INDUCERS OF INTERFERON AND HOST RESISTANCE .V. IN VITRO STUDIES
INDUCERS OF INTERFERON AND HOST RESISTANCE .V. IN VITRO STUDIES
复制标题
DOI：
10.1073/pnas.61.1.340
复制
发表时间：
1968-01-01
影响因子：
11.1
通讯作者：
HILLEMAN, MR
中科院分区：
文献类型：
--
作者：
FIELD, AK;TYTELL, AA;HILLEMAN, MR
材料和方法。-(I)聚肌苷酸(rI)和聚核糖胞苷酸(rC)购自印第安纳州埃尔克哈特的Miles Laboratories。 rI:rC复合物是通过在磷酸盐缓冲盐水(0.006M磷酸钠0.15M NaCl pH 7.0)中以等摩尔浓度混合rI和rC来制备的。 (2)细胞培养物：通过常规程序制备和维持表1中列出的各种原代、细胞株和细胞系培养物。 RK13培养物是兔肾细胞的稳定系，WI-38和HFL培养物是人胚胎肺的二倍体细胞株。 RK13 和 WI-38 培养物有详细记录，HFL 细胞株是由 C. Baugh 博士在这些实验室中开发的。根据 Field 等人的方法制备兔脾细胞悬液。 2 (3) 使用的病毒是从本实验室的种子储备中制备的。鼻病毒血清型是根据 Hamparian 等人的数字系统指定的。 7 (4) 用 rI: rC 过夜处理细胞培养物后测量对病毒感染的抵抗力的诱导。去除诱导剂后，通过空斑减少测定来测量对病毒复制的干扰。 1 (5)通过 rI 对原代兔肾细胞或兔脾细胞悬浮液中的干扰素诱导：通过将细胞上清液的系列稀释液转移至 Falcon 烧瓶中的 RK13 培养物来测定 rC。随后孵育过夜，然后取出并用水泡性口炎病毒（VSV）攻击，观察斑块形成的减少。使用 RK13 细胞进行测定，因为它们对 rI: rC 诱导的 VSV 抗性相对不敏感，需要超过 1 yg/ml。因此，这些细胞不受干扰素样品中少量残余 rI: rC 的影响。干扰素滴度是样品的最高稀释度，其导致噬菌斑形成至少减少50%。(6)文中描述了其他相关方法。结果.-(1) rI: rC 在体外诱导对病毒感染的抗性：(a) 细胞培养物中的抗病毒活性：细胞培养物对 rI: rC 的敏感性随来源物种以及它们是原代细胞、细胞株还是系细胞而变化。如表 1 所示，许多原代细胞培养物对 rI: rC 敏感，尽管小鼠胚胎细胞表现出不同的反应。 HFL二倍体人细胞株敏感，而同种细胞的WI-38株则不敏感。稳定细胞系的 RK13 培养物显示出不同的敏感性。造成这种变化和差异的原因尚不清楚。 (b)rI、rC和rI:rC制剂的抗病毒活性测试：如表2所示，从6个不同来源获得的rI:rC复合物均具有高活性，
Materials and Methods.-(I) Polyrzboinosinic acid (rI) and polyribocytidylic acid (rC) were purchased from Miles Laboratories, Elkhart, Indiana. The rI: rC complex was prepared by mixing rI and rC in equimolar concentration in phosphate-buffered saline (0.006 M sodium phosphate 0.15 M NaCl pH 7.0).(2) Cell cultures: The various pri-mary, cell strain, and line cell cultures listed in Table 1 were prepared and maintained by ordinary procedures. The RK13 culture is a stable line of rabbit kidney cells, and the WI-38 and HFL cultures are diploid cell strains of human embryoniclung. The RK13 and WI-38 cultures are well documented and the HFL cell strain was developed by Dr. C. Baugh in these laboratories. Rabbit spleen cell suspensions were prepared according to Field et al. 2 (3) The viruses used were prepared from the seed stocks of this laboratory. The rhinovirus serotypes were designated according to the number system of Hamparian et al. 7 (4) Induction of resistance to viral infection was measured after overnight treatment of cell cultures with rI: rC. Following removal of inducer, interference with virus replication was measured by the plaque-reduction assay. 1 (5) Interferon induction in primary rabbit kidney cells or in rabbit spleen cell suspensions by rI: rC was assayed by transfer of serial dilutions of the cell supernatant fluids to RK13 cultures in Falcon flasks. This was followed by overnight incubation prior to removal and challenge with vesicular stomatitis virus (VSV) with observation for reduction in plaque formation. RK13 cells were used for assay since they were relatively insensitive to induction of resistance to VSV by rI: rC, requiring more than 1 yg/ml. Hence, these cells were unaffected by the small residual amount of rI: rC in the interferon samples. The interferon titer was the highest dilution of sample which caused at least 50% reduction in plaque formation.(6) Other pertinent methods are described in the text. Results.-(1) Induction ofresistance in vitro to viral infection by rI: rC:(a) Antiviral activity in cell cultures: Cell cultures varied in their sensitivity to rI: rC with respect to species of origin and as to whether they were primary, cell strain, or line cells. As shown in Table 1, many primary cellcultures were sensitive to rI: rC, although mouse embryo cells showed variable response. The HFL diploid human cell strain was sensitive, while the WI-38 strain of the same kind of cells was not. The RK13 culture of stable line cells showedvariable sensitivity. The cause of such variability and differences isnot known.(b) Tests of antiviral activity ofrI, rC, and rI: rC preparations: As shown in Table 2, rI: rC complex obtained from six different sources were all highly active,