Fornabaio M, Kim Y S, Stackpole C W
Department of Pathology, New York Medical College, Valhalla 10595.
Cancer Lett. 1989 Mar;44(3):185-90. doi: 10.1016/0304-3835(89)90059-1.
Cells of the mouse B16 melanoma growing in monolayer culture and as tumors were fractionated by isopycnic density centrifugation in a linear-density (1.02-1.20 g/ml) metrizamide gradient. Cultured cells concentrated into one or two distinct bands, with densities of 1.02-1.04 g/ml and 1.06-1.10 g/ml, depending on growth conditions. Cells subjected to extreme hypoxia (less than 0.02% O2) banded predominantly at the lower density, and normally-oxygenated cells banded at the higher density. Fractionated tumor cells concentrated at both densities. Compared with cells at the higher density, lower-density cells incorporated more of the hypoxic cell radiosensitizer [14C]misonidazole and less [3H]thymidine in vivo, were less clonogenic but more resistant to X-irradiation in situ, and labeled to a lesser extent with intravenously-delivered Hoechst 33342 fluorochrome, a marker for cells proximal to tumor blood vessels. Lower-density tumor cells were, therefore, enriched in non-proliferating radioresistant hypoxic cells from tumor regions remote from blood vessels.
在单层培养中生长的小鼠B16黑色素瘤细胞以及肿瘤细胞，通过在线性密度（1.02 - 1.20 g/ml）的甲泛葡胺梯度中进行等密度离心进行分级分离。培养细胞根据生长条件浓缩成一条或两条不同的带，密度分别为1.02 - 1.04 g/ml和1.06 - 1.10 g/ml。经历极端缺氧（氧气含量低于0.02%）的细胞主要聚集在较低密度带，而正常氧合的细胞聚集在较高密度带。分级分离的肿瘤细胞在两种密度处均有聚集。与较高密度的细胞相比，较低密度的细胞在体内摄取更多的缺氧细胞放射增敏剂[14C]米索硝唑且摄取较少的[3H]胸苷，克隆形成能力较低，但对原位X射线照射更具抗性，并且用静脉注射的Hoechst 33342荧光染料（一种肿瘤血管附近细胞的标志物）标记的程度较低。因此，低密度肿瘤细胞富含来自远离血管的肿瘤区域的非增殖性耐辐射缺氧细胞。
