CAJ | 학술논문

目的克隆人乙醛脱氢酶2(ALDH2)基因,研究ALDH2基因导入慢性粒细胞白血病细胞系K562细胞后对其增殖和抗氧化损伤的影响.方法从肝细胞中克隆人ALDH2基因,构建真核表达载体,用脂质体法将其导入K562细胞中,用RT-PCR和Western blot法检测ALD-H2基因的表达,锥虫蓝拒染法和MTT法检测转基因组细胞的增殖水平及对氧自由基引起的氧化损伤的反应;在此基础上,利用RT-PCR以及荧光分光光度法进一步检测氧自由基诱导后转基因组细胞中血红素加氧酶-1(HO-1)的表达和细胞内活性氧类(ROS)的产生.结果成功克隆人ALDH2基因并将构建的真核表达载体转染K562细胞,RT-PCR和Western blot法检测到ALDH2的高表达.锥虫蓝拒染法和MTT结果显示转基因组细胞增殖水平明显高于对照组(P＜0.05),经基因修饰的细胞对H2O2耐受性增高,H2O2的IC50值提高了7.8倍(IC50值分别为12.3μnol/L和1.4 μmol/L,P＜0.01).HO-1的表达和ROS的产生随H2O2浓度的增大而增加,而一定浓度H2O2诱导后HO-1的表达和ROS的产生在转基因组中显著低于对照组(P＜0.05).结论 ALDH2基因导人K562细胞后可增加对氧自由基引起的细胞损伤的耐受性,起到保护作用,该过程伴随着ROS水平以及HO-1表达的降低.
목적 극륭인을철탈경매2(ALDH2)기인,연구ALDH2기인도입만성립세포백혈병세포계K562세포후대기증식화항양화손상적영향.방법 종간세포중극륭인ALDH2기인,구건진핵표체재체,용지질체법장기도입K562세포중,용RT-PCR화Western blot법검측ALD-H2기인적표체,추충람거염법화MTT법검측전기인조세포적증식수평급대양자유기인기적양화손상적반응;재차기출상,이용RT-PCR이급형광분광광도법진일보검측양자유기유도후전기인조세포중혈홍소가양매-1(HO-1)적표체화세포내활성양류(ROS)적산생.결과 성공극륭인ALDH2기인병장구건적진핵표체재체전염K562세포,RT-PCR화Western blot법검측도ALDH2적고표체.추충람거염법화MTT결과현시전기인조세포증식수평명현고우대조조(P＜0.05),경기인수식적세포대H2O2내수성증고,H2O2적IC50치제고료7.8배(IC50치분별위12.3μnol/L화1.4 μmol/L,P＜0.01).HO-1적표체화ROS적산생수H2O2농도적증대이증가,이일정농도H2O2유도후HO-1적표체화ROS적산생재전기인조중현저저우대조조(P＜0.05).결론 ALDH2기인도인K562세포후가증가대양자유기인기적세포손상적내수성,기도보호작용,해과정반수착ROS수평이급HO-1표체적강저.
Objective To construct a eukaryotic expression vector containing aldehyde dehydrogenase-2 (ALDH2) gene and investigate the effects and its possible mechanisms of ALDH2 gene on cell proliferation and anti-oxidative damage in the K562 cells. Methods An eukaryotic expression vector containing the ALDH2 gene cloned from human hepatocytes was constructed and transfected into K562 cells by liposome.RT-PCR and Western blot were used to evaluate the expression of ALDH2. MTT assay was used to check the cell proliferation and trypan blue exclusion to check K562 cells damage induced by hydrogen peroxide (H2O2). RT-PCR and fluorescence spectrophotometry were used to determine the expression of heme oxygenase-1 (HO-1) and the generation of intracellular reactive oxygen species (ROS) respectively. Results RTPCR and Western blot analysis showed distinct higher ALDH2 protein expression in gene transfected group.The latter group had a higher cell proliferation (P＜0.05) and survival rate against H2O2 induced-oxidative damage, being increased by 7.8 times ( ICao was 12.3 μmol/L and 1.4 μmol/L for K562-pcDNA3.1-ALDH2 and control cells, respectively, P ＜0. 01 ). The HO-1 mRNA expression and the generation of intracellular ROS were downregulated at a specific concentration of H2O2 in the ALDH2 gene transfected group. Conclusion ALDH2 gene transfection can protect K562 cells against oxidative damage, and the downregulation of HO-1 expression and intracellular ROS may be involved in this process.