Chadwick C A, Diggle S P, Young A R, Potten C S
CRC Department of Epithelial Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, U.K.
Br J Dermatol. 1996 Oct;135(4):516-22.
Grain counting by eye is a tedious and time-consuming technique but one with great potential in cell kinetics and for the study of DNA excision repair activity (unscheduled DNA synthesis or UDS). We have been investigating the levels of UDS in human skin sections exposed in situ to ultraviolet radiation using a short-term incubation in tritiated thymidine and autoradiography and the decline in UDS levels with time (repair kinetics). We have adapted an automated image analysis system automatically to assess the number of grains over epidermal cell nuclei in autoradiographs of sections of epidermis. An excellent correlation was observed between visual counting and machine measurement of the area (in pixels) occupied by silver grains. The levels of UDS declined with time as lesions are progressively repaired. The half time (+/- standard deviation) for the reduction in UDS is 7.25 +/- 0.18 h. The grain counts can be significantly increased by increasing the autoradiographic exposure, by increasing the concentration of tritiated thymidine and by increasing the incubation time.
肉眼计数颗粒是一种繁琐且耗时的技术，但在细胞动力学和DNA切除修复活性（非程序性DNA合成或UDS）研究中具有巨大潜力。我们一直在利用氚标记胸腺嘧啶核苷短期孵育和放射自显影技术，研究原位暴露于紫外线辐射的人体皮肤切片中的UDS水平，以及UDS水平随时间的下降情况（修复动力学）。我们采用了一种自动图像分析系统，自动评估表皮切片放射自显影片中表皮细胞核上的颗粒数量。在肉眼计数与银颗粒占据面积（以像素为单位）的机器测量之间观察到了极好的相关性。随着损伤逐渐修复，UDS水平随时间下降。UDS降低的半衰期（±标准差）为7.25±0.18小时。通过增加放射自显影曝光量、增加氚标记胸腺嘧啶核苷浓度和延长孵育时间，颗粒计数可显著增加。
