Imunofluorescência para monitorar a Incorporação celular de lactoferrina humana e sua Associated atividade antiviral contra o vírus da hepatite C

Summary

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Abstract

Imunofluorescência é uma técnica de laboratório vulgarmente usados ​​para estudar vários aspectos da biologia. É tipicamente usado para visualizar a distribuição e / ou a localização de uma molécula alvo em células e tecidos. Imunofluorescência baseia-se na especificidade de anticorpos fluorescentes marcado contra os seus antigénios correspondentes no interior de uma célula. Ambas abordagens directas e indirectas de imunofluorescência pode ser usado que se baseiam no uso de anticorpos ligados a um fluorocromo. A imunofluorescência direta é menos freqüentemente usado porque fornece sinal inferior, envolve custos mais elevados e menos flexibilidade. Em contraste, imunofluorescência indireta é mais comumente utilizado devido à sua alta sensibilidade e fornece um sinal amplificado desde mais de um anticorpo secundário pode anexar a cada anticorpo primário. Neste texto, tanto microscopia de epifluorescência e microscopia confocal foi utilizada para monitorizar a internalização da lactoferrina humana, um componente importante da resposta imunesistema, em células hepáticas. Além disso, nós monitorizado o potencial inibitório da hLF na replicação intracelular do vírus da hepatite C por meio de imunofluorescência. Ambas as vantagens e desvantagens associadas a estes métodos são discutidos.

Introduction

Immunofluorescence is a technique that uses a fluorescence microscope to visualize the distribution and/or localization of a target molecule in a biological sample. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell¹. It is typically used on tissue sections and cultured cell lines in order to analyze the distribution/localization of various biological molecules such as proteins, nucleic acids and glycans. It should be noted that immunofluorescence is often used in combination with other non-antibody methods of fluorescent staining such as the 4′,6-diamidino-2-phenylindole (DAPI) stains which are typically used to label DNA². Moreover, this technique involves fixation of the cells which allows the analysis of cells at a specific time.

Different types of microscopes can be used to analyze immunofluorescence samples. The simplest is the epifluorescence microscope (Figure 1) for which excitation of the fluorochrome and detection of the fluorescence are done through the same light path³. Because most of the excitation light is transmitted through the sample, only reflected excitatory light can reach the objective together with the emitted light. This approach unfortunately leadsto a frequent high signal to noise ratio.In contrast, confocal microscopy (Figure 2) offers a distinct advantage for increasing optical resolution and contrast by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light⁴. This approach allows the reconstruction of three-dimensional structures from the obtained images. However, since an important fraction of the light from the sample is blocked at the pinhole, long exposures are often required.

There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect). Direct immunofluorescence involves a primary antibody linked with a fluorochrome (Figure 3). This method is less frequently used because it provides lower signal, involves higher cost and less flexibility¹. Moreover, such antibodies are generally harder to find commercially. On the other hand, the direct attachment of the fluorochrome to the antibody significantly reduces the number of steps in the procedure, saving time and frequently reducing non-specific background signal. This also limits the possibility of antibody cross-reactivty.

Indirect immunofluorescence involves a primary unlabelled antibody which is specific for the epitope of interest¹. A secondary antibody which carries the fluorochrome then recognizes the primary antibody and binds to it (Figure 3). Although indirect immunofluorescence is more complex and time consuming than direct immunofluorescence, it is frequently used because of its high sensitivity and it also provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In addition, a vast array of commercial secondary antibodies is available at affordable prices.

Hepatitis C virus (HCV) is a major public-health problem with 130-170 million individuals chronically infected worldwide. In order to halt the epidemic, therapy against HCV will need to be both effective and widely available. Studies focusing on safe and affordable natural product active against HCV have revealed the antiviral activity of the human Lactoferrin (hLF) protein which binds and neutralizes the circulating virion⁵. In the current study, investigation of hLF activity on the HCV subgenomic replicon system, which is independent from viral entry and shedding, revealed a distinct antireplicative activity of hLF against HCV. This manuscript presents a study in which immunofluorescence assays were performed to monitor the internalization of hLF, an important component of the immune system⁶, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus (HCV).

Protocol

1. Células e Preparação de tratamento Numa placa de 24 poços, colocar uma tampa de vidro no fundo dos poços. Lavar cada poço com tampão fosfato salino (PBS). Semente células Huh-7 que suportam o replicão de VHC subgen�ico em cada poço a uma densidade de 5 x 10 4 células / poço. Se não houver tratamento a fazer, as células podem ser semeadas a uma densidade mais elevada. O meio de cultura é de Dulbecco Modified Eagle Médium (DMEM) suplementado com 10% de Soro Fetal Bov…

Representative Results

A capacidade do Huh-7 linha celular hepática para internalizar exogenamente administrada hLF foi monitorizada utilizando imunofluorescência em um miscroscope confocal. hLF foi adicionado ao meio de cultura e a internalização foi deixada por 24 horas, após o que, a hLF extracelular foi lavada com PBS e a membrana ligada a hLF residual foi degradada com um tratamento com tripsina 5 min (1 mL). As células foram re-semeadas e deixou-se voltar a aderir durante 18 h antes de coloração por imunofluorescência. A fim de…

Discussion

A epidemia de HCV permanece uma ameaça global, com 80% dos pacientes recém-infectadas desenvolvem uma infecção crônica, colocando-os em risco de cirrose, insuficiência hepática ou carcinoma hepatocelular. Por acção directa agentes antivirais combatem a replicação do HCV e maturação representam agentes anti-HCV de primeira linha, como recentemente demonstrado pela aprovação regulamentar de dois NS3 N-terminal inibidores da protease (boceprevir e telaprevir). A actividade anti-VHC hLF é actualmente acredit…

Materials

DMEM	Wisent	319-005-CL
PAF	BioShop	PAR070.1	Flammable solid, skin irritant, lungs and eyes.
PBS	Wisent	311-425-CL	Without Ca2+ & Mg2+
NGS	Wisent	053-150
AlexaFluor 488-labeled anti-mouse	Invitrogen	A11017
AlexaFluor 568-labeled anti-rabbit	Invitrogeb	A21069
Wheat germ agglutinin Alexa Fluor 488 conjugate (WGA)	Invitrogen	W11261	Potentially mutagenic
Anti-NS5A rabbit	Abcam	ab2594
Anti-hLF mouse	Abcam	ab10110
SlowFade	Invitrogen	S36937
Hoechst stain	Life Techn.	H1399	Potentially mutagenic and carcinogenic
hLF	Sigma	L0520
Nikon Eclipse visible/epifluorescence Microscope	Nikon	TE2000-E
epifluorescence/confocal microscope	Olympus	FV1000