C型肝炎ウイルスに対するヒトラクトフェリンの細胞取り込みおよびそれに関連する抗ウイルス活性を監視するために、免疫蛍光
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
免疫蛍光は、一般的に生物学の多くの側面を研究するために使用される実験技術です。それは、典型的には、細胞及び組織中の標的分子の分布および/または局在化を可視化するために使用されます。免疫蛍光は、細胞内でのそれらの対応する抗原に対する蛍光標識抗体の特異性に依存しています。直接的および間接的の両方の免疫アプローチは、蛍光色素に結合された抗体の使用に依存しているものを用いることができます。それは、より低い信号を提供し、より高いコスト、より少ない柔軟性を必要とするので、直接免疫蛍光法は、あまり頻繁に使用されます。対照的に、間接免疫蛍光法は、より一般的に、その高い感度を使用し、複数の二次抗体は、それぞれ一次抗体に付着することができるので、増幅された信号を供給する。本稿では、落射蛍光顕微鏡及び共焦点顕微鏡法の双方は、ヒトラクトフェリンの内在化、免疫の重要な構成要素を監視するために使用しました。肝細胞へのシステム。また、我々は、免疫蛍光法を用いて、C型肝炎ウイルスの細胞内複製にHLFの阻害可能性を監視しました。これらのアプローチに関連する利点と欠点の両方が議論されています。
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
Representative Results
Discussion
HCVの流行は、肝硬変、肝不全や肝細胞癌の危険にそれらを置くこと、新たに感染した患者の80％が慢性感染症を発症して、世界的な脅威のまま。最近2 NS3 N末端プロテアーゼ阻害剤（ボセプレビルおよびテラプレビル）の規制当局の承認によって実証されるようにHCV複製および成熟をターゲットに直接作用型抗ウイルス剤は、プライム抗HCV剤を表します。 HLF抗HCV活性は、現在、ビリオンを循?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by both the Canadian Institutes of Health Research and Natural Sciences and the Engineering Research Council of Canada. M. Bisaillon is a Chercheur Boursier Senior from the Fonds de Recherche en Santé du Québec and also a member of the Centre de Recherche Clinique du Centre Hospitalier Universitaire de Sherbrooke. We thank Dr. Ralf Bartenschlager for the generous gift of the HCV replicon system. We also thank Dr. Charles Rice and Dr. Daniel Lamarre for kindly providing the hepatic cell line. We also want to thank Guillaume Tremblay for technical assistance.
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 |
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