白蛋白/细胞角蛋白19启动子调控的红绿双色荧光蛋白报告载体的构建及其在肝干细胞分化研究中的应用

Construction of DsRed/EGFP expression vectors driven by albumin/cytokeratin 19 promoters and their utilization in monitoring differentiation of liver progenitor cells

目的:利用报告基因监测肝干细胞的分化走向。方法:通过PCR从小鼠基因组中克隆了细胞角蛋白19 启动子片段,构建了细胞角蛋白19启动子调控的绿色荧光蛋白(pCK19 EGFP)和白蛋白启动子调控的红色荧光蛋白报告载体(pAlbD sRed),并利用报告载体标记的肝干细胞(liver epithelial progenitor cells,LEPCs)观察了悬浮培养时LEPCs的分化去向。结果:白蛋白/细胞角蛋白19启动子调控的红绿双色荧光蛋白报告载体可以实时地显示肝原始细胞在不同的诱导环境下的分化走向。结论:双色荧光蛋白报告载体的成功构建为研究LEPCs的分化、筛选可诱导LEPCs定向分化的分子提供了便捷的工具。

Objective:To construct the fluorescence protein expression vectors as reporters for the differentiation of liver epithelial progenitor cells(LEPCs). Methods: The cytokeratin 19 promoter was amplified by PCR from mouse tail genomic DNA and suncloned into promoter-removed pEGFP C1 vector. Albumin promoter was recovered from pGEMAlbSVPA and cloned into the promoterless vector pDsRed1 1. By using the reporter genes tagging cells, the phenotype variation of LEPCs cultured as aggregates was studied. Results: DsRed/EGFP expression vectors driven by albumin/cytokeratin 19 promoters could monitor the differentiation of LEPCs in a real-time manner. Conclusion: The reporter vectors are useful for studying the LEPCs differentiation and for evaluating differentiation-promoting agents.