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目的探讨抗氧化酶Prdx6保护肺泡Ⅱ型上皮细胞(AECⅡ)抵抗过氧化氢(H2O2)诱导的细胞凋亡的作用。方法 AECⅡ分离纯化于野生小鼠(WT组)和Prdx6转基因过表达小鼠(Prdx6 TG组),并暴露在不同浓度(50～500μmol.L-1)H2O2下24 h。Nile Red染色用于鉴定AECⅡ;免疫印迹法探测Prdx6蛋白表达;应用钙黄绿素乙酰甲酯和乙啶二聚物染色法研究细胞生存率;应用膜联蛋白A(AV)和碘化丙啶(PI)染色法研究细胞凋亡情况。结果 Prdx6 TG组AECⅡPrdx6蛋白表达量是WT组的7倍,同时,H2O2可剂量相关性地诱导AECⅡPrdx6蛋白表达;AECⅡ生存率和H2O2呈剂量负相关,Prdx6 TG组细胞生存率显著高于WT组(P<0.05);AV和PI荧光染色显示细胞凋亡的程度H2O2治疗浓度呈正相关,同时,Prdx6 TG组细胞凋亡阳性率明显低于WT组(P<0.05)。结论抗氧化酶Prdx6可以保护AECⅡ抵抗H2O2诱导的细胞凋亡,是其作为抗氧化酶的主要功能之一。
Objective To investigate the protective effect of Prdx6 against apoptosis of alveolar type Ⅱ epithelial cells (AECⅡ) induced by hydrogen peroxide (H2O2). Methods AEC Ⅱ was isolated and purified from wild-type mice (WT) and Prdx6 transgenic mice (Prdx6 TG) and exposed to H 2 O 2 at different concentrations (50-500 μmol·L -1) for 24 h. Nile Red staining was used to identify AECⅡ; Immunoblotting was used to detect Prdx6 protein expression; Calcein acetylmethylacrylate and ethidium dyestuff staining were used to study the cell survival rate; Annexin A (AV) and Propidium Iodide (PI) ) Staining study of apoptosis. Results The expression of AECⅡPrdx6 in Prdx6 TG group was 7-fold higher than that in WT group. At the same time, H2O2 induced AECⅡPrdx6 protein expression in a dose-dependent manner. The survival rate of AECⅡwas negatively correlated with the dosage of H2O2, while the survival rate of Prdx6 TG group was significantly higher than that of WT group P <0.05); AV and PI staining showed that the degree of apoptosis was positively correlated with the concentration of H2O2, while the positive rate of apoptosis in Prdx6 TG group was significantly lower than that in WT group (P <0.05). Conclusion Antioxidant enzyme Prdx6 can protect AECⅡ against H2O2-induced apoptosis as one of the main functions of antioxidant enzymes.