Expression of PDX1 in human umblical cord mesenchymal stem cells mediated by adenovirus vector / 中华内分泌外科杂志

ABSTRACT

ObjectiveTo construct recombinant adenovirus vector containing human pancreatic and duodenal homeobox factor 1 (PDX1) and detect its expression in human umblical cord mesenchymal stem cells (HUCMSCs). MethodsPDX1 obtained by BgⅢ/XhoI enzyme digestion from pUC57-PDX1 was ligated into the recombinant shuttle vector pShuttle-GFP-CMV to obtain the recombinant shuttle plasmid pShuttle-GFP-CMVPDX1. pShuttle-GFP-CMV- PDX1 was shifted to pAdxsi vector to obtain pAdxsi-GFP-PDX1 virus plasmid. The recombinant plasmid was packaged and amplified in 293 cells. The expression of PDX1 gene and protein in HUCMSCs was detected by fluorescence microscopy, RT-PCB, immunofluorescence, immunohistochemistry, and Western Blot. ResultsPDX1 gene was inserted correctly into shuttle plasmid and the recombinant adenovirus vector was successfully constructed according to the results of sequence and enzyme digestion identification. The adenovirus was effectively transfected into HUCMSCs. RT-PCR verified that PDX1 mRNA was positively expressed in HUCMSCs. Expression of PDX1 protein in the nuclear of HUCMSCs was found by immunofluorescence assay, immunohistochemistry and Western Blot. ConclusionThe adenovirus vector containing PDX1 gene is successfully constructed and effectively expressed in HUCMSCs.