免疫监视人乳铁蛋白的细胞摄取及其相关抗病毒活性抗丙型肝炎病毒

摘要

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.

摘要

免疫是通常用于研究生物学的许多方面实验室技术。它通常用于可视化目标分子的细胞和组织中的分布和/或定位。免疫荧光依赖于对小区内其相应的抗原的荧光标记的抗体的特异性。直接和间接的免疫荧光方法可以用于其中依靠使用带有荧光联抗体。直接免疫是不经常使用的，因为它提供较低的信号，涉及到较高的成本和更少的灵活性。与此相反，间接免疫荧光更常用，因为其高灵敏度和提供放大的信号，因为一个以上的次级抗体可以附加到每个主抗体。在这份手稿，无论是荧光显微镜和共聚焦显微镜被用来监测人乳铁蛋白的内化，免疫的重要组成部分系统，进入肝细胞。此外，我们监测了使用免疫荧光的丙型肝炎病毒的细胞内复制的hLF的抑制潜能。都与这些方法相关的优点和缺点进行了讨论。

引言

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).

研究方案

1.细胞制备与处理

1. 在一个24孔板，把一个玻璃盖在孔的底部。用磷酸盐缓冲盐水（PBS）洗涤各孔。
2. 种子Huh-7细胞在每孔配套的HCV亚基因组复制到^5×10 4个细胞的密度/孔。如果没有治疗做，细胞可以在更高的密度接种。培养基是Dulbecco氏改良的Eagle培养基（DMEM），补充有10％胎牛血清（FBS），2mM L-谷氨酰胺，1mM丙酮酸钠和250毫克/毫升的G-418（以维持复制子）。
3. 在37℃和5％的CO _2。第二 ​​天，处理细胞以3微米的hLF的从人乳纯化。

2.多聚甲醛/蔗糖制备（12％的PAF和12％蔗糖）

1. 把500个毫升的PBS在一个烧杯中，并加热它在20℃至30℃。溶解60克蔗糖的PBS中。溶解60克的多聚醛（PAF）在PBS /蔗糖溶液中。慢慢加入氢氧化钠2N（3至7毫升）中获得澄清溶液为止。
2. 调节pH至7.4，用HCl。完整体积至500ml，用PBS。通过重力的滤纸过滤器过滤。在使用前，稀释用PBS溶液至4％的PAF和4％蔗糖（寄存4℃，1周最大）的终浓度。

3.免疫

1. 在所需的时间（0小时，2小时或24的治疗小时），洗涤细胞两次（1分钟），用PBS并用PBS / 4％PAF / 4％蔗糖固定20分钟，在室温。所述细胞通常是在30-40％汇合和1.5×10 ^5个细胞的使用。
2. 透化在PBS中的10％正常山羊血清（NGS）将细胞用0.15％的Triton X-100的PBS进行5分钟，在RT和块20分钟。
3. 稀释的感兴趣的蛋白质的一次抗体，在10％NGS（例如:主小鼠抗体hLF抗体稀释1:1000）。应用第一抗体的细胞。
4. 4小时后在室温下或O /北，4℃，洗细胞三次，持续5分钟，用10％NGS。细胞染色与标记荧光染料稀释在10％NGS的二级抗体（例如:荧光染料具有488纳米连接于抗体抗小鼠1的激发波长:1,000）在RT在黑暗1小时。
5. 一次5分钟，用PBS洗涤细胞，并将细胞染色用4'，6-二脒基-2-苯基吲哚（DAPI）（1微克/ ml）在室温15分钟在黑暗中。
6. 5分钟两次，用PBS洗涤细胞。盖山在SlowFade安装介质眼镜通过显微镜可视化之前。这些细胞是现在可以通过荧光或共聚焦显微镜分析。
7. 使用不同的控制，以确保检测的特异性。例如，所有抗体可为了检测自体荧光被省略。第一抗体可以被省略，以确定由所述第二抗体的非特异性染色。最后，如​​果可能，控制不这样做的细胞或组织表达也可用于感兴趣的分子。
8. 可视化使用适当的荧光显微镜（epifluorescence的或共焦）和过滤器组适合于所使用的荧光标记的样品。幻灯片也可以存储在<-20℃供以后检查一个滑动箱。

4.荧光显微镜

1. 保持样品在黑暗中以防止光漂白。打开所选择的显微镜（萤光或共焦）的光源。
2. 打开显微镜。打开相机的显微镜上。把滑动显微镜上进行可视化。添加浸油在滑动以增加物镜的数值孔径。
3. 选择适当的过滤器。调整的重点和合适的目标。调节百叶窗以便检测适当的荧光染料，但防止光漂白。请顶灯关在看样品，以尽量减少背景光。
4. 变更日ë滤波器可视化的各种荧光染料（例如DAPI和为488纳米的激发波长下）。拍照与摄像头。

结果

使用免疫荧光共聚焦miscroscope肝的Huh-7细胞系的内化外生有管理的体hLF的能力进行监测。的hLF加入到培养液中并内化被允许进行24小时，在这之后，细胞外的hLF用PBS洗涤和结合体hLF残留膜降解，5分钟胰蛋白酶处理（1ml）中。将细胞再接种，并使其重新附着的免疫荧光染色前1​​8小时。为了概括的胞质限制，将细胞的膜进行染色的488纳米缀合物的激发波长的麦胚凝集素（WGA）的荧光染料。所述的hLF?...

讨论

丙型肝炎病毒的流行仍然是一个全球性的威胁，有80％的新感染者发展为慢性感染的，这使他们肝硬化，肝衰竭或肝癌的危险。直接作用的靶向HCV复制和成熟抗病毒剂代表素抗HCV药物，如最近由两个NS3 N-末端蛋白酶抑制剂（用boceprevir和替拉瑞韦）的监管机构批准证明。所述的hLF抗HCV活性的目前认为表现为病毒进入抑制剂，结合循环病毒粒子和阻止它们进入到他们的肝细胞靶。我们对铁蛋白的研究?...

材料

Name	Company	Catalog Number	Comments
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