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目的探讨线粒体乙醛脱氢酶2(mitochondrial aldehyde dehydrogenase 2,ALDH2)在高氧引起肺泡上皮细胞损伤中的作用。方法采用pcDNA3.1-ALDH2转染A549细胞增加ALDH2的表达,采用ALDH2 siRNA转染A549细胞抑制ALDH2的表达,细胞分组如下:①对照组(A549细胞),②高ALDH2表达组(A549细胞+pcDNA3.1-ALDH2),③低ALDH2表达组(A549细胞+ALDH2 siRNA),将3组细胞均置于常氧(95%空气+5%CO2)和高氧(95%O2+5%CO2)中培养72 h。ALDH2表达量检测采用Western blot,ALDH2活性通过在酶标仪上测定A340nm下NAD+转化为NADH的含量变化来得到,细胞脂质过氧化程度检测采用硫代巴比妥酸法测定丙二醛(malondialdehyde,MDA)含量进行,细胞损伤情况通过检测细胞培养液和细胞内乳酸脱氢酶活力来计算,细胞凋亡检测采用AnnexinⅤ-FITC及PI染色后流式细胞术进行,细胞死亡率检测采用台盼蓝染色法。结果转染pcDNA3.1-ALDH2后A549细胞中ALDH2表达显著增多(P<0.05),活性显著增强(P<0.05),转染ALDH2 siRNA后A549细胞中ALDH2的表达显著减少(P<0.05),活性显著减弱(P<0.05),高氧暴露可显著降低ALDH2的表达及活性(P<0.05)。转染pcDNA3.1-ALDH2显著减轻了A549细胞高氧暴露后的脂质过氧化和细胞损伤程度(P<0.05),降低了细胞凋亡率及死亡率(P<0.05);转染ALDH2 siRNA则显著加重了A549细胞高氧暴露后的脂质过氧化和细胞损伤程度(P<0.05),增加了细胞凋亡率及死亡率(P<0.05)。结论 ALDH2可以减轻高氧引起的肺泡上皮细胞损伤,其机制可能与ALDH2清除了细胞脂质过氧化产物有关。
Objective To investigate the role of mitochondrial aldehyde dehydrogenase 2 (ALDH2) in alveolar epithelial cell injury induced by hyperoxia. Methods A549 cells were transfected with pcDNA3.1-ALDH2 to increase ALDH2 expression. ALDH2 siRNA was used to transfect A549 cells to inhibit the expression of ALDH2. The cells were divided into the following groups: ① control group (A549 cells), ② high ALDH2 expression group (A549 cells + pcDNA3 ALDH2, ALDH2, ALDH2, ALDH2, ALDH2, ALDH2, ALDH2, ALDH2, ALDH2) Cultivation 72 h. The expression of ALDH2 was detected by Western blot. ALDH2 activity was determined by measuring the change of NAD + to NADH at A340nm on a microplate reader. The degree of lipid peroxidation was measured by thiobarbituric acid method. Malondialdehyde , MDA). The cell damage was calculated by measuring cell culture fluid and intracellular lactate dehydrogenase activity. Cell apoptosis was detected by flow cytometry with Annexin V-FITC and PI staining. The cell death rate was measured by MTT assay Blue staining. Results After transfected with pcDNA3.1-ALDH2, the expression of ALDH2 in A549 cells was significantly increased (P <0.05) and significantly increased (P <0.05). The expression of ALDH2 in A549 cells was significantly decreased (P <0.05) (P <0.05). Hyperoxia exposure significantly decreased ALDH2 expression and activity (P <0.05). Transfection of pcDNA3.1-ALDH2 significantly reduced lipid peroxidation and cell injury (P <0.05) and decreased the apoptosis rate and mortality of A549 cells after hyperoxia exposure (P <0.05). Transfection of ALDH2 siRNA Significantly increased the level of lipid peroxidation and cell injury (P <0.05) after A549 cells hyperoxia exposure, and increased the rate of apoptosis and mortality (P <0.05). Conclusion ALDH2 can reduce alveolar epithelial cell injury induced by hyperoxia. The mechanism may be related to ALDH2 clearance of lipid peroxidation products.