OBJECTIVETo investigate the effects of adenoviral vector-mediated over-expression of Wnt3 on the apoptosis of hepatic progenitor cells in vitro.

METHODSHepatic progenitor cells transfected with Ad-GFP-Wnt3 vector or the control vector Ad-GFP were examined for cell apoptosis under fluorescence microscopy with Hoechst 33342 staining, and the proportion of apoptotic cells were determined by flow cytometric analysis with Annexin-PE/7-ADD staining. The mRNA and protein expressions of Bax, Bcl-2 and Bcl-xl in the cells were detected by real-time PCR and Western blotting, respectively.

RESULTSReal-time PCR and Western blotting showed a high expression of Wnt3 in Ad-GFP-Wnt3-transfected hepatic progenitor cells, which exhibited significantly decreased cell apoptosis as compared with the control group. The expressions of Bcl-2 and Bcl-xl mRNA and proteins increased significantly while Bax expression decreased obviously in Ad-GFP-Wnt3-transfected cells (P<0.05).

CONCLUSIONSAdenoviral vector-mediated over-expression of Wnt3 can suppress apoptosis of hepatic progenitor cells possibly through the Bcl-2 pathway.

Apoptosis ; Cells, Cultured ; Genetic Vectors ; Hepatocytes ; cytology ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stem Cells ; cytology ; Transfection ; Wnt3 Protein ; metabolism ; bcl-X Protein ; metabolism

In this study, we aimed to construct a non-replication mRNA platform and explore the side effects of electroporation-mediated delivery of mRNA on the mice as well as the expression features of the mRNA. With luciferase gene as a marker, in vitro transcription with T7 RNA polymerase was carried out for the synthesis of luciferase-expressed mRNA, followed by enzymatic capping and tailing. The mRNA was delivered in vivo by electroporation via an in vivo gene delivery system, and the expression intensity and duration of luciferase in mice were observed via an in vivo imaging system. The results demonstrated that the mRNA transcripts were successfully expressed both in vitro and in vivo. The electroporation-mediated delivery of mRNA had no obvious side effects on the mice. Luciferase was expressed successfully in all the mRNA-transduced mice, while the expression intensity and duration varied among individuals. Overall, the expression level peaked on the first day after electroporation and rapidly declined on the fourth day. This study is of great importance for the construction of non-replication mRNAs and their application in vaccine or antitumor drug development.
Animals ; Electroporation/methods* ; Gene Transfer Techniques ; Luciferases/metabolism* ; Mice ; RNA, Messenger/genetics*