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目的建立早产大鼠肺泡Ⅱ型上皮细胞(AECⅡ)原代培养高氧细胞损伤模型,研究高氧对泛素化蛋白的降解及AECⅡ凋亡的影响。方法早产大鼠原代AECⅡ体外培养,利用950 m L/L O2建立高氧细胞损伤模型并随机分为空气组和高氧组。空气或高氧暴露24、48、72 h后,采用异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双染色结合流式细胞术检测细胞凋亡,实时定量PCR检测泛素mRNA表达,Western blot法检测细胞内总泛素化蛋白及凋亡相关蛋白Bax/Bcl-2比值和caspase-3表达,利用特异性荧光底物检测20S蛋白酶体活性。结果与同时间点空气组相比,各时间点高氧组AECⅡ凋亡增加,AECⅡ中20S蛋白酶体活性降低,泛素mRNA和总泛素化蛋白表达增加,同时Bax/Bcl-2比值和caspase-3表达明显上调。结论高氧暴露引起AECⅡ中20S蛋白酶体活性降低,泛素化蛋白降解减少,通过上调Bax/Bcl-2比值和caspase-3表达诱导AECⅡ凋亡。
Objective To establish a primary culture model of hyperoxic cell injury of preterm rat alveolar type Ⅱ epithelial cells (AEC Ⅱ) and study the effect of hyperoxia on the degradation of ubiquitinated protein and the apoptosis of AEC Ⅱ. Methods The primary AECⅡ rat preterm model was cultured in vitro. The model of hypoxic cell injury was established with 950 m L / L O2 and randomly divided into air group and hyperoxia group. After 24, 48 and 72 h of exposure to air or hyperoxia, apoptosis was detected by double staining with annexin V-FITC / PI and labeled with fluorescein isothiocyanate (FITC) The expression of ubiquitin mRNA was detected by real-time quantitative PCR, the ratio of Bcl-2 / Bcl-2 and the expression of caspase-3 were detected by Western blot, and the activity of 20S proteasome was detected by specific fluorogenic substrate. Results Compared with air group at the same time point, the apoptosis of AECⅡ in hyperoxia group was increased at each time point. The activity of 20S proteasome in AECⅡwas decreased, the expression of ubiquitin mRNA and total ubiquitinated protein increased, while the ratio of Bax / Bcl-2 and caspase -3 expression was significantly increased. CONCLUSION: Exposure to hyperoxia reduces the activity of 20S proteasome and decreases the degradation of ubiquitinated protein in AECⅡ. Apoptosis of AECⅡwas induced by up-regulating Bax / Bcl-2 ratio and caspase-3 expression.