组织特异性启动子驱动荧光报告基因质粒转染hES建立药物胚胎毒性体外检测体系的研究

Drug embryotoxicity refers to the toxic effects of drugs on the embryo, and it was traditionally detected by in vivo tests using animals. In vivo tests are mostly time-consuming, difficult in making models, easily influenced by many factors, expensive and require large numbers of animals. The embryonic stem cell test (EST) is an in vitro alternative embryotoxicity tests and was established in 1997 by Spielmann et al. Two permanent murine cell lines (ES cells and 3T3 fibroblasts) were used in the EST to perform cytotoxicity assay and differentiation assay. Without sacrificing any animals, the EST can classify the embryotoxicity of compounds ,and it is currently the most promising in vitro alternative to the in vivo animal tests.However, there is species differences in the embryotoxicity of drugs, and there is a huge genetic distance between rodent and human, the test results of the mouse EST system may not be applicable to humans. In previous study, we have used cynomolgus monkey ES cells (cmES) and fibroblasts to initially establish primate EST systems, at the same time, we efficiently genetic modified cmES with lentiviral vectors by multisite gateway technique. Here, we intend to construct modular lentivectors containing the constitutive promoter of three different tissue-specific promoters (Tα1 of α-tubulin, AFP of alpha fetoprotein, α-MHC of alpha-myosin heavy chain) driving transcription of the hrGFP (humanized Renilla GFP) gene. The vectors are based on self-inactivating HIV-1 lentivirus and are used to transfected into H1 cells. After selected with puromysin, purified Ta1-hrGFP/ AFP-hrGFP/α-MHC-hrGFP H1 will be obtained. hEST system can be established with Ta1-hrGFP/AFP-hrGFP/α-MHC-hrGFP H1 and human embryonic fibroblasts. Tissue- specific embryotoxicity of drugs can classified with prediction model separately. The system will provide a powerful research tool to accurately detect human tissue-specific embryotoxicity drugs.

药物胚胎毒性是指药物对胚胎的毒害作用，传统的动物实验方法存在着耗时长、消费大等弊端。胚胎干细胞试验法（EST）是采用小鼠ES和成纤维细胞建立的胚胎毒性体外检测方法，是目前最理想的体外检测方法。由于药物胚胎毒性存在种属差异，而啮齿类动物与人类遗传距离较远，小鼠EST体系的检测结果未必适用于人类。申请人前期已利用食蟹猴细胞初步建立灵长类EST体系，并利用gateway技术成功转染食蟹猴ESC。本项目拟进一步采用gateway技术构建神经细胞Tα1、肝细胞AFP以及心肌细胞α-MHC驱动的hrGFP质粒，并转染hES细胞系H1，得到Tα1-hrGFP H1、AFP-hrGFP H1和α-MHC-hrGFP H1，将其与人胚胎成纤维细胞一起建立hEST体系，根据EST体系的预测模型可以将药物对不同组织的胚胎毒性进行分类。该体系的建立将为准确检测药物对人类组织特异性的胚胎毒性提供强有力的研究工具。

药物胚胎毒性是指药物对胚胎的毒害作用，其分级直接影响药物能否应用于临床。胚胎毒性传统的检测方法是采用动物实验，但由于其耗时长、花费大以及牺牲大量动物等弊端，不能满足新药研发的需要。胚胎干细胞试验法（EST）是采用两种小鼠永生细胞系胚胎干细胞和胚胎成纤维细胞3T3，进行两种细胞的细胞毒性实验以及胚胎干细胞的分化抑制实验。并根据预测模型对胚胎毒性进行分类。在众多体外替代实验中，由于不需要牺牲动物，是目前为止最理想的体外检测方法。由于药物胚胎毒性存在种属差异，而啮齿类动物与人类遗传距离较远，小鼠EST 体系的检测结果未必适用于人类，因此采用人类细胞建立EST体系尤为重要。本项目在以前的研究基础上，采用了gateway 技术成功构建神经细胞启动子Tα1、肝细胞AFP启动子 以及心肌细胞α-MHC 启动子驱动的hrGFP 质粒，并转染hES 细胞系H1，经过抗性药物的筛选后，得到了Tα1-hrGFP H1、AFP-hrGF PH1 和α-MHC-hrGFP H1。对转染得到的H1细胞进行了干细胞生物学标记及其全能性进行了鉴定，发现转染后的H1保持了人胚胎干细胞的生物学特性。将Tα1-hrGFP H1、AFP-hrGF PH1 和α-MHC-hrGFP H1与人胚胎成纤维细胞（HEF）一起建立hEST 体系。共选取了胚胎毒性已得到验证的六种不同胚胎毒性的药物，采用建立的hEST 体系进行HEF和H1的细胞毒性实验以及Tα1-hrGFP H1、AFP-hrGF PH1 和α-MHC-hrGFP H1的分化抑制实验，得到每个实验的反应终点，包括IC50HEF、IC50H1、ID50Neuro、ID50Liver和ID50Cardio。将这些数值带入EST的预测模型计算三个公式值的大小，并进行比较，从而成功对胚胎毒性进行了分级。结果发现建立的hEST体系对药物的胚胎毒性分级与体内实验胚胎毒性分级一致。这说明本研究建立的hEST体系可以准确地对药物的胚胎毒性进行分级，并排除了种属间的差异，可用于新药研发，同时可以指导临床用药，具有一定的科学意义和应用价值。