从小鼠在原代肝细胞培养物的炎性反应的诱导
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Consumables | |||
| Cell Strainer 70 μm Nylon cell strainer | Falcon | 352350 | |
| BD Insyte Autoguard | BD | 381412 | |
| 50 mL Polypropylene Conical Tube | Falcon | 352098 | |
| 100 6-inch Cotton Tipped Applicators | Puritan | 806-WC | |
| 1cc U-100 Insulin Syringe 28 G 1/2 | Becton Dickinson | 329420 | |
| Tissue Culture Dish 100 x 20 mm Style | Corning | 353003 | |
| 6 well Cell Culture Cluster | Costar | 3516 | |
| 5/0 Black Braided Surgical Silk (100 yards) | LOOK | SP115 | |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Minipuls 3 Perfusion Pump | Gilson | F155007 | |
| Hemacytometer Kits, Propper | VWR | 48300-474 | |
| Hemacytometer Cover Glasses, Propper | VWR | 48300-470 | |
| Surgical Scissers – Sharp/Blunt | F.S.T. | 14001-12 | |
| Iris Scissors-ToughCut Straight | F.S.T. | 14058-11 | |
| Dumont SS-45 Forceps | F.S.T. | 11203-25 | |
| Student Tissue Forceps | F.S.T. | 991121-12 | |
| Name | Company | Catalog Number | Comments |
| Reagents | |||
| Acetic acid solution, 2.0 N | Sigma | A8976-100ML | |
| Isoflurane, USP 250 mL | Piramal Healthcare | 66794-013-25 | |
| KetaVed 1000mg/10mL (100mg/mL) | VEDCO | 50989-161-06 | |
| Xylazine 100 mg/mL | AnaSed Injection | 139-236 | |
| Willams' Medium E (1x) | gibco | 12551-032 | |
| Liver Perfusion Medium (1x) | gibco | 17701-038 | |
| Liver Digest Medium (1x) | Life Technologies | 17703034 | |
| Primary Hepatocyte Thawing and Plating Supplements | Life Technologies | CM3000 | |
| Primary Hepatocyte Maintenance Supplements | Life Technologies | CM4000 | |
| Phosphate Buffer Saline (PBS) pH 7.4 | ThermoFisher scientific | 10010031 | |
| Collagen Type 1 | Corning | 354236 | |
| Trypan Blue Solution | VWR | 45000-717 |
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