人牙本质涎磷蛋白启动子驱动LacZ报告基因在人牙胚间充质细胞中表达

Expression of the reporter LacZ driven by human dentin sialophosphoprotein promoter in human dental mesenchymal cells

摘要:

牙本质涎磷蛋白 (DSPP) 的表达是细胞向成牙本质细胞分化的标志。试图分析人DSPP启动子及构建人DSPP启动子驱动的LacZ基因表达的报告体系，从而方便快捷检测细胞是否向成牙本质细胞分化。为了建立能表达DSPP的细胞体系，分离了人牙胚间充质细胞，并用地塞米松诱导培养液进行诱导，结果显示，该诱导培养液能有效地诱导人牙胚间充质细胞DSPP基因的表达。利用双荧光素酶报告系统对4段人DSPP基因5′上游区域 (?4 000?+54、?2 500?+54、?1 447?+54和?1 027?+54) 进行分析，结果显示?2 500?+54区域的启动子活性最高。5′上游区从-2 500 bp延长到-4 000 bp时，启动子活性下降；5′上游区从?2 500 bp缩短至?1 447 bp时，启动子活性下降；再次将?1 447 bp缩短至?1 027 bp时，启动子活性进一步下降。结果暗示在?4 000 bp至?2 500 bp区域存在转录抑制元件，?2 500 bp至?1 027 bp区域存在转录激活元件。用?2 500?+54启动子区域和LacZ基因构建phDSPP-LacZ慢病毒报告载体，并分别在人牙胚间充质细胞和永生化人牙胚间充质细胞系ihEDMC4上检测phDSPP-LacZ报告载体的功能，通过X-Gal染色，结果显示在2种细胞牙向分化过程中均可检测到LacZ基因的表达。研究构建的phDSPP-LacZ慢病毒报告载体可为诱导人源细胞牙向分化、牙齿发育、牙齿再生工程等研究中DSPP的表达检测提供一种更加便捷的手段。

Abstract:

The expression of dentin sialophosphoprotein (DSPP) is the marker for cells differentiated into odontoblasts. This study attempted to analyze the DSPP promoter and build the reporter LacZ expression system driven by this promoter, which allows convenient and quick detection of odontoblast cells. First, we separated the human dental mesenchymal cells in which the expression of DSPP can be effectively induced by dexamethasone. Second, four 5′ flanking regions of human DSPP gene (? 4 000?+54, ?2 500?+54, ?1 447?+54 and ?1 027?+54) were analyzed, the results showed that the highest promoter activity lied in the ?2 500?+54 region. The promoter activity is reduced when the 5′ flanking region was extended from ?2 500 to ?4 000 bp upstream from the transcription start site; The promoter activity are also decreased when the 5′ flanking regions were shorted from ?2 500 to ?1 447 bp and from ?1 447 to ?1 027 bp, indicating that potential suppresser elements are lied in the region between ?4 000 and ?2 500 bp and potential activator elements are lied in the region between ?2 500 and ?1 027 bp. Then we constructed the lentiviral report vector phDSPP-LacZ containing the ?2 500?+ 54 promoter region in front of the LacZ gene. The expression of LacZ was detected using X-Gal staining in both human dental mesenchymal cells and immortalized human dental mesenchymal cells infected with phDSPP-LacZ. The phDSPP-LacZ lentiviral vector may provide a more convenient method to detect the expression of DSPP in human odontogenic differentiation, tooth development and tooth regeneration studies.