论文部分内容阅读
目的研究组蛋白H2AX在高体积分数氧(高氧)暴露肺泡Ⅱ型上皮细胞(AECⅡ)中的动态表达,探讨其在高氧致肺细胞损伤中的作用。方法以原代培养的AECⅡ为研究对象,随机分为高氧组和空气组。在实验开始后6 h、12 h、24 h、48 h收集细胞,运用免疫荧光、Western blot及实时荧光定量PCR技术检测AECⅡ中H2AX的表达规律。结果高氧组H2AX蛋白平均荧光强度在实验开始后6 h、12 h均明显高于空气组(Pa<0.05),24 h与空气组比较差异无统计学意义(P>0.05),48 h明显低于空气组(P<0.05)。高氧组H2AX蛋白平均荧光强度随着高氧暴露时间延长渐下降(F=62.7,P<0.01),空气组无明显变化(F=0.3,P>0.05)。H2AX蛋白相对表达量和H2AX mRNA相对表达量的变化趋势与免疫荧光法结果基本相同,高氧暴露6 h后明显升高,随高氧暴露时间延长逐渐降低(F=126.6、244.4,Pa<0.01)。结论在高氧暴露早期,H2AX表达明显升高,随着高氧暴露时间的延长H2AX逐渐降低,这可能造成AECⅡ中DNA双链断裂修复障碍,导致细胞凋亡和坏死,提示H2AX可能与肺损伤密切相关。
Objective To investigate the dynamic expression of histone H2AX in alveolar type Ⅱ epithelial cells (AECⅡ) exposed to high volume fraction of oxygen (hyperoxia), and to explore its role in hyperoxia-induced lung injury. Methods AEC Ⅱ cultured in primary culture was randomly divided into hyperoxia group and air group. The cells were harvested at 6 h, 12 h, 24 h and 48 h after the start of the experiment, and the expression of H2AX in AECⅡ was detected by immunofluorescence, Western blot and real-time fluorescence quantitative PCR. Results The average fluorescence intensity of H2AX protein in hyperoxia group was significantly higher than that in air group at 6 h and 12 h (P <0.05), but no significant difference at 24 h and air group (P> 0.05) Lower than air group (P <0.05). The mean fluorescence intensity of H2AX decreased gradually with the time of hyperoxia exposure (F = 62.7, P <0.01) and no significant change in air group (F = 0.3, P> 0.05). The trend of relative expression of H2AX protein and relative expression level of H2AX mRNA was basically the same as that of immunofluorescence. After 6 h of hyperoxia exposure, the expression of H2AX protein increased significantly and decreased gradually with the prolongation of hyperoxia exposure (F = 126.6,244.4, Pa <0.01) ). Conclusions The expression of H2AX was significantly increased in the early stage of hyperoxia exposure. H2AX gradually decreased with the prolongation of hyperoxia exposure, which may result in the repair of DNA double-strand breaks in AECⅡ, leading to apoptosis and necrosis. It suggests that H2AX may be associated with lung injury closely related.