Construction of recombinant adenovirus Ad-rat PLC gamma 2 and its effects on apoptosis of rat liver cell BRL-3A in vitro

摘要

Although the role of PLC gamma(2) in apoptotic response has been reported, too little is known about whether PLC.2 induces liver cell apoptosis during liver regeneration. Therefore, this study firstly packaged Ad-PLC gamma(2) 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-PLC gamma(2) 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 1x10(10) 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 PLC gamma(2) 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-PLC gamma(2) group were significantly increased (P<0.01), and the cell cycle in Ad-PLC gamma(2) 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) inthe percentage of S and G2/M phase cells. It was concluded from above results that recombinant adenovirus AdPLC.2 was packaged successfully, and could promote cell apoptosis by arresting the transition from G1 to S phase of BRL-3A cells.