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目的探讨高氧状态下早产大鼠肺泡Ⅱ型上皮细胞(AEC2)中CCAAT增强子结合蛋白α(C/EBPα)和肺泡表面活性蛋白A(SP-A)、SP-B、SP-C、SP-D的表达及两者相关性。方法原代培养早产大鼠AEC2,利用950 mL/LO_2建立高氧细胞损伤模型,随机分为空气对照组和高氧实验组。分别于空气及高氧暴露后24、48、72 h,收获各组细胞。采用倒置相差显微镜观察各组细胞形态变化,实时荧光定量PCR及Western blot法分别检测C/EBPα和SP-A、SP-B、SP-C、SP-D mRNA及蛋白水平;CCK-8法检测细胞增殖。结果随培养时间延长,空气组细胞C/EBPαmRNA及蛋白表达逐渐降低,SP-A、SP-B、SP-C、SP-D mRNA和蛋白水平及AEC2增殖逐渐升高。高氧组细胞C/EBPα、SP-A、SP-B、SP-C、SP-D mRNA和蛋白水平及AEC2增殖呈先递增后递减趋势。与空气组相比,高氧组细胞C/EBPα、SP-A、SP-B、SP-C、SP-D mRNA和蛋白水平及AEC2增殖在高氧48 h明显增加。高氧组细胞C/EBPα蛋白水平与SP-A、SP-B、SP-C、SP-D蛋白水平及AEC2增殖呈正相关(r=0.96、0.98、0.92、0.97、0.90)。结论高氧暴露早期,C/EBPα可促进肺泡表面活性蛋白分泌,参与机体保护性调节作用,但随高氧暴露时间延长,丧失代偿保护作用。
Objective To investigate the expression of CCAAT enhancer binding protein α (C / EBPα) and alveolar surfactant protein A (SP-A), SP-B, SP-C and SP in premature rat alveolar type Ⅱ epithelial cells (AEC2) -D expression and the correlation between the two. Methods Primary cultured rat AEC2 cells were cultured in primary culture, and the model of hyperoxic cell injury was established with 950 mL / L LO 2. The models were randomly divided into air control group and hyperoxia group. The cells of each group were harvested 24, 48 and 72 h after air and hyperoxia exposure, respectively. The changes of cell morphology were observed by inverted phase contrast microscope. The mRNA and protein levels of C / EBPα and SP-A, SP-B, SP-C and SP-D were detected by real-time fluorescence quantitative PCR and Western blot respectively. Cell Proliferation. Results With the prolongation of culture time, the mRNA and protein expressions of C / EBPα in air group decreased gradually. The mRNA and protein levels of SP-A, SP-B, SP-C, SP-D and the proliferation of AEC2 increased gradually. The mRNA and protein levels of C / EBPα, SP-A, SP-B, SP-C, SP-D and the proliferation of AEC2 in the hyperoxia group increased first and then decreased. Compared with the air group, the mRNA and protein levels of C / EBPα, SP-A, SP-B, SP-C, SP-D and the proliferation of AEC2 cells in hyperoxia group increased significantly at 48 h of hyperoxia. The level of C / EBPα protein in hyperoxia group was positively correlated with the levels of SP-A, SP-B, SP-C, SP-D and AEC2 proliferation (r = 0.96,0.98,0.92,0.97,0.90). Conclusion C / EBPα can promote the secretion of surfactant protein in the early stage of hyperoxia exposure and play a protective role in the protection of the body. However, C / EBPα may lose its compensatory role as the hyperoxia exposure prolongs.