偿。进一步的特征是通过染色小鼠左叶的区域在术后56天, 与丝状肌动蛋白 (f 肌动蛋白), 以可视化细胞膜 (图 4A)。在70% 部分 hepatectomies 后14天, 将损伤部位远端和近端区域与未受伤的对照组和成人裂片进行比较。肝细胞被发现有类似的区域如未受伤的控制, 约 1.5-2 x 小于成年小鼠正在进行再生后, 经典70% 部分肝切除 (图 4B)。这表明肥大在再生方面没有作用。最后, 在 90% PBS 和10% 乙醇和1、3、5、7、14天的手术后, 注射了0.025 毫克 5-乙炔 2 '-脱氧尿苷 (教育局) 的新生小鼠。在手术后7天内, 从允许恢复的小鼠中计算出的教育局阳性细胞数 (图 4C)。与未受伤的对照组相比, 在受伤/再生左叶中发现的教育局阳性细胞数量显著增加, 表明细胞增殖有助于新生儿再生。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57302/57302fig1.jpg"> 图 1: 部分小叶肝切除概述.(A)在 P0 进行的新生儿肝切除术显示部分小叶切除的一般示意图和时间线. 分析是在 P7、P14、P35 或 P56 进行的。切除也在 P7 和 P14 受审。(B) 显示左叶切除范围的示意图, 标定20和30% 切除。(此数字已由蔡et修改)6. (C) 来自新生儿手术的图像显示: 右锁骨中段切口 (左、中心) 和左叶尖部 (右) 外露。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57302/57302fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57302/57302fig2.jpg"> 图 2: 部分小叶切除术后再生.P0 部分小叶 hepatectomies 的小鼠随访2、7、14、35、56天。肝的固定和染色的 H & E, 并指出再生的程度, 左尖。箭头表示 P0 小鼠再生的区域。刻度条为1厘米。这个数字是从蔡等. 修改的。6<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57302/57302fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57302/57302fig3.jpg"> 图 3: 部分小叶切除术后每裂片再生.(A) 对 P0 部分小叶 hepatectomies 的小鼠进行了手术后7、35和56天的分析。对小鼠进行了安乐死, 并与受损伤的小鼠 (红) 的所有裂片肿块进行比较, 并与未受伤对照组 (红) 的年龄相匹配。B对 P14 部分小叶 hepatectomies 的小鼠进行手术后7、35、56天的分析。从损伤小鼠 (红) 中取出所有裂片, 并与未受伤对照组 (红色) 进行比较。* = p < 0.05, ** p < 0.005, *** = p < 0.0005, NS = 不显著。这一推测已从蔡等. 修改。6<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57302/57302fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57302/57302fig4.jpg"> 图 4: 肝切除后再生的特征.(A) 对 P0 部分小叶 hepatectomies 的小鼠进行切除后56天的分析, 并对 F 肌动蛋白染色。图像显示的污点从近和远端地区的截肢, 以及从年龄匹配未受伤的控制, 并从成年小鼠14天后, 70% 部分肝切除。刻度条为100µm. (B) 在切除部位近端或远端受伤的肝细胞区, 与未受伤对照组和成人70% 部分 hepatectomies 的肝细胞区域进行比较. * = p < 0.05, ** = p< 0.005, ** = p < 0.0005, *** = p < 0.00005, NS = 不显著。(C) 在 P0 进行小叶 hepatectomies 的小鼠接受了治疗, 并对切除后7天进行了分析。显示左叶中的教育局+单元格。(缩放条, 100 µm)。(D) 在与对照组比较的部分小叶肝切除术7和14天后, 对小鼠的教育局+细胞进行量化。值是指电子扫描电镜。这一推测已经从蔡等。6<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57302/57302fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
图 S1: 幼年小鼠的不完全再生.P14 部分小叶 hepatectomies 的小鼠随访7、35、56天。肝的固定和染色的 H & E 和再生的程度, 左尖的更新。箭头表示 P0 小鼠再生的区域。刻度条为1厘米。这一推测是从蔡等. 修改的。6<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57302/Figure_S1.pdf">请单击此处下载此文件.</a>

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 ...

partial-lobular-hepatectomy-surgical-model-for-morphologic-liver

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.0K 次观看.  Stanford University School of Medicine. 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。

视频: Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

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

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

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

The objective of this report is to describe the protocols to derive the retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells using 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

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 ...

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.

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.

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 ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

鼠标子宫内膜上皮细胞和基质细胞的隔离<em&gt;体外</em&gt;蜕膜

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

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 abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

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

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 any type of injury. Common injury models in these animals remove large amounts of tissue, which damages multiple organs. To overcome this broad tissue damage, we describe here a method to selectively remove a single organ, the pharynx, in the planarian Schmidtea mediterranea. We achieve this by soaking animals in a solution containing the cytochrome oxidase inhibitor sodium azide. Brief exposure to sodium azide causes extrusion of the pharynx from the animal, which we call &#34;chemical amputation.&#34;&#160;Chemical amputation removes the entire pharynx, and generates a small wound where the pharynx attaches to the intestine. After extensive rinsing, all amputated animals regenerate a fully functional pharynx in approximately one week. Stem cells in the rest of the body drive regeneration of the new pharynx. Here, we provide a detailed protocol for chemical amputation, and describe both histological and behavioral methods to assess successful amputation and regeneration.

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

The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

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

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

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

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

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

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

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 ...