摘要
目的 探讨不同时间高浓度氧对肺泡Ⅱ型细胞(AECⅡ)内质网自噬与肺泡表面活性物质产生功能之间的关系.方法 将大鼠RLE-6TN细胞分为对照组(C组,21％O2常规培养72 h)、高氧H1、H2、H3、H4组(95％O2分别培养12、24、48、72 h).实时荧光定量PCR法检测内质网自噬受体ATL3、CCPG1、FAM134B、RTN3、SEC62的mRNA水平,Western blot法检测内质网自噬相关蛋白ATL3、CCPG1、FAM134B、RTN3、SEC62的水平,ELISA法检测各组二棕榈酰卵磷脂(DPPC)水平.结果 与C组相比,H1组ATL3、SEC62的mRNA水平、CCPG1的蛋白水平、DPPC 产量下降(P＜0.05),CCPG1、FAM134B、RTN3 的 mRNA 水平、ATL3、FAM134B、RTN3、SEC62 的蛋白水平显著下降(P 均＜0.01);H2 组 ATL3、CCPG1、FAM134B、RTN3、SEC62 的 mRNA 水平、ATL3、CCPG1、FAM134B、RTN3、SEC62的蛋白水平差异无统计学意义(P均＞0.05),DPPC产量增高(P＜0.05);H3、H4组ATL3、CCPG1、FAM134B、RTN3、SEC62 的 mRNA 水平、ATL3、CCPG1、FAM134B、RTN3、SEC62 的蛋白水平、DPPC 产量显著下降(P均＜0.01).与H1组相比,H2组CCPG1、FAM134B的mRNA水平、ATL3、RTN3的蛋白水平、DPPC产量显著增高(P＜0.01),SEC62的蛋白水平增高(P＜0.05);H3组ATL3、SEC62的蛋白水平显著下降(P＜0.01),RTN3的mRNA水平、RTN3的蛋白水平下降(P＜0.05);H4组ATL3的mRNA水平、CCPG1的蛋白水平下降(P＜0.05)、RTN3的mRNA水平、ATL3、RTN3、SEC62的蛋白水平显著下降(P＜0.01).与H2组相比,H3组、H4组ATL3、CCPG1、FAM134B、RTN3 的 mRNA 水平、ATL3、RTN3、SEC62 的蛋白水平、DPPC 产量显著下降(P均＜0.01);H4组 SEC62 的 mRNA 水平下降(P＜0.05);H3 组 CCPG1、FAM134B 的蛋白水平下降(P＜0.05);H4 组 CCPG1、FAM134B的蛋白水平显著下降(P＜0.01);与H3组相比,H4组ATL3、RTN3、SEC62的蛋白水平显著下降(P＜0.01);其余各组差异均无统计学意义(P均＞0.05).结论 高氧12 h AECⅡ内质网自噬水平、DPPC产量下降;高氧24 h AECⅡ内质网自噬水平恢复到基础值,DPPC产量达到峰值,48 h后内质网自噬水平、DPPC产量再次下降,72 h后AECⅡ内质网自噬水平、DPPC产量衰减仍保持较低水平,AECⅡ内质网自噬与其功能密切相关.
Abstract
Objective To investigate the effect of hyperoxia on endoplasmic reticulum(ER)autophagy and surfac-tant production in alveolar type Ⅱ cells at different times.Methods Rat RLE-6TN cells were divided into control group(group C,cultured in 21％O2 for 72 h),hyperoxia groups H1,H2,H3 and H4(cultured in 95％O2 for 12 h,24 h,48 h and 72 h respectively).The mRNA levels of ER autophagy receptors ATL3,CCPG1,FAM134B,RTN3,and SEC62 were detected by real-time fluorescent quantitative PCR.The levels of ER autophagy-related proteins CCPG1 and FAM134B were detected by Western blot.ELISA was used to measure the level of dipalmitoyl lecithin(DPPC)in each group.Results Compared with group C,the mRNA levels of ATL3 and SEC62,CCPG1 protein level and DPPC production in group H1 decreased(P＜0.05).The mRNA levels of CCPG1,FAM134B and RTN3 and the protein levels of ATL3,FAM134B,RTN3 and SEC62 were significantly decreased(all P＜0.01).In group H2,the mRNA levels of ATL3,CCPG1,FAM134B,RTN3 and SEC62 and the protein levels of ATL3,CCPG1,FAM134B,RTN3 and SEC62 were not significantly different(all P＞0.05),while the DPPC production was increased(P＜0.05).The mRNA levels of ATL3,CCPG1,FAM134B,RTN3 and SEC62,the protein levels of ATL3,CCPG1,FAM134B,RTN3 and SEC62,and the DPPC production in the group H3 and H4 were significantly decreased(all P＜0.01).Compared with group H1,the mRNA levels of CCPG1 and FAM134B,protein levels of ATL3 and RTN3,and DPPC production in group H2 were significantly increased(P＜0.01),and the protein level of SEC62 was increased(P＜0.05).The protein levels of ATL3 and SEC62 in group H3 decreased significantly(P＜0.01),and the mRNA and protein levels of RTN3 were decreased(P＜0.05).The mRNA level of ATL3 and the protein level of CCPG1 in group H4 were decreased(P＜0.05).The mRNA level of RTN3 and the protein levels of ATL3,RTN3 and SEC62 decreased significantly(P＜0.01).Compared with the group H2,the mRNA levels of ATL3,CCPG1,FAM134B and RTN3,the protein levels of ATL3,RTN3 and SEC62,and the DPPC production in the group H3 and H4 were significantly decreased(all P＜0.01).The mRNA level of SEC62 in group H4 was decreased(P＜0.05).The protein levels of CCPG1 and FAM134B in group H3 were de-creased(P＜0.05).The protein levels of CCPG1 and FAM134B in group H4 were significantly decreased(P＜0.01).Compared with the group H3,the protein levels of ATL3,RTN3 and SEC62 in the group H4 were significantly de-creased(P＜0.01).There was no significant difference in other groups(all P＞0.05).Conclusion The level of endo-plasmic reticulum autophagy and DPPC production in alveolar type Ⅱ epithelial cells decreased after 12 h of hyperoxia.The level of ER autophagy in type Ⅱ alveolar epithelial cells returned to the basic level and DPPC production reached the peak after 24 h of hyperoxia.After 48 h of hyperoxia,the level of ER autophagy and DPPC production decreased again,the level of ER autophagy and DPPC production of type Ⅱ alveolar epithelial cells remained at a lower level after 72 h of hyperoxia.
NETL
NSTL
万方数据