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目的构建HSP70启动子与绿色荧光蛋白重组真核表达载体,并在人Chang’s肝细胞表达,建立一种新的体外药物肝毒性早期预测细胞模型。方法提取人Chang’s肝细胞基因组DNA,钓取HSP70启动子基因,构建重组载体,经脂质体转染人Chang’s肝细胞,用G418筛选稳定转染细胞,用荧光显微镜观察绿色荧光表达情况,用实时荧光定量PCR检测EGFP基因表达量的变化。结果成功钓取HSP70启动子基因并导入真核表达载体pEGFP-N1;G418以300 ng/mL的终浓度筛选出稳定转染的单克隆细胞;荧光显微镜观察到肝毒性药物酮康唑刺激人Chang’s肝细胞后,可诱导HSP70启动子调控增强型绿色荧光蛋白发出绿色荧光,药物刺激组EGFP mRNA相对表达量与正常对照组比较有统计学意义(t=-14.21,P<0.05)。结论成功构建HSP70启动子与绿色荧光蛋白共表达真核表达载体,获得稳定转染单克隆细胞,并证实HSP70在肝毒性药物刺激下的应激表达,为建立新的体外药物肝毒性早期预测细胞模型进行高通量筛选新药奠定了基础。
OBJECTIVE: To construct a recombinant eukaryotic expression vector of HSP70 promoter and green fluorescent protein (EGFP) and express in human Chang’s hepatocytes to establish a novel in vitro model of hepatotoxicity. Methods Genomic DNA of human Chang’s hepatocytes was extracted and the HSP70 promoter was obtained. The recombinant vector was constructed and transfected into human Chang’s hepatocytes by lipofectamine 2000. The transfected cells were stably transfected with G418. The expression of green fluorescence was observed by fluorescence microscopy. Fluorescent quantitative PCR detection of EGFP gene expression changes. Results The gene of HSP70 promoter was successfully obtained and introduced into the eukaryotic expression vector pEGFP-N1. Stably transfected monoclonal cells were screened by G418 at a final concentration of 300 ng / mL. Fluorescence microscopy showed that the hepatotoxicity drug ketoconazole stimulated Chang’s The expression of EGFP mRNA in hepatocytes stimulated by HSP70 promoter enhanced the green fluorescent protein (EGFP) expression. Compared with the normal control group, the relative expression of EGFP mRNA in the drug-stimulated group was statistically significant (t = -14.21, P <0.05). Conclusions The co-expression of HSP70 promoter and green fluorescent protein (EGFP) in eukaryotic expression vector was successfully constructed, and stable transfected monoclonal cells were obtained. The expression of HSP70 was confirmed under the stimulation of hepatotoxic drugs. In order to establish a new in vitro hepatotoxicity assay, The model lays the foundation for high-throughput screening of new drugs.