Name: isolation of regenerating hepatocytes after partial hepatectomy in mice
Uploaded: 2022-12-02T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice at JoVE.com

这项研究得到了瑞士国家基金（项目赠款310030_189262）的支持。

所有动物实验均符合瑞士联邦动物法规，并经苏黎世兽医局批准（n° 007/2017，156/2019），确保人类护理。将10-12周龄的雄性C57BL / 6小鼠保持在12小时的昼夜循环中，并自由获取食物和水。每个实验组由六到八只动物组成。有关本协议中使用的所有材料、设备和试剂的详细信息，请参阅 材料表 。
1.小鼠肝部分切除术
<ol><li>对于标准肝切除术（70%），结?...

<ol>
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J., Cervantes-Alvarez, E., Navarro-Alvarez, N., Gayam, V., Engin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Liver Pathology. , (2019).</li><li>Charni-Natan, M., Goldstein, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+primary+mouse+hepatocyte+isolation.">Protocol for primary mouse hepatocyte isolation.</a> STAR Protocols. 1 (2), 100086 (2020).</li><li>Smedsr&#248;d, B., Pertoft, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+pure+hepatocytes+and+reticuloendothelial+cells+in+high+yield+from+a+single+rat+liver+by+means+of+Percoll+centrifugation+and+selective+adherence.">Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence.</a> Journal of Leukocyte Biology. 38 (2), 213-230 (1985).</li><li>Mederacke, I., Dapito, D. H., Aff&#242;, S., Uchinami, H., Schwabe, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+and+high-purity+isolation+of+hepatic+stellate+cells+from+normal+and+fibrotic+mouse+livers.">High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers.</a> Nature Protocols. 10 (2), 305-315 (2015).</li><li>Berry, M. N., Friend, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+preparation+of+isolated+rat+liver+parenchymal+cells:+a+biochemical+and+fine+structural+study.">High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study.</a> Journal of Cell Biology. 43 (3), 506-520 (1969).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+liver+cells.+I.+Effect+of+Ca+2++on+enzymatic+dispersion+of+isolated,+perfused+liver.">Preparation of rat liver cells. I. Effect of Ca 2+ on enzymatic dispersion of isolated, perfused liver.</a> Experimental Cell Research. 74 (2), 450-454 (1972).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods in Cell Biology. 13, 29-83 (1976).</li><li>Trotter, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fine+structure+study+of+lipid+in+mouse+liver+regenerating+after+partial+hepatectomy.">A fine structure study of lipid in mouse liver regenerating after partial hepatectomy.</a> Journal of Cell Biology. 21 (2), 233-244 (1964).</li><li>Kachaylo, 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=PTEN+down-regulation+promotes+&#946;-oxidation+to+fuel+hypertrophic+liver+growth+after+hepatectomy+in+mice.">PTEN down-regulation promotes &#946;-oxidation to fuel hypertrophic liver growth after hepatectomy in mice.</a> Hepatology. 66 (3), 908-921 (2017).</li><li>Jung, Y., Zhao, M., Svensson, K. J. <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+functional+analysis+of+hepatocytes+from+mice+with+fatty+liver+disease.">Isolation, culture, and functional analysis of hepatocytes from mice with fatty liver disease.</a> STAR Protocols. 1 (3), 100222 (2020).</li><li>Dold, 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=Portal+hyperperfusion+after+extended+hepatectomy+does+not+induce+a+hepatic+arterial+buffer+response+(HABR)+but+impairs+mitochondrial+redox+state+and+hepatocellular+oxygenation.">Portal hyperperfusion after extended hepatectomy does not induce a hepatic arterial buffer response (HABR) but impairs mitochondrial redox state and hepatocellular oxygenation.</a> PLoS One. 10 (11), 0141877 (2015).</li><li>Boyce, S., Harrison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+methodology+of+partial+hepatectomy+in+the+mouse.">A detailed methodology of partial hepatectomy in the mouse.</a> Laboratory Animals. 37 (11), 529-532 (2008).</li><li>Mitchell, C., Willenbring, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reproducible+and+well-tolerated+method+for+2/3+partial+hepatectomy+in+mice.">A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice.</a> Nature Protocols. 3 (7), 1167-1170 (2008).</li><li>Chen, T., Oh, S., Gregory, S., Shen, X., Diehl, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+omics+analysis+reveals+functional+diversification+of+hepatocytes+during+liver+regeneration.">Single-cell omics analysis reveals functional diversification of hepatocytes during liver regeneration.</a> JCI Insight. 5 (22), (2020).</li><li>Chembazhi, U. V., Bangru, S., Hernaez, M., Kalsotra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+plasticity+balances+the+metabolic+and+proliferation+dynamics+of+a+regenerating+liver.">Cellular plasticity balances the metabolic and proliferation dynamics of a regenerating liver.</a> Genome Research. 31 (4), 576-591 (2021).</li><li>Fiegel, H. C., Kaufmann, P. M., Kneser, U., Kluth, D., Rogiers, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming+of+hepatocytes+for+cell+culture+by+partial+hepatectomy+prior+to+cell+isolation.">Priming of hepatocytes for cell culture by partial hepatectomy prior to cell isolation.</a> Journal of Tissue Engineering. 6 (6), 619-626 (2000).</li><li>Roche, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Liberase TM Research Grade. , (2020).</li><li>Giugliano, 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=Hepatitis+C+virus+infection+induces+autocrine+interferon+signaling+by+human+liver+endothelial+cells+and+release+of+exosomes,+which+inhibits+viral+replication.">Hepatitis C virus infection induces autocrine interferon signaling by human liver endothelial cells and release of exosomes, which inhibits viral replication.</a> Gastroenterology. 148 (2), 392-402 (2015).</li><li>Shetty, 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=Common+lymphatic+endothelial+and+vascular+endothelial+receptor-1+mediates+the+transmigration+of+regulatory+T+cells+across+human+hepatic+sinusoidal+endothelium.">Common lymphatic endothelial and vascular endothelial receptor-1 mediates the transmigration of regulatory T cells across human hepatic sinusoidal endothelium.</a> The Journal of Immunology. 186 (7), 4147-4155 (2011).</li><li>Edwards, S., Lalor, P. F., Nash, G. B., Rainger, G. E., Adams, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+traffic+through+sinusoidal+endothelial+cells+is+regulated+by+hepatocytes.">Lymphocyte traffic through sinusoidal endothelial cells is regulated by hepatocytes.</a> Hepatology. 41 (3), 451-459 (2005).</li><li>Helling, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+failure+following+partial+hepatectomy.">Liver failure following partial hepatectomy.</a> HPB. 8 (3), 165-174 (2006).</li><li>Saran, U., Humar, B., Kolly, P., Dufour, J. F. <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+and+lifestyles.">Hepatocellular carcinoma and lifestyles.</a> Journal of Hepatology. 64 (1), 203-214 (2016).</li><li>Park, 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=Sugar-sweetened+beverage,+diet+soda,+and+nonalcoholic+fatty+liver+disease+over+6+years:+the+Framingham+Heart+Study.">Sugar-sweetened beverage, diet soda, and nonalcoholic fatty liver disease over 6 years: the Framingham Heart Study.</a> Clinical Gastroenteroly and Hepatology. , (2021).</li><li>Pocha, C., Kolly, P., Dufour, J. 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已发布的方案提供了一种可靠且直接的方法来分离高产量的正常和脂肪变性小鼠肝细胞，用于单细胞下游分析或FACS分选后的细胞批量分析。与密度梯度纯化相比，其明显优势在于细胞脂质含量对肝细胞的有效产量基本上没有影响。因此，脂肪变性肝细胞的比例将被保留并包含在下游分析中。这不仅对于脂肪源性肝病的研究至关重要，而且对于主要肝切除术后的任何过程分析也至关重要，其中肝细...

即使在组织大量流失后，肝脏也可以自我再生。这种独特的再生能力在部分（70%）肝切除术的实验模型中得到了明确说明，希金斯和安德森于1931年首次在大鼠中描述了1。在这个模型中，通过手术从动物身上切除70%的肝脏，方法是通过剪掉较大的肝叶。然后，剩余的叶通过代偿性肥大生长，在手术后约1周内恢复原始肝脏肿块，尽管没有恢复原始肝脏结构2，3。已经开发了具有不同组织切除量的其他肝切除术，例如86%扩展的肝切除术，其中肝脏残留物太小而无法恢复，最终导致肝切除术后肝衰竭（PHLF）和随后30%-50%的动物死亡4，5，6。这些模型能够研究正常和失败的肝脏再生，具体取决于切除组织的数量（图1）。
尽管肝脏切除术的小鼠模型已经成功使用多年，但直到最近才有更先进的分析方法允许在单细胞水平上更深入地了解。然而，对于大多数这些方法，单个肝细胞的存在是一个基本的先决条件。大多数分离原代肝细胞的方案基于两步胶原酶灌注技术和随后的密度梯度纯化，以从碎片和非实质以及死细胞中分离活肝细胞7，8，9。这种方法首先由Berry和Friend在196910中描述，并由Seglen及其同事在197211，12中进行了改编。然而，由于梯度离心依赖于细胞的密度和大小，因此在标准纯化过程中通常会丢失富含脂质的肝细胞。虽然对于许多研究问题来说，这种损失可以忽略不计，但它是早期肝脏再生的一个关键方面。在前2天，再生小鼠肝脏内的肝细胞积累脂质，从而增大大小并降低密度。这种短暂的再生相关脂肪变性（TRAS）用于提供再生燃料，并且是暂时的，但部分重叠了主要的增殖阶段，并且在肝小叶（肝脏的功能单位）内分布不均匀13，14。然而，在延长86%肝切除术后，TRAS也发生但持续存在，因为再生停滞并且脂质没有被氧化14。因此，在70%或86%肝切除术后对肝细胞进行梯度纯化将产生不具代表性的组分，因为大多数含脂质的肝细胞由于其低密度而丢失15。
在这种改良的分离方案中，通过经典的两步胶原酶灌注方法在肝切除术后24-48小时分离来自C57BL / 6小鼠的肝细胞。通常，用于细胞分离的残余物的插管和灌注是通过门 静脉完成的 。然而，大切除后留下的小残留物的门血管阻力高达16，因此灌注很微妙。由于腔静脉不受肝切除术的影响，因此可以通过插管 腔静脉轻松地 在逆行方向进行灌注。标准蠕动泵通过导管插入的下腔静脉 将 加热的溶液驱动到肝脏残余物中，使用逆行灌注并通过门静脉流出（补充图S1）。肝细胞被胶原酶解离并从Glisson的胶囊中释放出来。在使用低速离心方法逐步分离和仔细处理活肝细胞后，肝细胞可用于任何下游分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Zombie green</td>
			<td>BioLegend</td>
			<td>423111</td>
			<td>Amine-reactive viability dye</td>
		</tr>
		<tr>
			<td>Attane Isoflurane ad us. vet. 99.9%</td>
			<td>Provet AG</td>
			<td>QN01AB06</td>
			<td>CAUTION: needs ventilation</td>
		</tr>
		<tr>
			<td>EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>E8008-100ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>V001229</td>
			<td>Dilute with water to 70%</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FCS)</td>
			<td>Gibco</td>
			<td>A5256701</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS), Ca2+, Mg2+, phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>H9269-6x600ML</td>
			<td>For digestion/preservation</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt solution (HBSS), w/o Ca2+, w/o Mg2+, no phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>H6648-6x500ML</td>
			<td>For perfusion buffer</td>
		</tr>
		<tr>
			<td>HEPES solution, 1 M</td>
			<td>Sigma-Aldrich</td>
			<td>83264-100ML-F</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Histoacryl tissue adhesive (butyl-2-cyanoacrylate)</td>
			<td>B. Braun</td>
			<td>1050052</td>
			<td>For stabilization of cannulation site</td>
		</tr>
		<tr>
			<td>Hoechst 33258 Staining Dye Solution</td>
			<td>Abcam</td>
			<td>ab228550</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Liberase Research Grade</td>
			<td>Roche</td>
			<td>5401119001</td>
			<td>Lyophilized collagenases I/II</td>
		</tr>
		<tr>
			<td>NaCl 0.9% 500 mL Ecotainer</td>
			<td>B. Braun</td>
			<td>123</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paralube Vet Ointment</td>
			<td>Dechra</td>
			<td>17033-211-38</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>A1286301</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sudan IV &#8211; Lipid staining</td>
			<td>Sigma-Aldrich</td>
			<td>V001423</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Temgesic (Buprenorphine hydrochloride), Solution for Injection 0.3 mg/mL</td>
			<td>Indivior Europe Ltd.</td>
			<td>345928</td>
			<td>Narcotics. Store securely.</td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4%, sterile-filtered</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>Williams&#8217; Medium E</td>
			<td>Sigma-Aldrich</td>
			<td>W4128-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL serological pipette, Greiner Cellstar</td>
			<td>Merck</td>
			<td>P7865</td>
			<td>-</td>
		</tr>
		<tr>
			<td>50 mL Falcon tubes</td>
			<td>TPP</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BD Neoflon, Pro IV Catheter 26 G</td>
			<td>BD Falcon</td>
			<td>391349</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cell scraper, rotating blade width 25 mm</td>
			<td>TPP</td>
			<td>99004</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Falcon Cell Strainer 100 &#181;m Nylon</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fenestrated sterile surgical drape</td>
			<td>-</td>
			<td>-</td>
			<td>Reusable cloth material</td>
		</tr>
		<tr>
			<td>Filling nozzle for size 16# tubing (ID 3.1 mm)</td>
			<td>Drifton</td>
			<td>FILLINGNOZZLE#16</td>
			<td>To go into the tubes</td>
		</tr>
		<tr>
			<td>Flow cytometry tubes, 5 mL</td>
			<td>BD Falcon</td>
			<td>352008</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Male Luer to Barb, Tubing ID 3.2 mm</td>
			<td>Drifton</td>
			<td>LM41</td>
			<td>Connection tube to syringe</td>
		</tr>
		<tr>
			<td>Petri dishes, 96 x 21 mm</td>
			<td>TPP</td>
			<td>93100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Prolene 5-0</td>
			<td>Ethicon</td>
			<td>8614H</td>
			<td>To retract the sternum</td>
		</tr>
		<tr>
			<td>Prolene 6-0</td>
			<td>Ethicon</td>
			<td>8695H</td>
			<td>For skin suture</td>
		</tr>
		<tr>
			<td>Prolene 8-0</td>
			<td>Ethicon</td>
			<td>EH7470E</td>
			<td>Ligature gall bladder</td>
		</tr>
		<tr>
			<td>Tube 16#, WT 1.6 mm, ID 3.2 mm, OD 6.4 mm</td>
			<td>Drifton</td>
			<td>SC0374T</td>
			<td>Perfusion tube</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRFortessa Cell Analyzer Flow Cytometer</td>
			<td>BD</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Isis rodent shaver</td>
			<td>Aesculap</td>
			<td>GT421</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Isofluran station</td>
			<td>Provet</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Low-speed centrifuge &#8211; Scanspeed 416</td>
			<td>Labogene</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Neubauer-improved counting chamber</td>
			<td>Marienfeld</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Oxygen concentrator &#8211; EverFlo</td>
			<td>Philips&#160;</td>
			<td>1020007</td>
			<td>0 &#8211; 5 L/min</td>
		</tr>
		<tr>
			<td>Pipetboy &#8211; Pipettor Turbo-Fix</td>
			<td>TPP</td>
			<td>94700</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Shenchen perfusion pump &#8211; YZ1515x</td>
			<td>Shenchen</td>
			<td>YZ1515x</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Surgical microscope &#8211; SZX9</td>
			<td>Olympus</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>ThermoLux warming mat</td>
			<td>Thermo Lux</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Vortex Genie 2, 2700 UpM</td>
			<td>NeoLab</td>
			<td>7-0092</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Water bath &#8211; Precision GP 02</td>
			<td>Thermo scientific</td>
			<td>-</td>
			<td>Adjust to 42 &#176;C</td>
		</tr>
	</tbody>
</table>

isolation of regenerating hepatocytes after partial hepatectomy in mice

部分肝切除术已被广泛用于研究小鼠的肝脏再生，但分离高产量的活肝细胞用于下游单细胞应用具有挑战性。在小鼠正常肝脏再生的前2天观察到再生肝细胞内脂质的显着积累。这种所谓的短暂再生相关脂肪变性（TRAS）是暂时的，但部分与主要增殖期重叠。密度梯度纯化是大多数现有原代肝细胞分离方案的支柱。由于梯度纯化依赖于细胞的密度和大小，因此它将非脂肪性肝细胞群与脂肪性肝细胞群分开。因此，脂肪肝细胞经常丢失，产生不具代表性的肝细胞分数。 
所提出的方案描述了一种简单可靠的方法，用于 体内 分离再生肝细胞，无论其脂质含量如何。通过经典的两步胶原酶灌注方法在肝切除术后24-48小时分离来自雄性C57BL / 6小鼠的肝细胞。标准蠕动泵使用逆行灌注技术将加热的溶液 通过 导管下腔静脉驱动到残余静脉中，并通过门静脉流出。肝细胞被胶原酶解离，从Glisson的胶囊中释放出来。洗涤和仔细离心后，肝细胞可用于任何下游分析。总之，本文描述了一种直接且可重复的技术，用于分离小鼠部分肝切除术后代表性的再生肝细胞群体。该方法也可能有助于脂肪肝疾病的研究。

TRAS在肝切除术后16小时达到峰值，并在标准肝切除术后32-48小时逐渐消失，但在延长肝切除术后持续超过48小时。在肉眼上，TRAS很容易看到肝脏残留物的苍白肤色（图1F），并且可以在手术后16小时至48小时之间在肝切除小鼠中观察到。
估计的最终产量为70%肝切除术后10-15×106 肝细胞和延长86%肝切除术后4-9×106 肝细胞，平均最终生存能力分...

Watch this Scientific Journal Video about Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice at JoVE.com

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

富含脂质的肝细胞是肝脏再生所固有的，但通常在密度梯度离心时丢失。在这里，我们提出了一种优化的细胞分离方案，该方案保留了脂肪变性肝细胞，在小鼠部分肝切除术后产生具有代表性的再生肝细胞群。

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 ...

该协议的总体目标是提出一种优化的方法，用于在部分肝切除术后小鼠中分离肝细胞，而无需密度梯度离心。我们通常在早期再生期间看到很大比例的脂质肝细胞。由于密度低，通常在密度梯度离心时丢失的那些脂肪性肝细胞通过该方案保留。要准备灌注设备，请使用鲁尔锁连接器将 26 号IUI插管连接到管道的出口端。然后冲洗管路并将管子的入口端插入水浴中预热的灌注缓冲管中。用温热的灌注缓冲液以每分钟三毫升的泵速灌注。在进行手术之前，请确保小鼠已充分麻醉。然后用手术磨砂液和70%乙醇清洁腹部。然后切断缝合线以重新打开先前为部分肝切除术制作的中线切口，并轻轻地将伤口边缘拉开。如果肝切除术超过 24 至 48 小时，请拆下缝合线并用剪刀或手术刀割破皮肤。使用牵开器或简单的夹子保持腹部开放。腹腔应尽可能暴露，以优化通道和可视化。将5-0聚丙烯缝合线固定在胸骨上。用头拉它并将其固定在这个位置。使用无菌棉签将肠道向右移动，以显示门静脉和腔静脉。用湿布保留肠道。在鼠标后腿附近放置一个大约两厘米高的重物，例如金属配重环。然后将带有连接的 26 号IUI插管的管子放在物体上，并将针头小心地放在腔静脉顶部。根据需要调整管子的长度。将胶原酶储备溶液转移到预热的消化缓冲管中。每只动物准备10至20毫升的消化缓冲液。将泵速调整为每分钟三毫升后打开泵。缓冲液到达针头后，丢弃前两到三毫升预热的灌注缓冲液。要对下腔静脉进行插管，请使用无菌棉签轻轻地将腔静脉向穿刺下方的尾部拉动，以便提供的张力有助于将套管插入静脉。将 26 号IUI插管以浅角度插入肾脏下方的下腔静脉，同时缓冲液穿过针头。确保针斜面朝上。当针头进入管腔时，在导管的闪光腔中寻找血液。将针头再推进两到三毫米，以确保导管尖端进入静脉。将塑料导管滑过针头并再滑入腔静脉五毫米。缓慢而小心地取出针头。两到三秒后，寻找肝脏中形成的白点或门静脉肿胀，这表明灌注缓冲液流经肝脏并从中央静脉进入肝小叶。等待门静脉在肝脏表面形成白点的一到两秒内明显肿胀。用剪刀尽可能远离肝门切断门静脉。将流速增加到每分钟四到七毫升，具体取决于动物的体重、肝脏大小和初始肝切除术的程度。用镊子或血管夹夹住门静脉 7 到 10 秒钟。确保没有液体通过。大约 30 秒后进行第二次钳夹，并确保肝脏肿胀和放松。继续冲洗动物三到四分钟，观察从门静脉流出的透明缓冲液。在消化缓冲液到达肝脏之前夹住门静脉三到四秒钟。确保在释放钳夹时肝脏放松，并且灌注液保持清澈。一旦消化缓冲液到达肝脏，再次夹住门静脉。释放钳夹后，肝脏不应放松。以每分钟五毫升的流速消解约四分钟。随着消化的进展，寻找肝脏开始肿胀的迹象和肝脏表面的小透明切片。观察肝脏呈现出湿布的质地，看起来几乎湿透。用湿的无菌棉签小心地触摸稠度，以探测稠度。继续灌注，直到可以观察到肝脏表面纹理的显着差异。观察到肝脏呈现非常浅的颜色和气泡外观，表明Glisson的囊与实质分离。一旦肝脏获得这些特性，就停止消化。在空气进入肝脏之前取下针头。用镊子抓住叶之间的中央结缔组织，并将其作为锚点轻轻向上提起。切断肝脏与其他器官的所有连接并切除胆囊。轻轻地从腹腔中取出肝脏。要小心，因为在这个阶段它会很脆弱。将肝脏放入冰冷的保存缓冲液中。将肝脏转移到10厘米的培养皿中，并在肝脏表面的几个位置加入10毫升冰冷的威廉姆斯中等E.破裂Glisson胶囊。用两把镊子抓住中央部分。慢慢将它们拉开，让胶囊撕裂而不损坏肝细胞，并通过轻轻摇动胶囊来释放细胞。通过 100 微米细胞过滤器将 5 毫升肝髓过滤到 50 毫升管中。用10毫升新鲜冰冷培养基冲洗过滤器，并通过细胞过滤器过滤剩余的5毫升纸浆。在 4 摄氏度下以 50 G 离心提取物五分钟。吸出大部分上清液，留下一毫升通过轻轻旋转管来重悬细胞。加入40毫升冷威廉姆斯E培养基，重复该步骤三次。按照文本手稿中的说明继续分离肝细胞。在小鼠肝切除术70%后，最终肝细胞产量约为10至15×10至第6，平均最终活力为78%。在延长86%肝切除术后，肝细胞产量为4至9×10至第6，平均存活率为65%。部分肝切除术后，与正常肝脏相比，肝细胞显示出与脂质含量增加相对应的增加的细胞大小。在肝部分切除术后24小时观察到小鼠肝细胞中脂质含量增加，这被视为粒度增加或通过增大的肝细胞内存在脂质囊泡直接观察到。在使用经典的密度梯度纯化方法时，上清液中丢失了大的脂肪肝细胞，细胞沉淀中仅收集了较小的瘦肝细胞。通过改进的方案，脂质填充的肝细胞不会丢失，并且所有肝细胞都被沉淀。重复夹紧有助于冲洗过程。这样，肝脏就可以最好地清除可能抑制消化酶的剩余血液成分。

Research

JoVE Journal

Medicine

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

一个型号的不安流致动脉粥样硬化小鼠颈动脉部分结扎术和颈动脉内皮细胞的RNA分离的简单方法

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

科研

医学

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

局部或全身药物干预后评估的变化小鼠中挥发性全身麻醉药敏感性

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

之后，正常小鼠70％部分肝切除血管和实质再生的可视化

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

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

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 ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

静脉大鼠部分肝移植的体内血管定向70% 肝切除术

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

脂肪组织干细胞移植对大鼠部分结肠切除术后渗漏的减少

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Complete and Partial Aortic Occlusion for the Treatment of Hemorrhagic Shock in Swine

完全性和部分主动脉闭塞治疗猪失血性休克

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

非放射性 l-azidohomoalanine 标记法分离原发性小鼠肝细胞对新生蛋白合成的研究

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

猪自体移植肝细胞外细胞外细胞基因治疗

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...