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  

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

作　　者：鲁娣 宋殿荣[1] 张崴[1] 郭洁[1] 王雅楠[1] 杜文欣[1] LU Di;SONG Dianrong;ZHANG Wei;GUO Jie;WANG Yanan;DU Wenxin(Department of Gynecology,Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine,Tianjin 300150,China)

机构地区：[1]天津中医药大学第二附属医院妇科,天津300150

出　　处：《药物评价研究》2022年第9期1786-1794,共9页Drug Evaluation Research

基　　金：国家自然科学基金资助项目(81573740)。

摘　　要：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1×10^(6) 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 1×10^(6) 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 c

关 键 词：胚胎毒性 高内涵 多能性基因 方法建立 增殖毒性 

分 类 号：R965.2[医药卫生—药理学]