Goldberg J L, Zanella C L, Janssen Y M, Timblin C R, Jimenez L A, Vacek P, Taatjes D J, Mossman B T
Department of Pathology, University of Vermont, Burlington 05405, USA.
Am J Respir Cell Mol Biol. 1997 Sep;17(3):265-71. doi: 10.1165/ajrcmb.17.3.2991.
We developed in situ dual-fluorescence detection techniques for measuring apoptosis and proliferation simultaneously in single dishes of cells. The deoxyribonucleic acid (DNA)-specific labeling method, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), first was used in conjunction with a 4',6-diamidino-2-phenylindole (DAPI) counterstain to detect and measure morphologic characteristics of apoptotic rat pleural mesothelial (RPM) cells isolated from Fischer 344 rats and exposed to 300 microM hydrogen peroxide (H2O2). For this purpose, 100 TUNEL-positive nuclei were measured while being viewed with DAPI counterstaining for area, perimeter, longest diameter, and average diameter, using imaging software and an image-collection apparatus. We then exposed cells to a range of concentrations of crocidolite asbestos and putative apoptotic and mitogenic agents. Exposure to crocidolite asbestos (5 microg/cm2) caused a striking dose-dependent apoptotic response at 24 h, 48 h, and 72 h. The nonfibrous crocidolite analogue riebeckite failed to induce apoptosis. At 24 h, tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml) caused an increase in apoptotic nuclei. A second method, utilizing an antibody to 5'-bromodeoxyridine (BrdU) and oxazole yellow homodimer (YOYO), showed a dose-dependent increase in proliferation occurring in cells exposed to asbestos (5 microg/cm2) at 48 h and 72 h. In addition, increased numbers of rat pleural mesothelial (RPM) cells exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA), TNF-alpha, and epidermal growth factor (EGF) exhibited incorporation of BrdU at these time points, although total numbers of cells per unit area were unchanged. Results indicate a dynamic balance between apoptosis and increased DNA synthesis after exposure of mesothelial cells to asbestos.
我们开发了原位双荧光检测技术，用于在单个细胞培养皿中同时测量细胞凋亡和增殖。首先，将脱氧核糖核酸（DNA）特异性标记方法，即末端脱氧核苷酸转移酶（TdT）介导的脱氧尿苷三磷酸缺口末端标记（TUNEL），与4',6-二脒基-2-苯基吲哚（DAPI）复染剂结合使用，以检测和测量从Fischer 344大鼠分离并暴露于300微摩尔过氧化氢（H2O2）的凋亡大鼠胸膜间皮（RPM）细胞的形态特征。为此，使用成像软件和图像采集设备，在DAPI复染的情况下测量100个TUNEL阳性细胞核的面积、周长、最长直径和平均直径。然后，我们将细胞暴露于一系列浓度的青石棉和假定的凋亡及促有丝分裂剂中。暴露于青石棉（5微克/平方厘米）在24小时、48小时和72小时引起了显著的剂量依赖性凋亡反应。非纤维状青石棉类似物钠闪石未能诱导凋亡。在24小时时，肿瘤坏死因子-α（TNF-α）（10纳克/毫升）导致凋亡细胞核增加。第二种方法，利用抗5'-溴脱氧尿苷（BrdU）抗体和恶唑黄同二聚体（YOYO），显示在48小时和72小时暴露于石棉（5微克/平方厘米）的细胞中增殖呈剂量依赖性增加。此外，暴露于12-O-十四酰佛波醇-13-乙酸酯（TPA）、TNF-α和表皮生长因子（EGF）的大鼠胸膜间皮（RPM）细胞数量增加，在这些时间点显示出BrdU掺入，尽管每单位面积的细胞总数没有变化。结果表明间皮细胞暴露于石棉后凋亡与DNA合成增加之间存在动态平衡。
