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目的 建立FKBP12 /BAX双聚体可诱导细胞凋亡生物效应的细胞模型 ,并利用阳性药物对其进行鉴定。方法 RT PCR扩增鼠源性BAX全长基因 ,插入pUC18载体进行测序鉴定。将鉴定正确的mBAX插入载体pC4FV1E中 ,构建pC4FV1E/BAX表达质粒。将表达质粒与pcDNA3.1共转染HEK2 93细胞 ,建立pC4FV1E/BAX neo和neo基因稳定表达的细胞模型。利用形态学方法、流式细胞术等对化学药物诱导FKBP12 /BAX双聚体介导的细胞凋亡进行定性及定量的分析。结果 RT PCR、Westernblot法分别从mRNA及蛋白水平检测到FKBP12 mBAX融合基因的整合及表达 ;阳性化合物AP2 0 187作用 4～ 6h后 ,Giemsa染色可见明显的凋亡小体 ;钙依赖核酸内切酶亦被激活 ,出现典型的“DNAladder” ;天然产物FK5 0 6可竞争性地抑制这种阳性药物诱导的细胞凋亡。结论 FKBP12 /BAX双聚体可诱导细胞凋亡生物效应的细胞模型的建立 ,为FK5 0 6类小分子化合药物的高通量筛选提供可应用的技术平台。
OBJECTIVE: To establish a cell model of FKBP12 / BAX dimer that can induce the biological effects of apoptosis, and identify the cell model by positive drugs. Methods The full-length murine BAX gene was amplified by RT-PCR and inserted into pUC18 vector for sequencing. The correct mBAX was inserted into the vector pC4FV1E to construct pC4FV1E / BAX expression plasmid. The expression plasmid was co-transfected into HEK2 93 cells with pcDNA3.1 to establish a cell model stably expressing pC4FV1E / BAX neo and neo genes. Morphological methods, flow cytometry and other chemical drugs-induced FKBP12 / BAX dimer-mediated apoptosis were qualitative and quantitative analysis. Results The integration and expression of FKBP12 mBAX fusion gene were detected by RT-PCR and Western blotting respectively from the mRNA and protein levels. After treated with AP2 0 187 for 4 ~ 6 h, obvious apoptotic bodies were observed by Giemsa staining. Calcium-dependent endonuclease Is also activated, the typical “DNAladder” appears; natural product FK506 can competitively inhibit this positive drug-induced apoptosis. Conclusions FKBP12 / BAX dimer can induce the cell model of biological effects of apoptosis, and provide an applicable technology platform for the high-throughput screening of FK506 small molecule compounds.