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

Este trabalho foi financiado pelo NIH (R01HL128714 para CF) e (F31DK10236101 para KS) e da American Heart Association (CF e AG).

Todos os protocolos de animais e procedimentos experimentais foram aprovados pelo Comitê Institucional de Animal Care and Use (IACUC) da Universidade de Miami Miller School of Medicine. 1. Preparação <ol><li> Pré-aqueça meios de perfusão hepática e fígado digerir mídia em um banho de água a 37 ° C. </li><li> Configurar uma bomba de perfusão (30 mL / min). Cuidadosamente prefill o sistema de tubulação da bomba com a mídia perfusão hepática. Evitar as bolhas de ar no sistema...

<ol>
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Isolamento de hepatócitos primários a partir de ratinhos é uma ferramenta rápido, barato e fiável para estudar as respostas inflamatórias ex vivo. Se realizado corretamente, os resultados podem ser facilmente gerada e reproduzida de forma atempada e eficiente. Os seguintes pontos devem ser cuidadosamente avaliada, a fim de garantir um isolamento bem sucedido. A incisão cirúrgica ea canulação da VCI deve ser realizada sob anestesia geral e não após a eutanásia. Um invest...

Doença renal crónica (IRC) pode ser definida como um estado de inflamação aguda e crónica 1. Em pacientes com DRC, os níveis séricos do fator fosfatúrico hormona de crescimento de fibroblastos 23 (FGF23) aumentará progressivamente, a fim de manter a homeostase do fosfato sérico 2. Níveis FGF23 soro aumentados estão independentemente associadas com morbidade e mortalidade cardiovascular entre os pacientes que estão começando o tratamento de hemodiálise 3, 4. Além disso, vários estudos clínicos demonstraram uma correlação entre os níveis elevados e FGF23 níveis séricos de proteína C-reactiva (CRP), interleucina-6 (IL-6) e Tumor Necrosis Factor de α (TNFa) 5, 6. Além disso, em um estudo experimental, que demonstraram recentemente que FGF23 pode ter como alvo directamente hepatócitos e causa uma resposta inflamatória, aumentando PCR e IL-6 Produção no fígado 7. Assim, FGF23 pode atuar como um fator circulante que contribui para a inflamação sistêmica em CKD. No início dos anos 70, os hepatócitos primários foram isolados e estudados pela primeira vez 8. Desde então, as células hepáticas cultivadas primárias têm sido amplamente utilizados para examinar transformação metabólica, função hormonal, o metabolismo de drogas e toxicidade, bem como a imunidade e respostas inflamatórias 9, 10. Protocolos anteriores descreveram-se principalmente o isolamento enzimático de hepatócitos primários a partir de tecido de figado humano 11, 12. Enquanto um excelente modelo, este deixa de fora a possibilidade de estudar como a manipulação genética afeta os mecanismos de sinalização hepática complexos, bem como consequências funcionais sobre diferentes tipos de estímulos. No que se segue, descreve-se o isolamento de hepatócitos primários de murino. Notavelmente, o efeito de vários mediadores da resposta de fase aguda hepática, tais como lipopolissacáridos (LPS), IL-6 e FGF23 podem ser analisadas de uma forma fácil, rápida e reprodutível 13. Este trabalho apresenta um protocolo para o isolamento enzimático de hepatocitos de ratos adultos, e que demonstram que os indutores estabelecidos de inflamação, tais como LPS e IL-6, bem como novos mediadores inflamatórios, tais como FGF23, pode estimular directamente a expressão e secreção de citoquinas inflamatórias, tais como PCR e IL-6 em hepatócitos em cultura.

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induction of an inflammatory response in primary hepatocyte cultures from mice

O fígado desempenha um papel decisivo na regulação da inflamação sistêmica. Na doença renal crónica, em particular, o fígado reage em resposta ao meio urémico, o stress oxidativo, endotoxemia e a diminuição da depuração da circulando citocinas pró-inflamatórias através da produção de um grande número de reagentes de fase aguda. ferramentas experimentais para estudar a inflamação eo papel subjacente dos hepatócitos são cruciais para compreender a regulação ea contribuição de citocinas hepáticas a uma resposta de fase aguda sistêmica e um cenário pró-inflamatória prolongada, especialmente em um ambiente complexo, tais como doença renal crónica. Uma vez que o estudo dos mecanismos complexos de inflamação in vivo permanece um desafio, uso intensivo de recursos e geralmente requer a utilização de animais transgénicos, hepatócitos isolados primários fornecer uma ferramenta robusta para obter insights mecanísticos para a resposta de fase aguda hepática. Desde esta técnica in vitro apresenta custos moderados, alta reprodutibilidade e conhecimento técnico comum, hepatócitos isolados primários podem também ser facilmente usados ​​como uma abordagem de triagem. Aqui, nós descrevemos um método baseado em enzimática para isolar os hepatócitos primários de murino, e que descrevem a avaliação de uma resposta inflamatória nestas células usando ELISA e quantitativa PCR em tempo real.

Histologia As imagens de microscopia de luz representativas de células isoladas e cultivadas primárias estão representados na Figura 1A. Análise imunocitoquímica demonstra que hepatócitos isolados altamente expressar albumina (vermelho), bem como do receptor de factor de crescimento de fibroblastos 4 (FGFR4) (verde). Os núcleos são corados com 4 &#39;, 6-diamidino-2-phenylindole (DAPI) (azul). (Fi...

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

Aqui, mostramos uma abordagem enzimática para isolar hepatócitos primários a partir de ratos adultos, e descreve-se a quantificação de uma resposta inflamatória por meio de ELISA e PCR em tempo real.

A indução de uma resposta inflamatória em culturas primárias de hepatócitos de ratinhos

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 ...

The overall goal of this procedure is to successfully isolate functional murine hepatocytes for studying the expression and secretion of inflammatory cytokines such as CRP and IL6 in response to mediators of the hepatic acute phase response. This method could help answer key questions of hepatocellular function and the contribution of hepatic cytokines to a prolonged inflammatory environment as found in chronic kidney disease. The main advantage of this technique is that it provides a robust tool for a simple, fast, and reproducible analysis of hepatic metabolomics and inflammation as well as a response to drugs.To begin, preheat the liver perfusion medium and liver digest medium in a water bath at 37 degrees Celsius. Set up a perfusion pump as shown here. Then use liver perfusion medium to carefully pre-fill the tubing system of the pump.Try avoiding any air bubbles in the system. Prepare the stereotactic microscope for the perfusion procedure. Then after anesthetizing a donor mouse according to the text protocol, place the anesthetized animal on an absorbent pad.Use adhesive tape to fix the mouse's extremities. Next, shave and disinfect the abdomen of the mouse. Then use surgical scissors with sharp blunt tips to perform a ventral laparotomy from the pubis to the cranial border of the liver.Incise the abdominal wall on both sides at a position caudal to the diaphragm. Expose the inferior vena cava or IVC by carefully moving the intestine to the right side using sterile cotton swabs. With fine surgical scissors and sharp sharp tips, make lateral incisions across the suprahepatic diaphragm and open the thoracic cavity.Then use 5-0 surgical silk to ligate the thoracic IVC. With a shielded IV catheter, cannulate the infrarenal IVC. Connect the perfusion pump and start perfusing the liver.If performed properly, the liver should immediately begin to blanch and swell. Next, with fine surgical scissors, transect the portal vein, allowing suitable drainage of blood and perfusion medium. After perfusing the liver with approximately 30 milliliters of liver perfusion medium, stop the perfusion pump.Switch to liver digest medium and resume the pump. Once perfusion is complete, remove the cannula. Gently expose the central region of the liver and locate the connecting tissue linking the liver lobes.Grab the central connecting fibers and carefully dissect all the tissues holding the liver in place. Then gently remove the liver. Immediately transfer the organ into a 50 milliliter polypropylene conical tube containing 15 milliliters of liver digest medium.While working under a cell culture hood, transfer the liver from the 50 milliliter polypropylene conical tube into a sterile tissue culture dish. With sterile forceps, gently tear the four lobes of the liver. If performed properly, the digestion medium should become turbid duet to the isolation of hepatocytes from the liver.Using a 25 milliliter sterile pipette tip, filter the turbid digestion medium through a 70 micrometer nylon cell strainer into a new 50 milliliter polypropylene conical tube to remove undigested tissue particles and cell debris. Centrifuge the combined filtrate at 50 times g in four degrees Celsius for two minutes, then aspirate the supernatant containing excess cell debris and use 25 milliliters of cold 1X William's Medium E to gently resuspend cells. After repeating the filtering and washing of the hepatocytes three times, aspirate the supernatant to remove excess cell debris.Resuspend the cells in 25 milliliters of warm 1X William's Medium E that contains primary hepatocyte thawing and plating supplements and filter the suspension through a new 70 micrometer nylon cell strainer into a 50 milliliter conical tube. Once the cells have been counted, plate them in two milliliters of William's Medium E that contains primary hepatocyte thawing and plating supplements on a collagen-coated six well cell culture cluster. Following two changes of medium to eventually starve the cells, treat the cells with PBS as vehicle, 25 nanograms per milliliter of FGF23, 100 micrograms per milliliter of LPS, or 50 nanograms per milliliter of IL6 in serum-free medium.Incubate the cultures for 24 hours. After preparing RNA using a spin column kit according to the manufacturer's instructions, add 200 nanograms of the isolated RNA to a PCR tube. Then add 14 microliters of endonuclease-free water and four microliters of reverse transcriptase to the tube.Place the PCR tube into the thermocycler and run the following reverse transcription program to generate cDNA. To carry out quantitative PCR, combine the following QPCR master mix components in a tube on ice. Vortex the master mix.Then aliquot nine microliters of the solution into each well of a 96 well plate. Carefully add one microliter of cDNA into each well, preparing three wells for each sample. Finally, run the samples in a real-time PCR instrument and analyze the data according to the text protocol.A representative light microscopy image of primary isolated and cultured cells are depicted here. Immunocytochemical analysis demonstrates that isolated hepatocytes highly express albumin as well as fibroblast growth factor receptor 4. In this experiment, primary isolated hepatocytes were treated with FGF23, LPS, or IL6 for 24 hours and mRNA levels of CRP and IL6 were analyzed by quantitative real-time PCR.LPS, IL6, and FGF23 significantly increased expression of CRP and IL6. As shown here, cell culture supernatants from isolated primary hepatocytes were analyzed by ELISA for CRP levels. As demonstrated with QPCR analysis, LPS, IL6, and FGF23 treatments significantly increased CRP levels in cell supernatants when compared to PBS-treated cells.Once mastered, this technique can be done in just a few hours if it is performed properly. While attempting this procedure, it's important to remember to maintain a sterile environment. Due to working with primary culture, minimizing the risk of possible contamination is most important for achieving optimal results.After watching this video, you should have a good understanding of how to successfully isolate murine hepatocytes for studying various hepatocellular functions such as expression and secretion of hepatic cytokines.

Research

JoVE Journal

Immunology and Infection

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Indução de hipofisite Auto-Imune Experimental em Ratos SJL

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

Imunologia e Infecção

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

Um método simples e eficiente para isolar Macrófagos de Mixed culturas primárias de células do fígado Adulto

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

Geração de Culturas Raft organotípicas de queratinócitos humanos primários

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

Isolamento Microglia primária a partir de cultura mista de células gliais de tecido de cérebro de rato neonatal

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

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

Diferenciando papéis funcionais da expressão do gene de células imunes e não-imunes na colite Rato por Transplante de Medula Óssea

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

Isolamento e injecção intravenosa de Murino de Medula Óssea Derivado monócitos

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

Indução de Murino inflamação intestinal por transferência adotiva de Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; Células T em imunodeficientes Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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

Ensaios de caracterização de romance endoteliais reguladores envolvidos na resposta inflamatória de triagem

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

Indução de paralisia e lesão do sistema Visual em camundongos por específicos de células T para Neuromielite Óptica Autoantigen Aquaporin-4

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

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Efeitos de gosto sinalização Abolishment de proteína na inflamação do intestino em um modelo de Mouse de doença inflamatória intestinal

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...