高氧导致20S蛋白酶体活性降低诱导早产大鼠Ⅱ型肺泡上皮细胞凋亡  

Hyperoxia induces AECⅡ apoptosis of premature rats by reducing the activity of 20S proteasome

作　　者：张燕雨 卢红艳[1] 鞠慧敏[1] 常明[1] 王秋霞[1] 张强[1] 

机构地区：[1]江苏大学附属医院儿科,江苏镇江212000

出　　处：《细胞与分子免疫学杂志》2016年第12期1600-1604,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81370746);镇江市社会发展项目(SH2015071)

摘　　要：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 hyperoxia-exposed cell damage model of primary cultured premature rat alveolar type Ⅱ epithelial cells （AEC H s） and investigate the effect of hyperoxia on ubiquitinated protein degradation and apoptosis of AEC Ⅱs, Methods Primary cultured premature rat AEC Ⅱ s in vitro were divided into air group and hyperoxia group, which were exposed to air and 950 mL/L O2, respectively. After 24-, 48- and 72-hour exposure, isothiocyanate-labeled annexinV/ propidium iodide （annexinV-FITC/PI） double staining combined with flow cytometry was utilized to detect the apoptosis of AEC Ⅱ s. Real-time quantitative PCR and Western blotting were performed to observe the mRNA expression of ubiquitin and the protein levels of total ubiquitinated proteins, Bax/Bcl-2 ratio and caspase-3. Specific fluorescence substrate was used to measure the activity of 20S proteasome. Results Compared with the air group, the apoptosis of AEC Ⅱ s in the hyperoxia group increased at each time point. The activity of 20S proteasome inAEC Ⅱ s in the hyperoxia group decreased dramatically, while the levels of ubiquitin mRNA and total ubiquitinated protein increased. At the same time, the expression of caspase-3 and the Bax/Bcl-2 ratio were significantly raised. Conclusion Exposure to hyperoxia decreases the activity of 20S proteasome in AEC Ⅱ s and the degradation of ubiquitinated proteins, which may induce the apoptosis of AEC Ⅱs by upregulating the expression of caspase-3 and the Bax/Bcl-2 ratio.

关 键 词：泛素化蛋白 20S蛋白酶体 高氧 凋亡 Ⅱ型肺泡上皮细胞 

分 类 号：R392.33[医药卫生—免疫学] Q255[医药卫生—基础医学]