这项工作得到了印度政府生物技术部向新德里国家免疫学研究所提供的核心赠款的支持。巴塔查尔吉博士目前的讲话是胃肠病学、肝病和营养科, 洛杉矶儿童医院。

本议定书所载程序已由新德里国家免疫学研究所机构动物伦理委员会批准。审批的串行参考编号为 IAEC#319/13.
注: 一般手术程序有很好的资源20和针对啮齿动物手术的特定协议21。对于那些第一次做动物外科手术的人来说, 建议在对动物进行手术前, 广泛地练习使用假的程序。
1. 准备
<ol><li>在试验之前, 保持无菌磷酸盐缓冲盐水 (PBS) 或盐水准备好。</li>
	<li>组装一个包括剪刀, 锯齿钳, 组织钳, 棉花, 棉花芽, 尼龙螺纹, 和不同的微型针持有人的手术套件。高压釜的手术套件。如果免疫受损的点头, 应特别小心。协议中包含了 "免疫" 鼠标。</li>
	<li>执行完整的实验程序, 从准备到手术结束, 在生物安全类 I 型橱柜。</li>
	<li>重点头。手术前6-8 周大鼠。在这项研究中使用了体重在14-18 克之间的老鼠。</li>
	<li>用剪毛机刮上中央和有疑的腹部区域的小鼠。将脱毛膏均匀涂抹在整个区域, 用刮刀彻底除去修剪过的毛发。2-5 分钟后, 用一块湿不育棉轻轻地去除毛发。</li>
	<li>将鼠标放在异氟醚腔内, 并解锁氧气缸的阀门。保持氧流的速率为4升/分和异氟醚汽化4% 诱导麻醉。<ol>
<li>通过轻柔的脚趾捏, 确保鼠标被正确地麻醉。</li>
	</ol>
</li>
	<li>在生物安全柜内放置一个手术板。把动物放在手术板上, 这样鼠标的腹部就会朝上, 老鼠的前部被放在与异氟醚和氧气供应相连的鼻锥内。</li>
	<li>减少异氟醚汽化至 2%, 并保持整个手术过程。</li>
	<li>对老鼠的皮肤进行消毒, 用70% 乙醇浸泡的无菌棉擦拭消毒。</li>
</ol>2. 手术方法
<ol><li>
部分肝切除术
	<ol>
<li>在胸骨下方的皮肤上进行横向切口, 垂直于剑突过程, 并与胸腔平行, 在直操作剪刀的帮助下。</li>
		<li>用镊子或无菌湿棉小贴士轻轻地将与腹部肌肉层相连的皮肤与皮肤和腹部肌肉层分开。用无菌棉花小贴士浸泡皮区, 避免脱水。</li>
		<li>通过在剑突下方的腹膜层进行横切口, 使左叶的面积平滑。使用两个湿润的棉花小贴士来暴露和举起肝脏的左叶。</li>
		<li>将棉花尖放在切口的腹部一侧, 再将另一条棉花尖放在隔膜侧。轻轻按下放置在隔膜上的尖端, 并给出一个滑动推的另一尖端, 以解除肝脏左叶。</li>
		<li>通过在微钳或棉花小窍门的帮助下, 通过在左叶上滑动一条带环的尼龙线, 并将环滑向靠近脐部的左叶底部。轻轻地向下推尼龙线循环到底部的左叶。</li>
		<li>用显微外科针架和微钳将尼龙线的两端系在左叶顶部。在另一侧增加两个结节。</li>
		<li>用剪刀把绑的裂片解剖出来。不要试图切断非常接近线程。如果手术时间超过5分钟, 保持腹膜腔和器官湿润与无菌 PBS, 以避免因液体流失而脱水。</li>
		<li>用4-0 线线缝合法缝合腹膜。随后, 通过不连续缝合, 尽快关闭皮肤。</li>
	</ol>
</li>
	<li>
细胞移植 注: 根据李et(C57BL/6-Tg) 所描述的程序, 荧光蛋白表达肝细胞与转基因 gfp 小鼠 (30 Scha/J) 分离。22和沈et al。23与单核细胞 (24 ) 分化的人源肝样细胞 (NeoHep) 也用于移植。然而, 从任何其他来源获得的细胞也可以在议定书中使用。<ol>
<li>在 Iscove 修饰 Dulbecco 的培养基 (IMDM) 的大约50µL 中悬浮20万个可行细胞, 并将其吸入1毫升胰岛素注射器, 30G 针。把注射器放在冰上, 把它放凉。</li>
		<li>放置鼠标的方式, 左侧面部分朝上执行手术的人。确定脾区, 横向解剖周围的忧郁症地区的皮肤, 其次是通过腹膜层的短切口, 只是为了暴露脾脏。</li>
		<li>轻轻抬起脾脏, 并在两个 PBS 湿棉花小贴士的帮助下, 把它放在腔外。</li>
		<li>一只手用两根棉花尖小心翼翼地握住脾脏, 将注射器的针头精确地垂直于脾脏。轻轻刺穿脾脏, 将针慢慢地推入体内;针不应该比2毫米更深。</li>
		<li>慢慢推下注射器的活塞, 将细胞注入脾脏。移植后, 保持注射器针稳定, 并从脾脏中慢慢取出, 以避免出血或细胞丢失。</li>
		<li>用棉尖将脾脏放回腹膜腔后, 通过连续缝合4-0 条线缝合腹膜层。用同样的缝线缝合皮肤间断。避免使用伤口夹来闭合皮肤;相反, 用4-0 缝合线关闭它。伤口剪辑限制了鼠标的自然运动, 经常剪辑变得松散, 迅速出来。</li>
	</ol>
</li>
</ol>3. 术后护理
<ol><li>关闭皮肤后, 使用无菌棉尖, 用碘溶液 (betadine) 擦拭缝合部位的周围环境。</li>
	<li>注射一剂量抗生素头孢噻肟为600毫克/千克体重 (通常12毫克头孢噻肟100µL 生理盐水/小鼠) 腹腔使用1毫升注射器。</li>
	<li>每日剂量止痛 Meloxicam 为1毫克/千克体重 (通常12µg Meloxicam 100 µL 生理盐水/小鼠) 到动物腹腔, 在手术后三天。</li>
	<li>手术完成后, 停止异氟醚气体的流动, 把鼠标放回单独通风的笼子里。</li>
</ol>4. Euthanization 和刻画
<ol><li>实验结束后 (1 天10天后手术), 弄死小鼠根据机构动物伦理准则。</li>
	<li>收集血液由末期流血的动物, 刺穿眼球末端丛。</li>
	<li>从血液中分离出血清。</li>
</ol>

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</ol>

作者没有什么可透露的。

部分肝切除术是一种研究肝再生的方法, 据报道过度肝切除是模拟 AHF 模型。在 AHF 动物模型中, 啮齿动物, 特别是小鼠, 是研究最多的模型。为了获得小鼠肝脏损伤模型, 多达70% 的肝切除已报告具有良好的存活率25,26。然而, 在裸和其他 immunodeficient 鼠, 70% 例肝切除被报告为致命, 动物死亡24小时内27。
米切尔和 Willenbr...

目前, 肝细胞移植是一种替代全器官移植治疗严重肝病患者。据信, 它可以把病人移植到全身器官,1。除了同种异体肝细胞2外, 异体肝细胞3和干细胞 (4 ) 的肝细胞也正在动物模型中进行研究。在这一背景下, 受体移植细胞的归巢和植入电位是急性肝功能衰竭 (AHF) 细胞治疗的重要标准。
为研究肝细胞或肝样细胞的移植5, AHF 是由手术的6或药理7程序在动物模型中创建的, 其次是移植细胞。通过药理试剂制作 AHF 动物模型, 许多 hepatotoxins 如 d-乳糖8、乙酰氨基酚9、四氯化碳10、硫乙酰胺11、刀豆蛋白 12、脂多糖13已使用、等。从这个列表中, 每个试剂为 AHF 生成一组独特的功能, 但不幸的是, 没有单一试剂模仿人类 AHF。此外, hepatotoxins 诱发的 AHF 时间长, 使动物处于慢性应激状态, 难以获得重现性结果。
另一方面, 部分肝切除术 (phx-空港) 的手术方法是技能依赖性的, 在培养所需技能后, 可重现性结果容易获得。仅以手术干预诱发 AHF, 需要切除超过70% 的肝脏;然而, 70% 的肝切除术仍可用于研究肝脏移植细胞的植入和增殖, 以分析其在肝损伤期间的治疗能力14。肝细胞移植术后经腹膜切除后, 经腹腔15、尾静脉16、肝静脉17或脾18。目前, 肝静脉输液和肝细胞脾移植是首选的程序, 因为它们更容易繁殖。
在本文中, 我们已经描述了30% 部分肝切除在点头的程序。(点头。CB17-Prkdcscid/J) 肝左叶切除的小鼠。其次是移植 20万 GFP 表达小鼠 (C57BL/6-Tg (不列颠哥伦比亚) 30 scha/J) 肝细胞以及人类起源 NeoHep19在脾脏。这个过程导致肝脏移植细胞的植入。这个程序是最不具侵入性和轻微疼痛的技术。

<table><tbody><tr><td>Gas Anesthesia System</td><td>Ugo Basile; Italy</td><td>211000</td><td></td></tr><tr><td>Weighing machine</td><td>Goldtech ; India</td><td></td><td>Local Procurement</td></tr><tr><td>Biological safety cabinet ( Class I)</td><td>Kartos international;&amp;nbsp; India</td><td></td><td>Local Procurement</td></tr><tr><td>Hair Trimmer</td><td>Panasonic ;&amp;nbsp; Japan&amp;nbsp;</td><td>ER-GY10&amp;nbsp;</td><td></td></tr><tr><td>Straight operating scissor with sharp /sharp blades</td><td>Major Surgicals; India</td><td></td><td>Local Procurement</td></tr><tr><td>Forceps with Serrations</td><td>Major Surgicals; India</td><td></td><td>Local Procurement</td></tr><tr><td>Micro needle holders&amp;nbsp; straight &amp;amp; curved&amp;nbsp;</td><td>Mercian ;&amp;nbsp; England&amp;nbsp;</td><td>BS-13-8</td><td></td></tr><tr><td>1 ml insulin syringe with 30G *5/16 needles&amp;nbsp;</td><td>Dispo Van; India</td><td></td><td></td></tr><tr><td>1 ml syringe with 26 G * 1/2 needle</td><td>BD ; US&amp;nbsp;</td><td>REF 303060</td><td></td></tr><tr><td>Nylon Threads&amp;nbsp;&amp;nbsp;</td><td>Mighty ; India</td><td></td><td>(1-0) Local Procurement</td></tr><tr><td>MERSUTURES 4-0 Sterilised Surgical Needled Suture</td><td>Ethicon, Johnson &amp;amp; Johnson, India</td><td>NW 5047</td><td></td></tr><tr><td>TRUGUT 76 cm 4-0 absorbable surgical suture</td><td>Sutures India Pvt. Ltd; India</td><td>SN 5048</td><td>Sterilised Surgical Needled Suture Catgut Chromic</td></tr><tr><td>Cotton Buds</td><td>Pure Swabs Pvt Ltd ;&amp;nbsp; India</td><td></td><td>Local Procurement</td></tr><tr><td>Surgical Tape</td><td>3M India ; India</td><td>1530-1</td><td>Micropore Surgical Tape</td></tr><tr><td>Microtome</td><td>Histo-Line Laboratories, Italy</td><td>MRS3500</td><td></td></tr><tr><td>Shandon Cryotome E Cryostat</td><td>Thermo Electron Corporation ; US</td><td></td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Carl Zeiss ; Germany&amp;nbsp;</td><td>LSM 510 META</td><td></td></tr><tr><td>Bright Field Microscope</td><td>Olympus, Japan</td><td>LX51</td><td></td></tr><tr><td>Automated analyser</td><td>Tulip, Alto Santracruz, India</td><td>Screen Maaster 3000</td><td>Biochemical analyser for liver functional test</td></tr><tr><td>Flow Cytometer</td><td>BD ; US&amp;nbsp;</td><td>BD FACSverse</td><td>Assesment of presence of cells post transplantation</td></tr><tr><td>Veet hair removal cream&amp;nbsp;</td><td>Reckitt Benckiser , India</td><td></td><td></td></tr><tr><td>FORANE</td><td>Abbott ; US</td><td></td><td>isoflurane USP 99.9%&amp;nbsp;</td></tr><tr><td>Taxim</td><td>AlKem ; India</td><td></td><td>cefotaxime sodium injection</td></tr><tr><td>Povidone-Iodine solution&amp;nbsp;</td><td>Win-Medicare;&amp;nbsp; India</td><td></td><td>Betadine</td></tr><tr><td>Paraformaldehyde</td><td>Himedia; India</td><td>GRM 3660</td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>Life technologies, Thermo Fisher scientific ; US</td><td>12200-036</td><td></td></tr><tr><td>Sucrose</td><td>Sigma ; US</td><td>S0389</td><td></td></tr><tr><td>Tissue-Tek</td><td>Sakura; US</td><td>25608-930</td><td>O.C.T compound</td></tr><tr><td>DAPI</td><td>Himedia; India</td><td>MB 097</td><td></td></tr><tr><td>anti-Albumin goat Polyclonal</td><td>Thermo Scientific,Pierce, US</td><td>PA126081</td><td></td></tr><tr><td>anti-connexin 32/GJB1 Polyclonal</td><td>abcam, UK</td><td>ab64609-500</td><td></td></tr><tr><td>antiGFP rabbit polyclonal&amp;nbsp;</td><td>Santa Cruz biotechnology; US</td><td>SC 8334</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-goat</td><td>&amp;nbsp;Molecular Probes , Thermo Fisher Scientific ;&amp;nbsp; US</td><td>A11058</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-sheep</td><td>&amp;nbsp;Molecular Probes , Thermo Fisher Scientific ;&amp;nbsp; US</td><td>A11015</td><td></td></tr><tr><td>Alexa Fluor 594 chicken anti rabbit</td><td>&amp;nbsp;Molecular Probes , Thermo Fisher Scientific ;&amp;nbsp; US</td><td>A21442</td><td></td></tr><tr><td>Goat anti rabbit IgG HRP</td><td>Invitrogen, Thermo Fisher Scientific; US&amp;nbsp;</td><td>65-6120</td><td></td></tr><tr><td>anti-Ki67 antibody</td><td>abcam, UK</td><td>ab15580</td><td></td></tr><tr><td>Antigen Unmasking Solution, Citric acid base</td><td>Vector laboratories, US</td><td>H-3300</td><td></td></tr><tr><td>ProLong Diamond antifade mountant</td><td>Life technologies, Thermo Fisher scientific ; US</td><td>P36966</td><td></td></tr><tr><td>SGOT (ASAT) KIT</td><td>Coral Clinical System, India</td><td></td><td></td></tr><tr><td>SGPT (ALAT) KIT</td><td>Coral Clinical System, India</td><td></td><td></td></tr><tr><td>Alkaline Phosphatase Kit (DEA)</td><td>Coral Clinical System, India</td><td></td><td></td></tr><tr><td>Hematoxylin Solution, Mayer&#039;s</td><td>Sigma ; US</td><td>MHS16</td><td></td></tr><tr><td>Eosin Y solution, alcoholic</td><td>Sigma ; US</td><td>HT110132</td><td></td></tr><tr><td>DPX Mountant&amp;nbsp;</td><td>Sigma ; US</td><td>6522</td><td></td></tr><tr><td>Melonex (Pain Killer)</td><td>Intas Pharmaceuticals Ltd; India</td><td></td><td>Meloxicam injection&amp;nbsp;</td></tr><tr><td>DAB enhanced liquid substrate system tetrahydrochloride</td><td>Sigma ; US</td><td>D3939</td><td></td></tr></tbody></table>

intrasplenic transplantation of hepatocytes after partial hepatectomy in nod.scid mice

部分肝切除术是研究肝再生和细胞基础疗法在各种病理条件下的作用的一种多功能和可再生的方法。部分肝切除也促进移植细胞的植入和增殖, 加速新生血管和细胞向肝脏迁移。在这里, 我们描述了一个简单的协议, 执行30% 肝切除和移植细胞在脾脏的非肥胖糖尿病/严重联合 immunodeficient 点头。(点头。CB17Prkdc的/J) 鼠标。
在这个过程中, 做了两个小切口。第一切口是暴露和切除肝脏的左叶, 另一小切口被做暴露脾脏为脾移植细胞。这个程序不需要任何专门的外科手术技能, 它可以在5-7 分钟内完成, 减轻压力和疼痛, 更快的恢复, 更好的生存。我们已经展示了从绿色荧光蛋白 (gfp) 中分离出的肝细胞移植, 表达小鼠 (转基因 C57BL/6-Tg (不列颠哥伦比亚) 30 scha/J), 以及人源性肝细胞 (NeoHep) 在部分 hepatectomized 点头。老鼠。

30% 部分肝切除术后肝细胞增殖:用免疫组化 (IHC) 染色 Ki-67 细胞增殖标记物, 观察30% 肝切除术后剩余肝脏中肝细胞的增殖。一天后肝切除, 小鼠被安乐死, 其余肝叶切除, 石蜡切片获得。切片染色的 Ki-67 抗体, 其次是标签与辣根过氧化物酶 (HRP) 共轭二级抗体。以二氨基联苯胺为基质, 用于开发褐色色素以识别染色细胞。细胞核被用苏木精染色, 并在20X 物镜下观察。图 1</str...

Watch this Scientific Journal Video about Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice at JoVE.com

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

我们已经描述了一个协议, 执行部分肝切除 (phx-空港) 和细胞移植通过脾脏点头。(点头。CB17-Prkdc有缺陷的/J) 鼠标。在本议定书中, 切口是为了揭露和切除肝脏的左叶, 其次是另一个切口的脾移植细胞。

肝细胞部分肝切除术后脾移植。小鼠

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

intrasplenic-transplantation-hepatocytes-after-partial-hepatectomy

Research

JoVE Journal

Medicine

13.9K 次观看.  National Institute of Immunology. 我们已经描述了一个协议, 执行部分肝切除 (phx-空港) 和细胞移植通过脾脏点头。(点头。CB17-Prkdc有缺陷的/J) 鼠标。在本议定书中, 切口是为了揭露和切除肝脏的左叶, 其次是另一个切口的脾移植细胞。

视频: Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

在肝炎症和纤维化环境中产生的肝细胞癌的致癌肝细胞诱导正交小鼠模型

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

科研

癌症研究

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

医学

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

部分小叶切除术: 形态学肝再生的外科模型

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

发育生物学

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 ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

利用人体肝干细胞人源化小鼠肝脏生成

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

建立与艾滋病毒相关的肝脏发病机制的双人性化TK-NOG小鼠模型

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

免疫与感染

A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone splenectomy regardless of precipitating causes are prone to develop an overwhelming post-splenectomy infection and experience increased risks of deep venous thrombosis and malignancies. Recently, epidemiological studies indicated that splenectomy might be associated with the occurrence of cardiovascular diseases, suggesting that physiological functions of the spleen have not yet been fully recognized. Here, we introduce a mouse model of vascularized heterotopic spleen transplantation, which not only can be utilized to study the function and behavioral activity of splenic immune cell subsets in different biologic processes, but also can be a powerful tool to test the therapeutic potential of spleen transplantation in certain diseases. The main surgical steps of this model include donor spleen harvest, the removal of recipient native spleen, and spleen graft revascularization. Using congenic mouse strains (e.g., mice with CD45.1/CD45.2 backgrounds), we observed that after syngeneic transplantation, both donor-derived splenic lymphocytes and myeloid cells migrated out of the graft as early as post-operative day 1, concomitant with the influx of multiple types of recipient cells, thus generating a unique chimera. &#160;Despite relatively challenging techniques, this procedure can be performed with &#62;90% success rate. This model allows tracking the fate, longevity, and function of splenocytes during steady state and in a disease setting following a spleen transplantation, thereby offering a great opportunity to discover the distinct role for spleen-derived immune cells in different disease processes.

This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity.

血管化异位脾移植小鼠模型在脾细胞生物学和移植免疫研究中的应用

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

尽量减少小鼠肝内胰岛移植期间输注后门静脉出血

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

小鼠肝部分切除术后再生肝细胞的分离

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

小鼠肝移植和同种异体移植物通畅性分析

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

作者聚焦:通过小鼠模型中的 MicroCT 血管造影增强移植研究 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

利用显微计算机断层扫描血管造影成功对小鼠进行原位肝移植

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...