高内涵筛选技术联合多能性基因Oct4、Sox2和Nanog检测建立药物胚胎毒性快速筛查方法

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。

Establishment of a rapid screening method for drug embryotoxicity by high content screening combined with detection of pluripotency genes Oct4, Sox2 and Nanog

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.