目的 通過高內涵篩選技術評價測試藥對PA-1細胞的增殖毒性，并聯(lián)合分化毒性的評價指標——多能性基因（Oct4、Sox2和Nanog）表達檢測，初步建立藥物胚胎毒性快速篩查方法。方法 選擇維生素C、青霉素G、維生素E作為無胚胎毒性的陰性測試藥，甲氨蝶呤、5-氟尿嘧啶、環(huán)磷酰胺作為具有胚胎毒性的陽性測試藥。以測試藥臨床治療劑量下的最大血藥濃度（C_max）為基礎設置加藥濃度，陰性測試藥設置濃度梯度為：1/2C_max、C_max、2C_max、3C_max、4C_max；陽性測試藥設置濃度梯度為：1/8C_max、1/4C_max、1/2C_max、C_max、2C_max。測試藥物在維持培養(yǎng)液（培養(yǎng)液加入1×10⁶ U·L^-1的白血病抑制因子）中作用于PA-1細胞24 h后，基于高內涵篩選技術，應用DAPI、Calcein-AM、PI共染PA-1細胞，通過共聚焦成像檢測熒光強度，計算細胞存活率；測試藥物在分化培養(yǎng)液（不加入白血病抑制因子）中作用于PA-1細胞24 h后，實時熒光定量PCR（qRT-PCR）技術檢測多能性基因Oct4、Sox2和Nanog的表達變化。結果 DAPI、Calcein-AM、PI 3者共染可以很好地區(qū)分活細胞及死細胞；陰性測試藥維生素C、維生素E、青霉素G對PA-1細胞的增殖能力均無影響；與對照組比較，陽性測試藥5-氟尿嘧啶、甲氨蝶呤1/4C_max及以上濃度引起PA-1細胞存活率顯著下降（P＜0.05）；環(huán)磷酰胺1/8C_max及以上濃度引起PA-1細胞存活率顯著下降（P＜0.05）。陰性測試藥維生素C、維生素E、青霉素G對PA-1細胞多能性基因Sox2、Oct4、Nanog的表達均無影響。與對照組比較，陽性測試藥5-氟尿嘧啶1/8C_max及以上濃度使PA-1細胞多能性基因Sox2、Oct4、Nanog的表達顯著下降（P＜0.05）；甲氨蝶呤1/8C_max及以上濃度使Oct4、Nanog的表達顯著增加（P＜0.05），但當濃度達到2C_max時對Sox2的表達依然沒有影響；環(huán)磷酰胺1/8C_max及以上濃度使Sox2、Oct4、Nanog的表達均顯著增加（P＜0.05）。結論 高內涵篩選技術（DAPI、Calcein-AM、PI共染）聯(lián)合多能性基因的檢測可初步建立藥物胚胎毒性快速篩查方法。

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

國家自然科學基金資助項目（81573740）