动物造模 z-bio 2022-03-09 482

　　Objective: To investigate the regulatory effects of nuclear transcription factors TRB, RXR and agonist T4 on Cip4 gene expression in gerbils.

　　Methods: The sequence of the promoter region of the Cip4 gene of gerbils was synthesized, and the reporter vector pGL3-Cip4 was constructed; the transcription factors TRB and RXR were cloned into the pcDNA3.1 expression vector. The pGL3-Cip4, pcDNA3.1-TRB, pcDNA3.1 -RXR and pRL-TK (reference plasmid) vectors were co-transfected into HEK-293 cells to construct a luciferase dual reporter system for Cip4 gene. The effects of plasmid dosage and TRB agonist thyroxine T4 on transcriptional activity were observed.

　　Results: The dual-luciferase reporter gene system using the Cip4 promoter region of gerbils as the promoter sequence had the highest transfection efficiency when (promoter + transcription factor): pRL-TK=20∶1 and the total plasmid amount was 2μg. And has the highest luciferase activity. The test results show that adding TRB alone does not change the transcription level of Cip4 (P>0.05), only adding RXR can increase the transcription level of Cip4 (P<0.05), and the combined action of RXR and T4 can up-regulate Transcription level of Cip4 (P<0.001). The co-action of TRB and T4 could down-regulate the activity of Cip4 (P<0.05). When RXR, TRB and T4 coexisted, the transcription level of Cip4 was significantly down-regulated (P<0.01).

　　Conclusion: This study confirmed that T4, TRB and RXR co-regulated the transcription of Cip4 gene in gerbils, mimicked the activation of the transcription of target gene Cip4 by the ligand-activated transcription factor TRB/RXR, and confirmed the transcriptional activation of Cip4 and RXR signaling. The pathways are closely related, which lays the foundation for revealing the mechanism of thyroxine in human NAFLD. This reporter gene system can be used for the mechanism analysis of ligand-activated transcription factor regulation of Cip4 gene expression and the screening of related drugs.