RESEARCH ON MECHANISNS OF HSV-1 LATENT INFECTION BY IN SITU HYBRIDIZATION

原位杂交研究HSV-1潜伏感染机制

基本信息

批准号：
62570356
负责人：
NAKAMURA Shozo
金额：
$ 1.41万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62570356/
关键词：
Herpes simplex encephalitis antigens nucleic acid sequences 核酸免疫組織化学法 in situ hybridization法 in situ ハイブリダイゼーション

项目摘要

In order to elucidate the correlation between herpes simplex virus(HSV-1) and the central nervous system tissue, we performed the detection of viral antigens and RNA in the brain tissue sections from a patient with herpes simplex virus encephalitis using immunocytochemistry and in situ hybridization.In the present study the hybridization protocol reported by Haase AT et al. were applied for the detection of viral RNA and antigens with a few modifications.The sections were first immunocytocehmically stained to detect HSV-1 antigens by ABC method, and then hybridized with ^3H-labelled HSV-1 cDNA probe for the detection of RNA after the acetylation of sildes for the prevention of nonspecific bibdings of isotope to slides.HSV-1 infacted Vero cells as positive control demonstrated both antigens and RNA. However, no hybridization signals and color deposits were observed in uninfacted Vero cells.In frozen brain tissue sections of herpes simplex encephalitis the viral antigen(VA) positive cell … More s, containg brown-colored deposits located primarily in the cytoplasm, were widely distributed throughout the lesions of a part of gray matter. Such VA positive cells were morphologically identified to be swollen neurons, glial cells and endothelial cells. In addition, some of VA positive cells have also hybridization signals over the nuclei, namely VA and RNA positive cells which were diffusely located colse to VA positive cells. Moreover, only RNA postive cells with numerous silver grains over the nuclei were predominantly distributed at the neighborhood of VA positive cells and at the white matter.In quantitative analysis of HSV-1 infecgted cells of a part of gray matter, cells positive with viral antigen were counted 23.2% of total cells.Both VA and RNA cells and only RNA cells were detected 13.4% and 18.3% respectively. In addition, in a part of white matter VA and RNA cells and only RNA cells were found 9.3% and 41.7% although there were was not detected VA positive cells.We concluded that the herpes simplex virus (HSV) infects the virious kind of cells composed of brian tissues and shows the virious stages of infection, namely; VA positive cells, VA and Rna positive cells and only RNA positive cells. Moreover, it may be considered that these various stages of infacted cells are developed according to many factors such as different kind of cells, infactious routes and host immune responses. Less

为了阐明单纯疱疹病毒（HSV-1）与中枢神经系统组织的相关性，我们采用免疫细胞化学和原位杂交技术对一名单纯疱疹病毒脑炎患者脑组织切片中的病毒抗原和RNA进行了检测。在本研究中，Haase AT 等人报告的杂交方案经过一些修改后用于检测病毒 RNA 和抗原。首先对切片进行免疫细胞染色以检测。采用ABC法提取HSV-1抗原，然后与^3H标记的HSV-1 cDNA探针杂交，乙酰化后检测RNA，以防止同位素对玻片的非特异性结合。HSV-1感染Vero细胞为阳性对照显示了抗原和RNA，然而，在未感染的Vero细胞中没有观察到杂交信号和颜色沉积。在单纯疱疹性脑炎的冷冻脑组织切片中。病毒抗原（VA）阳性细胞，主要位于细胞质中，含有棕色沉积物，广泛分布于部分灰质病变部位，形态学鉴定为肿胀的神经元、胶质细胞。此外，一些VA阳性细胞在细胞核上也有杂交信号，即VA和RNA阳性细胞，其分布在VA阳性细胞附近，而且只有RNA阳性细胞上有大量银粒。细胞核主要分布在VA阳性细胞附近和白质。对部分灰质HSV-1感染细胞进行定量分析，病毒抗原阳性细胞占总细胞数的23.2%。VA和RNA细胞和仅RNA细胞分别被检测出13.4%和18.3%。此外，在白质VA的一部分中，虽然存在RNA细胞和仅RNA细胞，但仅检测到9.3%和41.7%。未检测到 VA 阳性细胞。我们得出结论，单纯疱疹病毒 (HSV) 感染由脑组织组成的病毒类细胞，并显示出感染的病毒阶段，即：VA 阳性细胞、VA 和 Rna 阳性细胞以及仅 RNA 阳性细胞。而且，可以认为，感染细胞的这些不同阶段是根据不同细胞种类、感染途径和宿主免疫反应等多种因素而发展的。