摘要
我们根据 N-乙酰-β-D-氨基葡糖酶(下简称 NAG 酶)在活细胞参与下的呈色反应首次建立了人表皮细胞生长的 NAG 酶反应快速比色测定法。结果显示 OD 值与细胞数量呈对应关系,作图得一 S 形曲线.表皮细胞量在一定范围内与 OD 值基本上呈一线性关系。用此方法测定在不同培养基和培养条件下人表皮细胞的贴壁、生长和生长曲线,包括一些生长因子对人表皮细胞增殖作用的检测.实验结果用其他方法验证较为满意。此方法的优点在于简便、快速、灵敏.不需要同位素即能定量地分析、处理大量的培养标本,为表皮细胞培养和移植的研究提供了一个有用的检测方法。
Attachment and growth of human epidermal keratinocyte culture can be assayedt by counting cells or measuring incorporation of radioactive nucleotides(~3H-TdR)during cell pro- liferation.In this study a rapid calorimetric assay for human epidermal keratinocyte growth and viability has been developed based on the colour reaction of NAG(p-nitrophenol- N-acetyl-β-D-glucosaminide).The results can be read on a scanning multiwell spectrophoto- meter and show a high degree of precision.The data of experiments show that the absor- bance(OD)is directly proportional to the number of cells.10~3-10~5 cells per well(1.5cm^2) can be assayed by controlling the time of colour reaction.This method was used to mea- sure keratinocyte proliferation in different culture systems or different culture conditions, growth factor and keratinocyte growth-promoting activity stimulations.The results were supported by counting cells,measuring of ~3H-TdR incorporation,or analysing the area of keratinocyte confltteats stained with Rhodanile blue.The main advantages of the colorime- trio assay are its rapidity and precision,the avoidance of any radioisotope,and it is capa- ble of handling large numbers of culture.
