Name: induction of an inflammatory response in primary hepatocyte cultures from mice
Uploaded: 2017-03-10T15:00:00
Description: Watch this Scientific Journal Video about Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice at JoVE.com

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><p class="jove...

<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. 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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. 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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。

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			<td height="21" style="height:21px;width:363px;">Consumables</td>
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			<td>329420</td>
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			<td height="21" style="height:21px;">Tissue Culture Dish 100 x 20 mm Style</td>
			<td>Corning</td>
			<td>353003</td>
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			<td height="21" style="height:21px;">6 well Cell Culture Cluster</td>
			<td>Costar</td>
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			<td>14058-11</td>
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			<td>F.S.T.</td>
			<td>11203-25</td>
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			<td height="22" style="height:23px;">Student Tissue Forceps</td>
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			<td>991121-12</td>
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			<td>Sigma</td>
			<td>&#160;&#160;&#160; A8976-100ML</td>
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			<td>&#160;&#160;&#160; 50989-161-06</td>
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			<td>139-236</td>
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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 ...

Research

JoVE Journal

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Induction of Experimental Autoimmune Hypophysitis in SJL Mice

SJL小鼠诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

科研

免疫与感染

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

从小学的人角质形成细胞器官筏文化的代

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

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Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

葡萄糖-6-磷酸异构酶（G6PI）诱导RA小鼠中iNKT细胞的采用免疫治疗

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...