Enhancement or inhibition of PLC2 expression in rat hepatocytes by recombinant adenoviral vectors that contain full-length gene or siRNA

摘要

We investigated the effects of recombinant adenovirus vectors that overexpress or silence PLC2 on the expression of this gene during hepatocyte proliferation. Hepatocytes were isolated, identified by immunofluorescent cytochemical staining and infected by previously constructed Ad-PLC2 and Ad-PLC2 siRNA1, siRNA2 and siRNA3. Green fluorescent protein (GFP) expression was observed by fluorescence microscopy. Infection percentage was calculated by flow cytometry. mRNA and protein levels of PLC2 were detected by quantitative reverse transcription-PCR (qRT-PCR) and western blotting, respectively. The viability of the infected hepatocytes was measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. We found that nearly 97% of cells were positive for the hepatocyte marker, CK18. After infection of Ad-PLC2 and Ad-PLC2 siRNA, more than 99% of hepatocytes expressed GFP significantly, and mRNA and protein expression of PLC2 was up-regulated significantly in Ad-PLC2 infected hepatocytes, but down-regulated in Ad-PLC2 siRNA2 infected cells. The cell proliferation rate decreased in PLC2-overexpressing cells, while the rate increased in PLC2-silencing cells. We verified that recombinant Ad-PLC2 and Ad-PLC2 siRNA2 were constructed successfully. These two recombinant vectors promoted or decreased the expression of PLC2 in rat hepatocytes and affected the cell proliferation rate, which provides a useful tool for further investigation of the role of PLC2 in hepatocyte apoptosis.