Construction of recombinant adenovirus Ad-rat PLCγ2 and its effects on apoptosis of rat liver cell BRL-3A in vitro

Abstract

Although the role of PLCγ2 in apoptotic response has been reported, too little is known about whether PLCγ2 induces liver cell apoptosis during liver regeneration. Therefore, this study firstly packaged Ad-PLCγ2 recombinant adenovirus and primarily evaluated its effect on apoptosis of rat liver cell BRL-3A in vitro. Following ten days of co-transfection of pHBAd-MCMV-GFP-PLCγ2 and pHBAd-BHG into HEK293 cells, viral cytopathic effect (CPE) was apparent. Following three rounds of amplification, tissue culture infectious dose 50 (TCID50) assay showed that the titer value reached 1í—1010 PFU/mL. After 24 h of transfection of recombinant adenovirus into BRL-3A cells, transfection efficiency of adenovirus into BRL-3A cells was above 90% when obsereved under fluorescent microscopy. qRT-PCR and Western blot assays showed mRNA and protein levels of PLCγ2 were significantly elevated in the transfected BRL-3A cells. Flow cytometric analysis showed that, compared with the control and Ad-GFP groups, cell apoptosis rate of Ad-PLCγ2 group were significantly increased (P<0.01), and the cell cycle in Ad-PLCγ2 group was arrested at G1 phase which was manifested by a marked increase (P<0.01) in the percentage of G1 phase cells and a great decrease (P<0.01) in the percentage of S and G2/M phase cells. It was concluded from above results that recombinant adenovirus Ad-PLCγ2 was packaged successfully, and could promote cell apoptosis by arresting the transition from G1 to S phase of BRL-3A cells.