FKBP12/BAX双聚体诱导细胞凋亡模型的建立

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HEK293细胞,建立pCAFV1E／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、Westem blot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AP20187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“DNA ladder”;天然产物FK506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FK506类小分子化合药物的高通量筛选提供可应用的技术平台。

Establishment and identification of a chemically inducible dimerization model for high throughout screening of drug

Objective To establish and identify an inducible apoptosis cell model for high throughout drug screening. Methods Full-length Bax gene amplified from 3T3 cell lines was determined by sequence analysis followed by cloning into pC4FV1E.The resulting recombinant plasmid pC4FV1E/BAX was then introduced into HEK293 by Lipofectin. Cell clones stably expressing FKBP12-mBAX fusion proteins were characterized by RT-PCR and Western blot. The effect of inducible apoptosis on cell clones by AP20187 was analyzed by morphological and flow cytometric detection. Above all, competition assays proved the feasibility of such a cell model as a technique for high throughout drug screening. Results The integration and expression of FKBP12-mBAX fusion gene were identified by RT-PCR and Western blot respectively. Giemsa staining showed apoptosis bodies after induced by AP20187 for 4-6 hours. On the other hand, the detection of typical "DNA ladder" proved the activation of CAD(caspase-activated DNase). FK506 could competitively block such cell apoptosis induced by AP20187. Conclusion The inducible apoptosis cell model was successfully established, which offered an applicable technical platform for the high throughout screening of FK506-like small molecular compounds.