摘要
目的探讨鞘氨醇激酶1（SphK1）对肿瘤坏死因子α（TNF—α）诱导的气道黏蛋白（MUC）5AC的调节机制。方法培养正常人气道上皮细胞HBE16，给予SphK1特异性抑制剂N，N-二甲基鞘氨醇（DMS）处理及转染SphK1的小干扰RNA（siRNA）后，再各施以TNF-α刺激，四甲基偶氮唑盐法检测各组细胞增殖活力，Westernblot法检测各组SphK1、环氧化酶2（COX-2）、磷酸化P38（P—P38）、磷酸化细胞外信号调节激酶（p-ERK）和IKBα蛋白相对含量，酶联免疫分析法检测各组MUC5AC、前列腺素E2（PGE2）和环单磷酸腺苷（cAMP）的相对含量，荧光素酶报告基因系统测定cAMP反应结合蛋白（CREB）和核因子-κB（NF-κB）的相对活性，RT-PCR检测各组SphK1和SphK1 mRNA的表达水平，免疫荧光法检测胞内MUC5AC含量。结果与对照组相比，TNF．d组的SphK1 mRNA和蛋白水平显著升高（均P〈0．01），COX-2、PGE2、cAMP、P—P38和p-ERK的含量明显增加（均P〈0．01），IkBα含量明显降低（P〈0．01），CREB和NF—KB的活性明显增强（均P〈0．01），MUC5AC mRNA和蛋白含量亦显著高于对照组（均P〈0．01）；与TNF-α组相比，给予DMS处理后COX-2、PGE2、cAMP、P—P38和p-ERK的含量明显降低（均P〈0．01），IkBα含量显著增加（P〈0．01），CREB和NF-κB的活性明显减弱（均P〈0．01），MUC5AC mRNA和蛋白含量也显著降低（均P〈0．01），转染SphK1 siRNA后上述指标出现同DMS处理一致的变化（均P〈0．01）。结论SphK1参与调节了TNF-α所诱导的MUC5AC高表达过程中的主要信号因子及转录因子的表达，可能是MUC5AC合成及分泌过程中的重要调节因子。
Objective To explore the effects of sphingosine kinase 1 （SphK1） on mucin （MUC） 5AC overexpression under the induction of tumor necrosis factor-α（TNF-α）. Methods The HBE16 airway epithelial cells were cultured, pretreated with SphKl-specific inhibitor DMS or transfected with SphK1 siRNA. Then each group was stimulated by a certain concentration of TNF-α. The relative contents of SphK1, COX-2, p-P38, p-ERK and IκBα were detected by Western blot while the levels of MUC5AC, PGE2 and cAMP analyzed by enzyme linked immunoassay. The transcription activities of cAMP-response- element-binding protein and nuclear factor κB （NF-κB） were detected by luciferase reporter gene detection system. The mRNA expressions of SphK1 and MUC5AC were detected by RT-PCR （reverse transcription- polymerase chain reaction ） And the intracellular MUC5AC protein was detected by immunofluorescence. Results The levels of protein and mRNA expression of SphK1 and MUC5AC were obviously elevated [（0.69 ±0.12, 0.86 ±0.07）, （0.60±0.09, 0.79 ±0.05）]. They were all significantly higher than those in the control group （0. 26± 0. 03, 0. 33 ± 0. 04, 0. 18 ±0. 06, 0. 22± 0. 10）. There was an elevation of COX-2, PGE2, cAMP, p-P38, p-ERK, CREB and NF-KB activity （all P 〈0. 01 ） ; the production of IKBα protein was lower than that in the control group （ P = 0. 003 ）. DMS decreased the levels of MUC5AC mRNA expression （0. 37 ±0. 06） and protein production （0. 27 ±0. 04）, lowered the production of COX-2, PGE2, cAMP, p-P38 and p-ERK, inhibited the activity of CREB and NF-KB and increased the IKBα production （ all P 〈0. 01 ）. Transfeetion of SphK1 siRNA showed the similar effects as DMS （ all P 〈 0. 01 ）. Conclusion SphK1 plays an important role in regulating the MUC5AC expression under the induction of TNF-o~. And the activation of critical signal factors in that process is dependent on SphK1.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2011年第6期391-395,共5页
National Medical Journal of China
基金
国家自然科学基金（30770951）
国家自然科学基金中俄国际合作项目（81011120108）
中俄政府间合作项目（2009：13-01）
关键词
鞘氨醇
磷酸转移酶类
肿瘤坏死因子Α
黏蛋白类
转录因子
Sphingosine
Phosphotransferases
Tumor necrosis factor-alpha
Mucins
Transcription factors
