这项工作是由美国国立卫生研究院（R01HL128714到CF）和（F31DK10236101到KS）和美国心脏协会（CF和AG）的支持。

所有动物方案和实验过程由医学迈阿密大学米勒医学院大学的机构动物护理和使用委员会（IACUC）的批准。 1.准备<ol><li>预热肝灌注介质和肝脏，在37℃消化介质中的水浴中。 </li><li>设置一个灌注泵（30毫升/分钟）。仔细预填肝灌注介质泵的管道系统。避免在系统中的任何气泡，并准备立体显微镜。 </li><li>使用根据制造商的协议我型胶原涂层细胞培养皿。 </li></ol> 2.肝恢复<ol><li> 麻醉用氧气/异氟醚（异氟烷4％/ 2升/分钟氧气）供体小鼠。 <ol><li>通过降低异氟烷以2-2.5％或通过注射氯胺酮维持麻醉（100mg / kg的体重腹膜内）和赛拉嗪（10毫克/公斤体重的intraperitoneally）。连续异氟烷麻醉优选自氯胺酮降低血压，并可能导致肝毒性。 </li></ol></li><li>放置麻醉小鼠上的吸收垫。用胶带固定鼠标的四肢。 </li><li>刮脸和消毒鼠标的腹部，并从耻骨进行剖腹探查腹用手术剪刀锋利/钝头肝脏的颅边框。切开腹壁向两侧，膜片的尾部。 </li><li>经肠道仔细移动到右侧暴露下腔静脉（IVC）。用消毒棉签拭子和角度的尖镊子仔细解剖周围的IVC脂肪组织。 </li><li>切开肝上下隔膜，并用细手术剪刀锋利/锋利的尖端两侧切口打开胸腔。使用胸外科IVC丝（5/0）结扎。 </li><li>使用屏蔽静脉导管导管插入下腹IVC，小心地取出针头，连接灌注泵，开始灌注肝脏。如果执行得当，肝应立即开始发白和膨胀。 </li><li>横切门静脉使血液和灌注媒体适当的排水用细手术剪。 </li><li>灌注用约30毫升肝灌注介质的肝脏，然后切换到肝脏消化介质（30毫升）中。关闭灌注泵并取出插管灌注一次完成。 </li><li>轻轻暴露肝脏的中央区域并定位在连接组织联的肝叶。抓住中央连接光纤，仔细剖析控股肝脏全部到位组织和轻轻取出肝脏。 </li><li>紧接在肝脏转移到含有15毫升的50mL聚丙烯锥形管肝脏消化介质。 注:有没有具体的时间线的这一步。灌注后，肝脏保持在消化介质输送到无菌细胞培养罩。所有Subsequent步骤应在无菌环境中进行。 </li></ol>细胞3.隔离治疗<ol><li>在细胞培养罩，转移从50毫升聚丙烯锥形管含有消化媒体肝脏进入无菌组织培养皿。 </li><li>使用无菌镊子轻轻撕肝脏的四个叶片。如果正确执行，消化中应该由于从肝脏分离肝细胞变浑浊。 </li><li>用25毫升无菌枪头，通过70微米尼龙细胞过滤混浊消化介质过滤到一个新的50毫升聚丙烯锥形管以除去未消化的组织颗粒和细胞碎片。 </li><li>重复步骤3.2和3.3，用无菌磷酸盐缓冲盐水（PBS）。这有助于从肝驱除残存肝细胞。 </li><li>离心机在50×g离心在4℃下2分钟。吸出上清液含有过量的细胞碎片，轻轻地再暂停25毫升冷的1×细胞威廉姆斯的中E. </li><li>用25毫升无菌枪头，通过一个新的70微米尼龙细胞过滤器过滤再悬浮到50mL聚丙烯锥形管以实现清洁悬浮液。 </li><li>重复步骤3.5和3.6使用冷威廉姆斯中等E 1X，以获得更清晰的细胞悬液三个额外的洗涤。 </li><li>最后洗涤步骤后，吸去上清液，以除去过量的细胞碎片。重悬和过滤器的细胞在25mL暖Williams的媒体E 1X包含原代肝细胞的解冻和电镀补充剂通过一个新的70微米尼龙细胞过滤到50mL聚丙烯锥形管中。 </li><li>算使用血球25毫升细胞悬浮液中的细胞。平均预期15 - 每位成人肝20000000细胞。使用台盼蓝染色测定细胞生存力。 </li><li>板块细胞威廉姆斯的2毫升媒体E 1X包含原代肝细胞解冻和电镀添加剂在胶原包被的6孔策LL培养簇（每孔9×10 5个细胞）。 </li><li> 4小时后，介质改为威廉姆斯的媒体E 1X包含原代肝细胞的维护补充剂。 </li><li> 24小时后，血清饥饿用Dulbecco改进的Eagle培养基（DMEM）培养基1×6小时的细胞。 </li><li>用PBS处理细胞作为溶媒，FGF23（25毫微克/毫升），脂多糖（100微克/毫升），或在无血清培养基中的IL-6（50毫微克/毫升），并培育24小时。 </li></ol> 4.隔离的RNA <ol><li>使用根据制造商的说明在商业自旋柱试剂盒制备的RNA。 </li></ol> 5.生成的cDNA通过逆转录RNA分离<ol><li>添加200毫微克分离的RNA样品以PCR管。 </li><li>从步骤5.1添加核酸内切酶游离H 2 O至PCR管的14微升。 </li><li>从步骤5.1添加4微升反转录酶对PCR管。 </li><li>轻轻重悬内容，以混合均匀;离心平缓。 </LI&gt; <li>放置装有该混合物成热循环PCR管。用于逆转录运行热循环:1个循环的25℃（5分钟），42℃（30分钟），85℃（5分钟），然后保持在4℃。最终产品将是所需cDNA。 </li></ol> 6.实时定量PCR细胞分析（定量PCR） <ol><li> 准备96孔板。 <ol><li>放置在冰上的qPCR反应的所有成分:1μL的cDNA，0.25微米的正向引物，0.25μM反向引物，5μL的SYBR绿，3.5微升的PCR级水，以10μL的总体积。使用的SYBR Green，准备预混一式三份运行的所有样本。 </li><li>一旦主结构完成后，旋涡，使均匀。等分试样9的qPCR的微升主混合物到96-WLL板的各孔中。后，小心加cDNA的1微升到每口井。反应总量将成为10微升。 </li><li>加载完成后，密封用光学96孔平板粘合膜和短暂离心96孔板（50×g离心1分钟）。 </li></ol></li><li> 运行样品在实时PCR仪。处密封保存96孔板为实时PCR仪和运行样品按照仪器制造商的建议。标准和快速循环的例子包括在下面。 <ol><li>对于标准循环参数，使用以下命令:预变性94℃120秒，然后94℃40个循环15秒，60℃60秒，然后有选择地在4℃举行。 </li><li>对于快速循环参数:使用以下命令:94℃预变性30秒，然后95℃40个循环5秒，58℃15秒，然后72℃10秒。 </li></ol></li><li>参考仪器手册，了解有关如何分析数据的指导。 </li></ol> 7.通过ELISA细胞上清液的分析注:根据制造商的协议执行的所有步骤。 </p&gt; <ol><li> 样品制备 <ol><li>稀释分装398微升到单独的试管。吸取2μL细胞上清液样品放入398微升稀释剂管。这将提供为200的稀释倍数。 </li><li>对每个样品重复步骤7.1.1进行测试。 </li></ol></li><li> 程序 <ol><li>吸取100μL的200倍稀释的样品和标准于96孔板的孔中。 </li><li>孵育96孔板在25℃下用在以150rpm微孔板摇床上45分钟。 </li><li>洗孔5次用1×洗涤缓冲液。一旦最后一次洗涤完成后，取出用吸水纸全部残留洗涤液。 </li><li>加入酶标试剂500微升到每口井。 </li><li>重复步骤7.2.2 - 7.2.3。 </li><li>吸取100μL的四甲基联苯胺（TMB）试剂到96孔板的每个孔中。 </li><li>轻轻在25℃的微量混合96孔板20分钟摇床在150rpm。通过直接加入100微升的终止溶液以每孔停止反应，并轻轻混合。一旦停止溶液的颜色应该变黄。这个通知的终止溶液已正确混合。 </li><li>使用微量滴定板读数器，确定第一个5分钟内450nm处的光密度。 </li></ol></li><li> 结果计算 <ol><li>计算每个样本和每个标准平均吸光度值（A 450）的表。 </li><li>在NG密谋反对它的标准浓度每个标准的A 450平均组装的标准曲线/毫升的线性图。允许x轴表示的浓度和y轴来表示A 450值。 </li><li>利用意味着从每个稀释样品A 450，从标准曲线确定毫微克/毫升所需蛋白的浓度。 </li><li>通过稀释事实上乘以样品浓度河这将允许用于确定细胞上清液样品中的所希望的蛋白质的实际浓度。 </li><li>绘制从样本构建表成线图。如果OD 450值在标准曲线的外部，样品应适当地稀释并重新测试。 </li><li>结果归为500,000个细胞。 </li></ol></li></ol>

<ol>
	<li>Silverstein, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+chronic+kidney+disease:+role+in+the+progression+of+renal+and+cardiovascular+disease.">Inflammation in chronic kidney disease: role in the progression of renal and cardiovascular disease.</a> Pediatr Nephro. 24 (8), 1445-1452 (2008).</li><li>Isakova, T., 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=Fibroblast+growth+factor+23+is+elevated+before+parathyroid+hormone+and+phosphate+in+chronic+kidney+disease.">Fibroblast growth factor 23 is elevated before parathyroid hormone and phosphate in chronic kidney disease.</a> Kidney Int. 79 (12), 1370-1378 (2011).</li><li>Faul, C., 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=FGF23+induces+left+ventricular+hypertrophy.">FGF23 induces left ventricular hypertrophy.</a> J Clin Invest. 121 (11), 4393-4408 (2011).</li><li>Guti&#233;rrez, O. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+23+and+mortality+among+patients+undergoing+hemodialysis.">Fibroblast growth factor 23 and mortality among patients undergoing hemodialysis.</a> N England J Med. 359 (6), 584-592 (2008).</li><li>Munoz Mendoza, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+23+and+Inflammation+in+CKD.">Fibroblast growth factor 23 and Inflammation in CKD.</a> Clin J Am Soc Nephrol. 7 (7), 1155-1162 (2012).</li><li>Hanks, L. J., Casazza, K., Judd, S. E., Jenny, N. S., Guti&#233;rrez, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+of+Fibroblast+Growth+Factor-23+with+Markers+of+Inflammation,+Insulin+Resistance+and+Obesity+in+Adults.">Associations of Fibroblast Growth Factor-23 with Markers of Inflammation, Insulin Resistance and Obesity in Adults.</a> PLoS ONE. 10 (3), e0122885 (2015).</li><li>Singh, 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=Fibroblast+growth+factor+23+directly+targets+hepatocytes+to+promote+inflammation+in+chronic+kidney+disease.">Fibroblast growth factor 23 directly targets hepatocytes to promote inflammation in chronic kidney disease.</a> Kidney Int. , 1-12 (2016).</li><li>Howard, R. B., Christensen, A. K., Gibbs, F. A., Pesch, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enzymatic+preparation+of+isolated+intact+parenchymal+cells+from+rat+liver.">The enzymatic preparation of isolated intact parenchymal cells from rat liver.</a> J Cell Biol. 35 (3), 675-684 (1967).</li><li>Moshage, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+interleukin-1,+interleukin-6+and+its+interrelationship+on+the+synthesis+of+serum+amyloid+A+and+C-reactive+protein+in+primary+cultures+of+adult+human+hepatocytes.">The effect of interleukin-1, interleukin-6 and its interrelationship on the synthesis of serum amyloid A and C-reactive protein in primary cultures of adult human hepatocytes.</a> Bioch Biophysi Res Commun. 155 (1), 112-117 (1988).</li><li>Soldatow, V. Y., LeCluyse, E. L., Griffith, L. G., Rusyn, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+for+liver+toxicity+testing.">In vitro models for liver toxicity testing.</a> Toxicol. Res. 2 (1), 23-39 (2013).</li><li>Lee, S. M. L., Schelcher, C., Demmel, M., Hauner, M., Thasler, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Human+Hepatocytes+by+a+Two-step+Collagenase+Perfusion+Procedure.">Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure.</a> J Vis Exp. (79), (2013).</li><li>Kegel, V., 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=Protocol+for+Isolation+of+Primary+Human+Hepatocytes+and+Corresponding+Major+Populations+of+Non-parenchymal+Liver+Cells.">Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells.</a> J Vis Exp. (109), (2016).</li><li>Moshage, H., 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=Cytokines+and+the+hepatic+acute+phase+response.">Cytokines and the hepatic acute phase response.</a> J Pathol. 181 (3), 257-266 (1997).</li><li>Klaunig, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+liver+cell+culture.+I.+Hepatocyte+isolation.">Mouse liver cell culture. I. Hepatocyte isolation.</a> In vitro. 17 (10), 913-925 (1981).</li><li>Lu, Y. C., Yeh, W. C., Ohashi, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPS/TLR4+signal+transduction+pathway.">LPS/TLR4 signal transduction pathway.</a> Cytokine. 42 (2), 145-151 (2008).</li><li>Schmidt-Arras, D., Rose-John, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-6+pathway+in+the+liver:+From+physiopathology+to+therapy.">IL-6 pathway in the liver: From physiopathology to therapy.</a> J Hepatol. 64 (6), 1403-1415 (2016).</li><li>Grabner, A., 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=Activation+of+Cardiac+Fibroblast+Growth+Factor+Receptor+4+Causes+Left+Ventricular+Hypertrophy.">Activation of Cardiac Fibroblast Growth Factor Receptor 4 Causes Left Ventricular Hypertrophy.</a> Cell Metab. 22 (6), 1020-1032 (2015).</li><li>Shulman, M., Nahmias, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+17,+Long-Term+Culture+and+Coculture+of+Primary+Rat+and+Human+Hepatocytes.">Chapter 17, Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes.</a> Methods Mol Biol. 945, 287-302 (2012).</li></ol>

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从小鼠中分离原代肝细胞是一种快速，廉价和可靠的工具来研究炎症反应体外。如果正确执行，结果可以很容易地生成并及时和具有成本效益的方式再现。以下几点应仔细评估，以确保成功隔离。 手术切口和下腔静脉的插管应在全身麻醉下，不安乐死之后进行。一个年轻的，缺乏经验的调查员将需要更多的时间在一开始就熟悉小鼠腹部的解剖学特征，并进行正确的插?...

慢性肾病（CKD）可以被定义为急性和慢性炎症1的状态。在慢性肾脏病患者中，phosphaturic激素成纤维细胞生长因子23（FGF23）的血清水平逐步上升，以维持血清磷酸盐稳态2。提高血清FGF23水平分别与心血管发病率和死亡率谁开始血液透析治疗3，4患者有关。此外，一些临床研究表明升高的FGF23水平与C-反应蛋白（CRP），白介素-6（IL-6）和肿瘤坏死因子α（TNFα）5,6的血清水平之间存在很强的相关性。另外，在实验研究中，我们最近表明，FGF23可以直接靶向肝细胞，并导致增加的CRP和IL-6 P的炎症反应roduction在肝脏7。因此，FGF23可能充当有助于全身炎症反应在CKD循环因素。 在70年代初，原代肝细胞分离并研究首次8。自那时以来，原代培养的肝细胞已被广泛用来研究代谢加工，激素功能，药物代谢和毒性，以及免疫和炎症反应9,10。先前协议已经主要描述原代肝细胞的酶活隔离从人肝脏组织11，12。虽然一个很好的模式，这留下了研究操纵基因是如何影响肝脏复杂的信号机制，以及在不同类型的刺激功能的后果的能力。在下面，我们描述了小鼠原代肝细胞的分离。值得注意的是，肝急性期反应的几种介质，如脂多糖（LPS），IL-6和FGF23的效果可在简单，快速和可重复的方式13进行分析。 在此，我们提出了从成年小鼠肝细胞的酶促隔离的协议，并且我们表明，炎症的建立诱导，如LPS和IL-6，以及新颖的炎症介质如FGF23，可直接刺激的表达和分泌炎性细胞因子，如在培养的肝细胞的CRP和IL-6。

<table><tbody><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Strainer 70 &amp;mu;m Nylon cell strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>BD Insyte Autoguard</td><td>BD</td><td>381412</td><td></td></tr><tr><td>50 mL Polypropylene Conical Tube</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>100 6-inch Cotton Tipped Applicators&amp;nbsp;</td><td>Puritan</td><td>806-WC</td><td></td></tr><tr><td>1cc U-100 Insulin Syringe 28 G 1/2</td><td>Becton Dickinson</td><td>329420</td><td></td></tr><tr><td>Tissue Culture Dish 100 x 20 mm Style</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>6 well Cell Culture Cluster</td><td>Costar</td><td>3516</td><td></td></tr><tr><td>5/0 Black Braided Surgical Silk (100 yards)</td><td>LOOK</td><td>SP115</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Minipuls 3 Perfusion Pump</td><td>Gilson</td><td>F155007</td><td></td></tr><tr><td>Hemacytometer Kits, Propper</td><td>VWR</td><td>48300-474</td><td></td></tr><tr><td>Hemacytometer Cover Glasses, Propper</td><td>VWR</td><td>48300-470</td><td></td></tr><tr><td>Surgical Scissers - Sharp/Blunt</td><td>F.S.T.</td><td>14001-12</td><td></td></tr><tr><td>Iris Scissors-ToughCut Straight</td><td>F.S.T.</td><td>14058-11</td><td></td></tr><tr><td>Dumont SS-45 Forceps</td><td>F.S.T.</td><td>11203-25</td><td></td></tr><tr><td>Student Tissue Forceps</td><td>F.S.T.</td><td>991121-12</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetic acid solution, 2.0 N</td><td>Sigma</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; A8976-100ML</td><td></td></tr><tr><td>Isoflurane, USP 250 mL</td><td>Piramal Healthcare</td><td>&amp;nbsp;&amp;nbsp; 66794-013-25</td><td></td></tr><tr><td>KetaVed 1,000 mg/10 mL (100 mg/mL)</td><td>VEDCO</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; 50989-161-06</td><td></td></tr><tr><td>Xylazine 100 mg/mL</td><td>AnaSed Injection</td><td>139-236</td><td></td></tr><tr><td>Willams&#039; Medium E (1x)</td><td>gibco</td><td>12551-032</td><td></td></tr><tr><td>Liver Perfusion Medium (1x)</td><td>gibco</td><td>17701-038</td><td></td></tr><tr><td>Liver Digest Medium (1x)</td><td>Life Technologies</td><td>17703034</td><td></td></tr><tr><td>Primary Hepatocyte Thawing and Plating Supplements</td><td>Life Technologies</td><td>CM3000</td><td></td></tr><tr><td>Primary Hepatocyte Maintenance Supplements</td><td>Life Technologies</td><td>CM4000</td><td></td></tr><tr><td>Phosphate Buffer Saline (PBS) pH 7.4</td><td>ThermoFisher scientific</td><td>10010031</td><td></td></tr><tr><td>Collagen Type 1</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Trypan Blue Solution</td><td>VWR</td><td>45000-717</td><td></td></tr></tbody></table>

induction of an inflammatory response in primary hepatocyte cultures from mice

肝脏在全身性炎症的调节决定性的作用。特别是在慢性肾脏疾病，肝脏响应于尿毒症环境，氧化应激，内毒素血症和通过产生大量的急性期反应物的循环促炎性细胞因子的清除率降低反应。实验工具来研究炎症和肝细胞的基本作用是理解的调节和肝细胞因子的贡献全身急性期反应和延长的促炎症的情况下，尤其是在复杂的环境至关重要如慢性肾脏疾病。自体内研究炎症的复杂的机制仍然是具有挑战性的，耗费资源的，通常需要转基因动物的使用，一次分离的肝细胞提供一个强大的工具来获得机械分析上市肝急性期反应。由于这种体外技术特点适中成本，高重可生产和技术常识，一次分离的肝细胞也可以很容易地用作筛选方法。这里，我们描述一个基于酶促法以分离原代鼠肝细胞，和我们描述在使用ELISA和定量实时PCR这些细胞的炎症反应的评估。

组织学一次分离和培养的细胞的代表性光显微镜图像在图1A中描绘。免疫细胞化学分析表明，分离的肝细胞中高度表达白蛋白（红色），以及成纤维细胞生长因子受体-4（FGFR4）（绿色）。细胞核用4&#39;，6-二脒基-2-苯基吲哚（DAPI）（蓝色）。 （ 图1B）。 定量?...

Watch this Scientific Journal Video about Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice at JoVE.com

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

这里，我们表明酶促方法来原代肝细胞从成年小鼠隔离，我们描述了使用ELISA和实时PCR的炎症反应的量化。

从小鼠在原代肝细胞培养物的炎性反应的诱导

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

induction-an-inflammatory-response-primary-hepatocyte-cultures-from

Research

JoVE Journal

Immunology and Infection

9.9K 次观看.  University of Miami Leonard M. Miller School of Medicine. 这里，我们表明酶促方法来原代肝细胞从成年小鼠隔离，我们描述了使用ELISA和实时PCR的炎症反应的量化。 

视频: Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

CYP2D6动物模型：如何诱导小鼠自身免疫性肝炎

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

科研

医学

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 ...

癌症研究

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

胆管结扎小鼠:感应炎性肝损伤和纤维化的阻塞性胆汁淤积

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

诱导BALB/c小鼠药物诱导的自身免疫性肝炎研究其致病机制

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

免疫与感染

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

非酒精性脂肪性肝炎合并2型糖尿病的一种先进的小鼠模型

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

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

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

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 ...

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD), &#160;which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and qPCR.&#160;

分化肝细胞中的血管性脂细胞病的诱导和特征

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

毛利精密切肝片作为肝脏生物学的Ex Vivo模型

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

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

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

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

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

慢性肝损伤小鼠胆碱缺乏、氨基补充的饮食模型

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

生物学

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

原原小鼠肝细胞的分离和3D胶原蛋白三明治培养，研究细胞骨架在胆汁形成中的作用

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...