Over-expression of aldehyde dehydrogenase-2 protects against H2O2-induced oxidative damage and apoptosis in peripheral blood mononuclear cells  

作　　者：Xiu-ying HU Qin FANG Ji-shi WANG Jian-qiong XIE Bai-sheng CHAI Fang-qiong LI Xin CUI Yuan YANG 

机构地区：[1]Department of Hematology [2]Department of Pharmaceutics, the Affiliated Hospital of Guiyang Medical College, Guiyang 550004, China [3]School of Pharmaceutics, Guiyang Medical College, Guiyang 550004, China

出　　处：《Acta Pharmacologica Sinica》2011年第2期245-252,共8页中国药理学报（英文版）

基　　金：Acknowledgements This study was supported by the National Natural Science Foundation of China （No 30460127, 30760276, and 81070444）.

摘　　要：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）. 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 （H2O2）. 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. RTPCR 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 H2O2-Jnduced oxidative damage was higher in the ALDH2 gene-transfected group. Moreover, apoptosis rates in gene-transfected PBMCs incubated with 50 and 75 pmol/L H2O2 decreased by 7% and 6%, respectively. The generation of intracellular ROS was also markedly downregulated. Conclusion： ALDH2 gene transfection can protect PBMCs against H2O2-induced damage and attenuate apoptosis, accompanied with a downregulation of intracellular ROS. ALDH2 functions as a protector against oxidative stress.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）. 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 （H2O2）. 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. RTPCR 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 H2O2-Jnduced oxidative damage was higher in the ALDH2 gene-transfected group. Moreover, apoptosis rates in gene-transfected PBMCs incubated with 50 and 75 pmol/L H2O2 decreased by 7% and 6%, respectively. The generation of intracellular ROS was also markedly downregulated. Conclusion： ALDH2 gene transfection can protect PBMCs against H2O2-induced damage and attenuate apoptosis, accompanied with a downregulation of intracellular ROS. ALDH2 functions as a protector against oxidative stress.

关 键 词：aldehyde dehydrogenase-2 oxidative damage TRANSFECTION peripheral blood mononuclear cells APOPTOSIS 

分 类 号：R[医药卫生]