【作者】 邢瑞婷；

【导师】 张巧；

【作者基本信息】 郑州大学 ， 卫生毒理学， 2010， 硕士

【摘要】 吸烟是呼吸系统疾病的重要危险因素。香烟烟雾刺激正常人呼吸道上皮细胞,可诱导白细胞介素-8(Interleukin-8, IL-8)的表达增加。IL-8基因顺式调控元件内有包括核转录因子-κB(nuclear factor-κB, NF-κB)和CCAAT/增强子结合蛋白-β(CCAAT/enhancer binding protein-β, C/EBPβ)在内的多个核因子结合位点。但有关NF-κB和C/EBPβ在香烟烟雾诱导IL-8基因转录活化的细胞内信号传导途径中的作用尚不清楚。目的该研究用香烟烟雾凝集物(cigarette smoke extracts, CSE)刺激人支气管上皮BEAS-2B细胞,观察CSE对BEAS-2B细胞的损害作用和对细胞炎症因子IL-8表达的影响。利用IL-8促进子上的NF-κB, C/EBPβ结合位点突变细胞株(IL-8mNF-κB-BEAS-2B cells,IL-8mC/EBPβ-BEAS-2B cells),细胞中被激活的NF-κB, C/EBPβ不能与IL-8促进子结合。通过比较CSE刺激后,NF-κB, C/EBPβ结合位点突变BEAS-2B细胞与野生型BEAS-2B细胞(IL-8 WT BEAS-2B cells)中与IL-8促进子相连的荧光素酶(luciferase,fLCF)基因表达量的变化,观察NF-κB和C/EBPβ转录因子在CSE引起的人支气管上皮细胞IL-8表达变化中的作用。为进一步探讨吸烟导致呼吸系统炎症发生机制提供实验依据。实验方法1 CSE溶液的配制CSE原液的制备参照Su Y的方法并改良,CSE原液用无血清RPMI-1640培养液稀释,配制好的CSE于30min之内用于实验。2 MTT比色法测CSE溶液对BEAS-2B细胞的半数抑制浓度用0%、2.5%、5.0%、7.5%、10.0%、12.5%的CSE溶液刺激BEAS-2B细胞,测定不同浓度的CSE对BEAS-2B细胞的抑制作用,并得出CSE溶液的半数抑制浓度。3 CSE处理正常BEAS-2B细胞及IL-8基因mRNA表达水平检测根据MTT测定的结果,分别以浓度0%、2.5%、5.0%、7.5%、10.0%的CSE作用正常BEAS-2B细胞,通过荧光定量PCR,分析不同浓度CSE处理后细胞IL-8 mRNA水平的变化。4 CSE处理3种突变型BEAS-2B细胞及其fLCF mRNA表达水平检测根据CSE处理正常BEAS-2B细胞后IL-8基因表达的变化,用7.5%CSE作用3种突变型BEAS-2B细胞,通过荧光定量PCR,分析CSE处理后,细胞fLCF mRNA水平的变化。5荧光素酶活性测定以试剂盒中报告基因细胞裂解液为空白对照,测定7.5%CSE作用的正常BEAS-2B细胞和3种突变型BEAS-2B细胞的荧光素酶活性。结果1 BEAS-2B细胞的形态学观察CSE刺激支气管上皮细胞来源的BEAS-2B细胞,可导致BEAS-2B细胞形态改变。CSE处理后,细胞胞浆疏松,漂浮细胞增多。随着CSE浓度的增加,细胞受损情况也越严重。2 7.5%CSE处理3种突变型BEAS-2B细胞,细胞绿色荧光的变化3种突变型BEAS-2B细胞受到CSE作用后,荧光显微镜下观察到绿色荧光细胞减少,细胞回缩变圆。3 MTT检测结果CSE对BEAS-2B细胞的生长有显著的抑制作用,F=25.06,P<0.001。且随着药物浓度的增加,CSE对细胞的抑制作用增强,呈剂量-效应关系。CSE对BEAS-2B细胞的半数抑制浓度约为7.5%。4 BEAS-2B细胞IL-8 mRNA在不同浓度CSE作用后的RT-PCR检测结果CSE作用BEAS-2B细胞,可导致其炎症因子IL-8表达增加,单因素方差分析表达差异有统计学意义(F=1808.1,P<0.001)。低浓度(2.5%、5.0%和7.5%)的CSE刺激增加IL-8 mRNA的表达(P<0.001),且具有浓度依赖性增高；但是10.0%CSE处理组大量细胞死亡,其IL-8 mRNA表达量与无CSE处理组间差异无统计学意义(P>0.05)。5 7.5%CSE作用3种突变型BEAS-2B细胞fLCF mRNA的RT-PCR检测结果7.5%CSE作用后,IL-8mNF-κB-BEAS-2B细胞和IL-8mC/EBPβ-BEAS-2B细胞的fLCF mRNA表达量都较IL-8 WT BEAS-2B细胞低,且IL-8 WT BEAS-2B细胞fLCF mRNA表达量是IL-8mNF-κB- BEAS-2B细胞fLCF mRNA表达量的1.32倍,是IL-8mC/EBPβ- BEAS-2B细胞的fLCF mRNA表达量的1.54倍。6荧光素酶活性测定相同浓度和作用时间的CSE刺激,IL-8mNF-κB- BEAS-2B细胞和IL-8mC/EBPβ- BEAS-2B细胞的荧光素酶活性都较IL-8WT BEAS-2B细胞低,且差异有统计学意义(P<0.001)。结论NF-κB转录因子参与CSE诱导的IL-8基因转录活化的细胞内信号传导过程。该研究首次证实C/EBPβ转录因子参与CSE诱导的IL-8基因转录活化。提不NF-κB和C/EBPβ转录因子通过参与炎症因子IL-8表达,促进吸烟导致的呼吸系统炎症反应。更多还原

【Abstract】 Cigarette smoke mediated oxidative stress and inflammatory events in the bronchial epithelium are important processes in the pathogenesis of smoking related pulmonary diseases. Lots of harmful substances generated by smoking including free radicals and oxides can trigger pro-inflammatory cytokines, which are increased in the lungs of smokers. The airway epithelium is the primary target for any inhaled environmental agents and plays a critical role in the release of pro-inflammatory mediators. Previous in vivo findings have supported that cigarette smoke can induce pro-inflammatory cytokine (Interleukin-8, IL-8) release in smokers and in rodent lungs. However, the precise molecular mechanisms as how cigarette smoke generates signals for pro-inflammatory cytokine release, particularly in bronchial epithelium are not yet clearly understood.ObjectiveHere we examined the acute effect of aqueous cigarette smoke extracts (CSE) in cultured normal human bronchial epithelial cell line (BEAS-2B), and detected the changes of the transcription of Interleukin-8. IL-8 is a multifunctional cytokine that has significant neutrophil chemoattractant and activating properties. It was reported that NF-κB and C/EBPβinvolve in IL-8 promoter transactivation as well as in its generation. Herein, we used three kinds of mutant BEAS-2B cells to determine effects of NF-κB and C/EBPβon the transcription of Interleukin-8 in cigarette smoke extract treated BEAS-2B cells. These three kinds of cells were transfected by luciferase plasmid and EGFP plasmid. Luciferase constructs under the control of the IL-8 promoter (IL-8 WT BEAS-2B cells), as well as mutants in the NF-κB site (IL-8mNFκB-BEAS-2B cells) or in the C/EBPβsite (IL-8mC/EBPβ-BEAS-2B cells).Methods1 Preparation of cigarette smoke extracts solution CSE was prepared by using an improved modification of the method described by Su Y, and diluted by RPMI1640 medium without FBS.2 Using MTT colorimetric methods to get IC50BEAS-2B cells were treated by 0%,2.5%,5.0%,7.5%,10.0%,12.5%. CSE for 24h and added in MTT to get the inhibitions of BEAS-2B cells under the pression of different CSE concentrations. And then we got the Inhibitory Concentration 50(IC50).3 CSE of different concentrations treated BEAS-2B cells, and we detected the transcription of IL-8 mRNA by fluorescent quantitative PCRBased on the results of the MTT colorimetric methods, BEAS-2B cells were treated with 0%,2.5%,5.0%,7.5%,10.0%CSE for 24h for each experimental condition, total RNA was extracted and 1μg of the resulting RNA was then reverse transcribed to cDNA. And fluorescent quantitative PCR assay was used to detect the transcription of IL-8 mRNA.4 7.5%CSE stimulated three kinds of mutant BEAS-2B cells, and we detected the transcription of fLCF mRNA by fluorescent quantitative PCRThree kinds of mutant BEAS-2B cells were treated by the 7.5%CSE for 24h for each experimental condition, total RNA was extracted and 1μg of the resulting RNA was then reverse transcribed to cDNA. And fluorescent quantitative PCR assay was used to detect the transcription of fLCF mRNA.5 Luciferase assays7.5%CSE treated normal BEAS-2B cells and three kinds of mutant BEAS-2B cells for 24h. And then we measured the luciferase activity.Results1 Morphologic change of the BEAS-2B cells treated with CSEAfter the stimulated by CSE, the shapes of BEAS-2B cells became irregular. There were more floating cells compared with cells without CSE. We observed a CSE dose dependant increase in cellular damage. And the cell damage became more seriously with the increase of CSE.2 Changes of green fluorescent protein (EGFP) in three kinds of mutant BEAS-2B cells before and after CSE treated CSE had an obvious cyto-inhibition. Cells with green fluorescent protein (EGFP) became fewer and round observed under the fluorescence microscope.3 Results of the MTT colorimetric methodCSE had an obvious cyto-inhibition (F=25.06, P<0.001). With the increase of the concentration of CSE, cyto-inhibition became higher. There was a dose-effect relationship between CSE and the cyto-inhibition. And Inhibitory Concentration 50 of CSE was 7.5%.4 Transcription of IL-8 mRNA in BEAS-2B cells treated with different concentrations of CSE detected by fluorescent quantitative PCRBEAS-2B cells were stimulated with 2.5%,5.0%,7.5%and 10.0%CSE for 24h. IL-8 mRNA levels were up-regulated by CSE (2.5%,5.0%and 7.5%), (P<0.001)and had a CSE dose dependent increase. However CSE at a higher concentration (10.0%) made lots of cells dead and did not increase IL-8 mRNA level (P>0.05).5 Transcription of fLCF mRNA in three kinds of mutant BEAS-2B cells treated with 7.5%CSE detected by fluorescent quantitative PCRThe transcriptions of fLCF mRNA of IL-8mNF-κB-and IL-8mC/EBPβ-BEAS-2B cells were lower than that of IL-8 WT BEAS-2B cells. The transcription of fLCF mRNA of IL-8 WT BEAS-2B cells was 1.32 times than that of IL-8mNF-κB-BEAS-2B cells and 1.54 times than that of IL-8mC/EBPβ-BEAS-2B cells.6 Luciferase assays7.5%CSE treated BEAS-2B cells and three kinds of mutant BEAS-2B cells for 24h. We found that the luciferase activities in IL-8mNF-κB-BEAS-2B cells and in IL-8mC/EBPp-BEAS-2B cells were lower than that in IL-8 WT BEAS-2B cells. And the discrepancy had a statistical significance (P< 0.05).ConclusionsNF-κB regulated the release of Interleukin-8 in BEAS-2B cells treated by cigarette smoke extract. And this study showed for the first time that C/EBPβtranscription factor involved in the precise molecular mechanisms of the release of Interleukin-8 in BEAS-2B cells treated by cigarette smoke extract.更多还原