C 형 간염 바이러스에 대한 인간 락토페린의 세포 흡수 및 관련 바이러스 활동을 모니터링하는 면역 형광
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
흔히 면역 생물학의 여러 측면을 연구하는 데 실험 기술이다. 이것은 전형적으로 세포 및 조직에서 표적 분자의 분포 및 / 또는 위치 파악을 시각화하기 위해 사용된다. 면역은 세포 내에서 해당 항원에 대한 형광 표지 항체의 특이성에 의존한다. 모두 직접 및 간접 면역 형광 방법은 형광 색소와 링크 된 항체의 사용에 의존 할 수있다. 그것은, 낮은 신호를 제공 높은 비용과 적은 유연성을 포함하기 때문에 직접 면역 형광이 자주 사용된다. 대조적으로, 간접 면역 형광은 더 일반적 때문에 고감도의 사용과 둘 이상의 이차 항체 각 차 항체에 부착 할 수 있기 때문에 증폭 된 신호를 제공한다. 이 논문에서는, 표면 형광 현미경 및 공 초점 현미경 모두 인간 락토페린의 내재화, 면역의 중요한 구성 요소를 모니터링하는 데 사용 된간 세포로 시스템. 또한, 우리는 면역 형광을 사용하여 C 형 간염 바이러스의 복제를 HLF 세포의 억제 가능성을 모니터. 이 두 가지 접근 방식과 관련된 장점과 단점이 논의된다.
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 전염병은 간경변, 간부전이나 간암의 위험에 그들을 가하고, 만성 감염을 개발 새로 감염된 환자의 80 %로, 글로벌 위협이 남아있다. 최근 두 NS3 N- 말단 단백질 분해 효소 억제제 (Boceprevir 및 Telaprevir)의 규제 당국의 승인에 의해 입증 된 바와 같이 – 직동 HCV 복제 및 성숙을 대상으로 항 바이러스제는 주요 항 HCV 에이전트를 나타냅니다. HLF 항 HCV 활성은 현재 비리 순환 그들의 타겟 간세포 내로 ?…
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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