目的 通过高内涵筛选技术评价测试药对PA-1细胞的增殖毒性，并联合分化毒性的评价指标——多能性基因（Oct4、Sox2和Nanog）表达检测，初步建立药物胚胎毒性快速筛查方法。方法 选择维生素C、青霉素G、维生素E作为无胚胎毒性的阴性测试药，甲氨蝶呤、5-氟尿嘧啶、环磷酰胺作为具有胚胎毒性的阳性测试药。以测试药临床治疗剂量下的最大血药浓度（C_max）为基础设置加药浓度，阴性测试药设置浓度梯度为：1/2C_max、C_max、2C_max、3C_max、4C_max；阳性测试药设置浓度梯度为：1/8C_max、1/4C_max、1/2C_max、C_max、2C_max。测试药物在维持培养液（培养液加入1×10⁶ U·L^-1的白血病抑制因子）中作用于PA-1细胞24 h后，基于高内涵筛选技术，应用DAPI、Calcein-AM、PI共染PA-1细胞，通过共聚焦成像检测荧光强度，计算细胞存活率；测试药物在分化培养液（不加入白血病抑制因子）中作用于PA-1细胞24 h后，实时荧光定量PCR（qRT-PCR）技术检测多能性基因Oct4、Sox2和Nanog的表达变化。结果 DAPI、Calcein-AM、PI 3者共染可以很好地区分活细胞及死细胞；阴性测试药维生素C、维生素E、青霉素G对PA-1细胞的增殖能力均无影响；与对照组比较，阳性测试药5-氟尿嘧啶、甲氨蝶呤1/4C_max及以上浓度引起PA-1细胞存活率显著下降（P＜0.05）；环磷酰胺1/8C_max及以上浓度引起PA-1细胞存活率显著下降（P＜0.05）。阴性测试药维生素C、维生素E、青霉素G对PA-1细胞多能性基因Sox2、Oct4、Nanog的表达均无影响。与对照组比较，阳性测试药5-氟尿嘧啶1/8C_max及以上浓度使PA-1细胞多能性基因Sox2、Oct4、Nanog的表达显著下降（P＜0.05）；甲氨蝶呤1/8C_max及以上浓度使Oct4、Nanog的表达显著增加（P＜0.05），但当浓度达到2C_max时对Sox2的表达依然没有影响；环磷酰胺1/8C_max及以上浓度使Sox2、Oct4、Nanog的表达均显著增加（P＜0.05）。结论 高内涵筛选技术（DAPI、Calcein-AM、PI共染）联合多能性基因的检测可初步建立药物胚胎毒性快速筛查方法。

Objective To evaluate the proliferative toxicity of test substance on PA-1 cells by high content screening technology, and to establish a rapid screening method for drug embryotoxicity by combining with pluripotency genes (Oct4, Sox2 and Nanog) as evaluation indexes. Methods Vitamin C, penicillin G and vitamin E were selected as negative test drugs without embryotoxicity, and methotrexate, 5-fluorouracil and cyclophosphamide were selected as positive test drugs with embryotoxicity. The dosing concentration was set based on the maximum blood drug concentration (C_max) under the clinical therapeutic dose of the test drug, and the concentration gradient of the negative test drug was 1/2C_max, C_max, 2C_max, 3C_max, 4C_max. The concentration gradient of positive test drug was 1/8C_max, 1/4C_max, 1/2C_max, C_max, 2C_max. The test drug was incubated in the maintenance medium (the medium was added with 1×10⁶ U·L^-1 leukemia inhibitory factor) in PA-1 cells for 24 h. Based on the high content screening technology, PA-1 cells were co stained with DAPI, Calcein-AM and PI. The fluorescence intensity was detected by confocal imaging and the cell survival rate was calculated. After the test drug incubated with PA-1 cells in differentiation culture medium (without leukemia inhibitory factor) for 24 h, the expression changes of pluripotent genes Oct4, Sox2 and Nanog were detected by real-time fluorescent quantitative PCR (qRT-PCR). Results The CO staining of DAPI, Calcein-AM and PI can distinguish living cells from dead cells. Vitamin C, vitamin E and penicillin G had no effect on the proliferation of PA-1 cells. Compared with control group, the positive test drugs 5- fluorouracil, methotrexate of 1/4C_max and above concentrations significantly decreased the survival rate of PA-1 cells (P < 0.05). Cyclophosphamide of 1/8C_max and above significantly decreased the survival rate of PA-1 cells (P < 0.05). The negative test drugs vitamin C, vitamin E and penicillin G had no effect on the expression of pluripotency genes Sox2, Oct4 and Nanog in PA-1 cells. Compared with control group, the positive test drug 5-fluorouracil of 1/8C_max and above significantly decreased the expression of pluripotent genes Sox2, Oct4 and Nanog in PA-1 cells (P < 0.05). Methotrexate of 1/8C_max and above significantly increased the expression of Oct4 and Nanog (P < 0.05), but when the concentration reached 2C_max, it still had no effect on the expression of Sox2. Cyclophosphamide of 1/8C_max and above significantly increased the expression of Sox2, Oct4 and Nanog (P < 0.05). Conclusion High content screening technology (DAPI, Calcein-AM, PI co-staining) combined with detection of pluripotent genes can establish a rapid screening system for drug embryotoxicity.

R965.2

国家自然科学基金资助项目（81573740）