CAJ | 학술논문

目的探讨丙烯醛调控的糖皮质激素受体(GRs)类小泛素(SUMO)化水平对气道黏液高分泌的影响.方法构建重组真核表达载体质粒pcDNA3.1-EGFP-flag-SUMO1,酶切电泳鉴定.常规培养16HBE细胞,随机分为9组:A:空白对照组；B:丙烯醛刺激组；C:地塞米松组；D:丙烯醛刺激+地塞米松组；E:SUMO相关修饰物1(SUMO1)转染组；F:空质粒转染组；G:SUMO1转染+丙烯醛刺激组；H:SUMO1转染+丙烯醛刺激+地塞米松组；Ⅰ:空质粒转染+丙烯醛刺激+地塞米松组.每个实验组设5复孔,重复4次实验用于统计学分析.荧光显微镜下观察转染成功率,通过免疫共沉淀以及Western印迹法分析各实验组GRs SUMO化水平的变化,反转录-PCR法比较各实验组中黏蛋白(MUC)5AC转录水平的差异.用酶联免疫吸附法测定细胞培养上清液中MUC5AC的分泌量.结果 B组GRs SUMO化水平(0.079±0.023)低于A组(0.446±0.068)(t=-23.13,P=0.000);D组GRs SUMO化水平(0.574±0.018)低于C组(0.843±0.028)(t=-36.34,P=0.000).H组的MUC5AC mRNA转录水平(0.330±0.063)低于D组(0.617±0.190)(q=-7.80,P=0.000).细胞培养上清液中MUC5AC分泌水平显示,G组与B组差异无统计学意义,而H组[(0.416±0.092) μg/L)]显著低于D组[(0.663±0.104) μg/L]和G组[(0.740±0.343)μg/L](q=-7.31,-9.59;P=0.001,0.000).结论丙烯醛可下调GRs SUMO化水平,并降低地塞米松对MUC5AC转录的抑制作用.
목적 탐토병희철조공적당피질격소수체(GRs)류소범소(SUMO)화수평대기도점액고분비적영향.방법 구건중조진핵표체재체질립pcDNA3.1-EGFP-flag-SUMO1,매절전영감정.상규배양16HBE세포,수궤분위9조:A:공백대조조；B:병희철자격조；C:지새미송조；D:병희철자격+지새미송조；E:SUMO상관수식물1(SUMO1)전염조；F:공질립전염조；G:SUMO1전염+병희철자격조；H:SUMO1전염+병희철자격+지새미송조；Ⅰ:공질립전염+병희철자격+지새미송조.매개실험조설5복공,중복4차실험용우통계학분석.형광현미경하관찰전염성공솔,통과면역공침정이급Western인적법분석각실험조GRs SUMO화수평적변화,반전록-PCR법비교각실험조중점단백(MUC)5AC전록수평적차이.용매련면역흡부법측정세포배양상청액중MUC5AC적분비량.결과 B조GRs SUMO화수평(0.079±0.023)저우A조(0.446±0.068)(t=-23.13,P=0.000);D조GRs SUMO화수평(0.574±0.018)저우C조(0.843±0.028)(t=-36.34,P=0.000).H조적MUC5AC mRNA전록수평(0.330±0.063)저우D조(0.617±0.190)(q=-7.80,P=0.000).세포배양상청액중MUC5AC분비수평현시,G조여B조차이무통계학의의,이H조[(0.416±0.092) μg/L)]현저저우D조[(0.663±0.104) μg/L]화G조[(0.740±0.343)μg/L](q=-7.31,-9.59;P=0.001,0.000).결론 병희철가하조GRs SUMO화수평,병강저지새미송대MUC5AC전록적억제작용.
Objective To explore the variation of small ubiquitin-related modification (SUMO) level of glucocorticoid receptors (GRs) exposed to acrolein stimulation as well as the influence of the variation on mucus hypersecretion.Methods The recombinant plasmid was constructed by inserting small ubiquitin-like modifier 1 (SUMO1) cDNA into eukaryotic expression plasmid pcDNA3.1-EGFP with a flag sequence marker.The recombinant plasmid pcDNA3.1-EGFP-flag-SUMO1 was identified by enzyme digestion analysis.The 16HBE cells were cultured and randomized divided into 9 following groups:A.control; B.acrolein stimulated; C.dexamethasone incubated; D.acrolein stimulated and dexamethasone incubated; E.SUMO1 transfected; F.pcDNA3.1-EGFP vector transfected; G.SUMO1 transfected and acrolein stimulated; H.SUMO1 transfected,acrolein stimulated and dexamethasone incubated; Ⅰ.pcDNA3.1-EGFP vector transfected,acrolein stimulated and dexamethasone incubated.Each group consisted of 5 parallel wells and experiments were repeated for 4 times for statistical analysis.The transfection efficiency was evaluated by the expression of enhanced green fluorescent protein (EGFP) via fluorescence microscope.The SUMO modification level of GRs was measured with co-immunoprecipitation and Western blot.The transcription level of mucin (MUC) 5AC was evaluated with reverse transcriptionpolymerase chain reaction (RT-PCR).The MUC5AC secreted in supernatant was measured by enzymelinked immunosorbent assay (ELISA).Results The SUMO modification level was much lower in group B (0.079 ± 0.023) than that in group A (0.446 ± 0.068) (t =-23.13,P =0.000) and in group D (0.574 ±0.018) than that in group C (0.843 ±0.028) (t =-36.34,P =0.000).The transcription level of MUC5AC was significantly lower in group H (0.330 ±0.063) than group D (0.617 ± 0.190) (q =-7.80,P =0.000).Through ELISA,there was no significance between groups G and B in the term of secretion level of MUC5AC.While the secretion level of MUC5AC was much lower in group H ((0.416 ±0.092) μg/L) than group D ((0.663 ±0.104) μg/L) and group G ((0.740±0.343) μg/L) (q=-7.31,-9.59 ; P =0.001,0.000).Conclusion Acrolein decreases the SUMO modification of GRs and reduces the inhibitory effect of dexamethasone on the transcription of MUC5AC.