CAJ | 학술논문

目的观察脂多糖(lipopolysaccharide,LPS)刺激大鼠肺泡巨噬细胞NR8383后乙酰胆碱酯酶(acetylcholinesterase,ACHE)的表达变化,为研究胆碱能抗炎通路的调控提供新的实验依据.方法将体外去致热源培养的NR8383细胞分为对照组及LPS(1μg/mL)刺激组,于刺激后3、6、12、24 h各时间点分别离心收集上清液及细胞沉淀,采用酶联免疫吸附法(enzyme-linkedimmunosorbent assay,ELISA)测定上清液中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的变化,实时定量PCR检测细胞中AChE mRNA的表达改变,蛋白质免疫印迹法(Western blot)检测细胞中AChE蛋白的表达变化,乙酰胆碱酯酶T-CHE测试盒检测上清液中AChE活性的变化.组间多重比较采用单因素方差分析,进一步采用LSD-t检验进行两两比较,P＜0.05为差异具有统计学意义.结果与对照组相比,TNF-α的含量在LPS刺激NR8383细胞后3h开始显著上升(P＜0.05),12 h达高峰,24 h下降但仍明显高于对照组水平(P＜0.05).与对照组相比,AChE mRNA于刺激后3h开始上升,升高(3.19±0.44)倍(P＜0.05),6h后达高水平并维持于此水平,6、12、24 h分别上调[(5.65±0.63)、(5.40±0.71)、(5.35±0.77)[倍(与对照组相比,均P＜0.05);AChE蛋白的表达及上清液中AChE的活性在LPS刺激后12 h与各自对照组相比均上升[AChE蛋白:(1.37±0.01) vs.(1.05±0.02),P＜0.05; AChE活性:(6.14±1.13) U/mLvs.(2.64±0.85) U/mL,P＜0.05],随后呈下降趋势,于LPS作用24 h时均低于各自对照组[AChE蛋白:0.65±0.05,P＜0.05,AChE活性:(0.56±0.19)U/mL,P＜0.05].结论 LPS刺激大鼠肺泡巨噬细胞后,AChE的表达增加,提示其可能参与肺泡巨噬细胞炎症反应的调控;LPS刺激后24 h,AChE mRNA仍处于高表达水平,AChE蛋白及活性却下降,提示在肺泡巨噬细胞炎症反应中可能存在AChE转录后水平的调控.
목적 관찰지다당(lipopolysaccharide,LPS)자격대서폐포거서세포NR8383후을선담감지매(acetylcholinesterase,ACHE)적표체변화,위연구담감능항염통로적조공제공신적실험의거.방법 장체외거치열원배양적NR8383세포분위대조조급LPS(1μg/mL)자격조,우자격후3、6、12、24 h각시간점분별리심수집상청액급세포침정,채용매련면역흡부법(enzyme-linkedimmunosorbent assay,ELISA)측정상청액중종류배사인자-α(tumor necrosis factor-α,TNF-α)적변화,실시정량PCR검측세포중AChE mRNA적표체개변,단백질면역인적법(Western blot)검측세포중AChE단백적표체변화,을선담감지매T-CHE측시합검측상청액중AChE활성적변화.조간다중비교채용단인소방차분석,진일보채용LSD-t검험진행량량비교,P＜0.05위차이구유통계학의의.결과 여대조조상비,TNF-α적함량재LPS자격NR8383세포후3h개시현저상승(P＜0.05),12 h체고봉,24 h하강단잉명현고우대조조수평(P＜0.05).여대조조상비,AChE mRNA우자격후3h개시상승,승고(3.19±0.44)배(P＜0.05),6h후체고수평병유지우차수평,6、12、24 h분별상조[(5.65±0.63)、(5.40±0.71)、(5.35±0.77)[배(여대조조상비,균P＜0.05);AChE단백적표체급상청액중AChE적활성재LPS자격후12 h여각자대조조상비균상승[AChE단백:(1.37±0.01) vs.(1.05±0.02),P＜0.05; AChE활성:(6.14±1.13) U/mLvs.(2.64±0.85) U/mL,P＜0.05],수후정하강추세,우LPS작용24 h시균저우각자대조조[AChE단백:0.65±0.05,P＜0.05,AChE활성:(0.56±0.19)U/mL,P＜0.05].결론 LPS자격대서폐포거서세포후,AChE적표체증가,제시기가능삼여폐포거서세포염증반응적조공;LPS자격후24 h,AChE mRNA잉처우고표체수평,AChE단백급활성각하강,제시재폐포거서세포염증반응중가능존재AChE전록후수평적조공.
Objective To observe the changes of acetylcholinesterase (AChE) in rat alveolar macrophages NR8383 stimulated by lipopolysaccharide (LPS) in order to provide a novel experimental evidence for studying the regulation of the cholinergic anti-inflammatory pathway.Methods The NR8383 cells cultured with pyrogen-free in vitro were divided into control group and LPS (1 μg/mL) stimulation group.Culture supernatants and cell pellets were collected by centrifugation at 3 h,6 h,12 h and 24 h after stimulation,respectively.The level of tumor necrosis factor-α (TNF-α) in the supernatant was assayed by using enzyme-linked immunosorbent assay (ELISA).The expression of AChE mRNA in cells was detected by using real time quantitative RT-PCR,The level of AChE protein in cells was analyzed by using Western blot,The activity of AChE in the supernatant was measured by using True Choline esterase assay kit (TCHE).One-way analysis of variance (ANOVA) was used for comparisons between two groups,and LSD-t test was performed for further comparison,and difference was statistically significant at P ＜ 0.05.Results The level of TNF-α began to increased significantly at 3 h after stimulation of NR8383 cells with LPS (P ＜ 0.05) compared with control group,and reached its peak at 12 h,then decreased until 24 h but the level was still significantly higher than that in control group (P ＜ 0.05) ; The expression of AChE mRNA began to elevate at 3 h after stimulation compared with control group [(3.19 ± 0.44) times,P ＜0.05],at 6 h reached the high level and then maintained this level at 12 h and 24 h [(5.65 ± 0.63),(5.40 ±0.71),(5.35 ± 0.77) times,compared with control group,all P ＜0.05] ; The level of AChE protein in cells and the activity of AChE in the supernatant increased at 12 h after LPS stimulation compared with control group [AChE protein:(1.37 ± 0.01) vs.(1.05 ± 0.02),P ＜0.05 ; AChE activity:(6.14 ±1.13) U/ml vs.(2.64 ± 0.85) U/ml,P＜0.05],followed by a downward trend,and were lower than those in control groups at 24 h after LPS stimulation [AChE protein:(0.65 ± 0.05),P ＜ 0.05 ; AChE activity:(0.56 ± 0.19) U/ml,P ＜0.05].Conclusions The expression of AChE mRNA increased after stimulation with LPS in rat alveolar macrophages,suggesting that AChE may be involved in regulation of alveolar macrophages inflammatory response; AChE mRNA expression is still high while its protein and activity are declined at 24 h after LPS stimulation,suggesting that there may be some mechanisms existed in post-transcriptional regulation of AChE in alveolar macrophages during inflammatory response.