KINETICS OF THE RELEASE OF POLIOMYELITIS VIRUS FROM SINGLE CELLS
KINETICS OF THE RELEASE OF POLIOMYELITIS VIRUS FROM SINGLE CELLS
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DOI：
10.1016/0042-6822(55)90010-6
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发表时间：
1955-01-01
期刊：
影响因子：
3.7
通讯作者：
LWOFF, M
中科院分区：
文献类型：
--
作者：
LWOFF, A;DULBECCO, R;LWOFF, M
描述了一种用于研究感染病毒的单个分离动物细胞的技术。从 Dulbecco 和 Vogt [thisBulletin, 1954, v. 29, 887] 描述的类型的单层猴肾培养物制备分离猴肾细胞的悬浮液，通过用胰蛋白酶处理和剧烈移液，在这种情况下将病毒添加到悬浮液中，或者通过用磷酸盐缓冲盐水中的维奈酸钠处理先前感染的单层细胞，这在温和搅拌下产生单细胞悬浮液。将培养基和细胞悬浮液的小滴包埋在载玻片上的石蜡油或二甲基硅氧烷中。通过稀释或通过在毛细管中从细胞悬浮液的液滴中选择分离的细胞，在一滴培养基中获得单个细胞。 de Fonbrune 微操纵器极大地促进了这些操作。在相差 5% 至 60% 的条件下观察分离的细胞。观察到细胞沉降并散布在玻璃上。猴肾细胞通常在 15 到 60 分钟之间扩散，而经过类似处理的 HeLa 细胞则需要 3 到 48 小时之间的任何时间。细胞在滴液中保持健康 1-6 天。每个细胞被大约 5 个病毒颗粒感染。用显微镜观察细胞，并以 1/2 小时的间隔对液滴中的液体进行取样。大约95%。首先去除 90% 的液体进行取样。滴，添加新鲜液体并去除 90%。的这个。通过噬菌斑计数方法滴定液滴的内容物[loc.1]。引用]。研究了感染 I 型脊髓灰质炎病毒 Brunhilde 株的四种猴肾细胞。第一个细胞在 5 1/2 小时内没有产生病毒，然后产生超过 120 个空斑形成颗粒。第二个单元在 7 小时内产生 89 个颗粒，第三个单元在 7 1/2 小时内产生约 80 个颗粒。大部分病毒在半小时内产生，但有少量病毒在接下来的1/2小时内出现。每隔 10 分钟对含有第四个细胞的液滴进行取样，并在半小时内以均匀的速度产生病毒。 9小时实验结束时，冷冻细胞和液体。即使之前1/2小时的样本呈阴性，这些样本中始终含有一些噬菌斑形成颗粒。在感染过程中，细胞发生了变化。在病毒释放前一小时，细胞开始收缩并将颗粒物质集中在细胞核周围的中心。外围有一个明显的“透明”区域，当病毒在营养液中积聚时，该区域会变成空泡。 [该技术的详细信息应查阅原始论文。]A． J.比尔.
A technique is described for the study of single isolated animal cells infected with virus. Suspensions of isolated monkey kidney cells were prepared from monolayer monkey kidney cultures of the type described by Dulbecco and Vogt [thisBulletin, 1954, v. 29, 887], either by treatment with trypsin and vigourous pipetting, in which case virus was added to the suspension, or by treatment of prior infected monolayers with sodium versenate in phosphate buffered saline, which yielded suspensions of single cells on mild agitation. Small droplets of medium and cell suspension were embedded in paraffin oil or dimethyl siloxane on a glass slide. A single cell was obtained in a drop of medium by dilution or by selecting an isolated cell in a capillary tube from the droplet of cell suspension. These manipulations were greatly facilitated by a de Fonbrune micro-manipulator.Isolated cells were observed under phase contrast and from 5 to 60 per cent. of cells were observed to settle and spread out on the glass. Monkey kidney cells spread out between 15 and 60 minutes as a rule whereas HeLa cells treated similarly took any time between 3 and 48 hours. Cells appeared healthy for 1-6 days in the drops.Cells were infected with about 5 virus particles per cell. The cells were observed microscopically and the fluid in the drops was sampled at 1/2 hour intervals. Roughly 95 per cent. of the fluid was sampled by first removing 90 per cent. of the drop, adding fresh fluid and removing 90 per cent. of this. The contents of the drop were titrated by the plaque counting method [loc. cit.]. Four monkey kidney cells infected with the Brunhilde strain of Type I poliomyelitis virus were studied. The first cell yielded no virus for 5 1/2 hours, and then produced more than 120 plaque-forming particles. The second cell produced 89 particles at 7 hours and the third cell about 80 particles at 7 1/2 hours. The majority of the virus was produced in half an hour though a small amount appeared in the next 1/2 hour. The drop containing the fourth cell was sampled at 10-minute intervals and appeared to produce virus at a uniform rate for half an hour. At the termination of the experiments at 9 hours the cell and fluid were frozen. These samples always contained a few plaque-forming particles even if the previous 1/2-hour sample was negative.During the process of infection changes occurred in the cells. One hour before the release of virus the cell started to contract and concentrate granular material in the centre around the nucleus. There was a clear "hyaline" zone at the periphery, which became vacuolated during the hour when virus was accumulating in the nutrient fluid. [The original paper should be consulted for the details of the technique.]A. J. Beale.