Department of Hematology, the Affiliated Hospital of Guiyang Medical College, Guiyang, China.
Acta Pharmacol Sin. 2011 Feb;32(2):245-52. doi: 10.1038/aps.2010.203.
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).
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.
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 H₂O₂-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 H₂O₂ decreased by 7% and 6%, respectively. The generation of intracellular ROS was also markedly downregulated.
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.
构建含醛脱氢酶-2（ALDH2）基因的真核表达载体，观察转染 ALDH2 基因能否对过氧化氢（H₂O₂）诱导的人外周血单个核细胞（PBMCs）氧化损伤起保护作用，以及减轻 PBMCs 凋亡或死亡。
用 RT-PCR 从人肝细胞中克隆 ALDH2 基因，构建真核表达载体，并通过脂质体转染入 PBMCs。用 RT-PCR、间接免疫荧光和 Western blot 检测目的基因在靶细胞中的表达。MTT 法和流式细胞术检测 ALDH2 对 H₂O₂损伤 PBMCs 的影响。荧光分光光度法检测细胞内活性氧（ROS）水平。
成功构建真核表达载体 pcDNA3.1/myc-His-ALDH2，并转染入 PBMCs。RT-PCR 结果显示，基因转染组 ALDH2 mRNA 表达明显高于空载体转染组和阴性对照组。间接免疫荧光和 Western blot 结果显示，基因转染组 ALDH2 蛋白表达明显增高。ALDH2 基因转染组对 H₂O₂诱导的氧化损伤具有更高的细胞存活率。此外，基因转染 PBMCs 在孵育 50 和 75 μmol/L H₂O₂时，凋亡率分别下降 7%和 6%。细胞内 ROS 的产生也明显下调。
ALDH2 基因转染可减轻 H₂O₂诱导的 PBMCs 损伤和凋亡，下调细胞内 ROS。ALDH2 具有抗氧化应激作用。
