Aim
To construct an eukaryotic expression vector containing the aldehyde dehydrogenase-2 (ALDH2) gene, and determine whether transfection with the ALDH2 gene can provide protection against hydrogen peroxide-induced oxidative damage, as well as attenuate apoptosis or cell death in human peripheral blood mononuclear cells (PBMCs).
Conclusion
ALDH2 gene transfection can protect PBMCs against H&sub2;O&sub2;-induced damage and attenuate apoptosis, accompanied with a downregulation of intracellular ROS. ALDH2 functions as a protector against oxidative stress.
Methods
The ALDH2 gene was cloned from human hepatocytes by RT-PCR. The eukaryotic expression vector containing the gene was constructed and then transfected into PBMCs via liposomes. RT-PCR, indirect immunofluorescence assay, and Western blot were used to evaluate the expression of the transgene in target cells. MTT assay and flow cytometry were used to detect the effects of ALDH2 on PBMCs damaged by hydrogen peroxide (H&sub2;O&sub2;). The level of intracellular reactive oxygen species (ROS) was determined by fluorescence spectrophotometry.
Results
The eukaryotic expression vector pcDNA3.1/myc-His-ALDH2 was successfully constructed and transfected into PBMCs. RT-PCR results showed higher mRNA expression of ALDH2 in the gene-transfected group than in the two control groups (empty vector-transfected group and negative control). Indirect immunofluorescence assay and Western blot indicated distinct higher protein expression of ALDH2 in the gene-transfected group. The cell survival rate against H&sub2;O&sub2;-induced oxidative damage was higher in the ALDH2 gene-transfected group. Moreover, apoptosis rates in gene-transfected PBMCs incubated with 50 and 75 μmol/L H&sub2;O&sub2; decreased by 7% and 6%, respectively. The generation of intracellular ROS was also markedly downregulated.