【作者】 杨敏；

【导师】 陈平；

【作者基本信息】 中南大学 ， 内科学， 2009， 博士

【摘要】 第一章COPD肺血管内皮细胞凋亡和COX表达与功能的改变及其发生机制初探目的:观察COPD患者肺组织中血管内皮细胞凋亡、COX功能和COXⅡ表达变化,探讨COPD可能的发病机制。方法:2007年9月至2008年5月在中南大学湘雅二医院胸外科行肺切除术的周围型肺癌患者病例20例,根据COPD诊断标准和WHO吸烟调查方法标准将其分成3组:A组（非吸烟非COPD组）,7例;B组（吸烟非COPD组）,7例;C组（吸烟并COPD组）,6例。TUNEL法检测肺血管内皮细胞凋亡指数,分光光度法检测COX活性,普通RT-PCR和荧光RT-PCR（RQ-PCR）检测COXⅡmRNA表达,以及免疫组织化学和WB法检测COXⅡ蛋白的分布和表达。使用SPSS 13.0统计软件进行数据分析,正态分布的各组计量资料以（?）±s表示,多组间比较采用单因素方差分析（One-Way ANOVA）,变量间采用单因素相关分析（Pearson）,P＜0.05差异有统计学意义。结果:C组肺血管内皮细胞凋亡指数[（13.8±1.9）%]较B组[（9.6±0.8）%]和A组[（5.9±1.0）%]均增高,任意两组间差异均有统计学意义（均P＜0.05）。A组COX活性、COXⅡmRNA的ΔΔCt值和COXⅡ蛋白OD值分别为（7.6±0.4）×10^-1U/mg、（0.86±0.20）和（0.99±0.14）;B组分别为（6.0±0.6）×10^-1U/mg、（0.8±0.15）和（0.81±0.09）;而C组分别为（4.4±0.7）×10^-1U/mg、（0.76±0.17）和（0.60±0.07）,各变量在A、B、C三组中依次下降,任意两组间差异有显著性（均P＜0.05）。经相关性分析,细胞凋亡指数与FEV₁/Pre呈负相关（r=-0.846,P＜0.01）,与吸烟指数正相关（r=0.794,P＜0.01）。COX活性与FEV₁/Pre呈正相关（r=0.842,P＜0.01）,与吸烟指数负相关（r=-0.783,P＜0.01）。结论:（1）COPD患者肺血管内皮细胞凋亡。（2）COPD患者肺组织COXⅡ表达抑制并且COX活性下降。（3）吸烟可能与肺血管内皮细胞凋亡、COXⅡ表达抑制及COX活性下降有关。（4）COX可能介导COPD肺血管内皮细胞凋亡。第二章COPD患者COXⅡ的甲基化状态及其与COPD的关系目的:探讨COPD患者COXⅡ甲基化状态及其与COPD的关系。方法:2007年9月至2008年5月在中南大学湘雅二医院胸外科行肺切除术的周围型肺癌患者病例20例,根据最新COPD诊断标准和WHO吸烟调查方法标准将其分成3组:A组（非吸烟非COPD组）,7例;B组（吸烟非COPD组）,7例;C组（吸烟并COPD组）,6例,分别收集患者肺组织。同期在该院呼吸内科住院的吸烟并COPD患者10例和健康体检者20例,分为3组:D组（非吸烟非COPD组）,10例;E组（吸烟非COPD组）,10例（入选时均处于稳定期）;F组（吸烟并COPD组）,10例,分别收集其外周血5ml。提取肺组织和血标本中基因组DNA,亚硫酸盐修饰后MSP检测COXⅡ甲基化。使用SPSS13.0统计软件进行数据分析,计量资料以（?）±s表示,多组间均数比较采用单因素方差分析（One—Way ANOVA）。计数资料以率表示,组间COXⅡ甲基化阳性率差异用Fisher确切概率法进行比较。P＜0.05为差异有统计学意义。结果:A、B、C三组肺组织标本中COXⅡ甲基化阳性率分别为71.4%（5/7）、71.4%（5/7）和66.7%（4/6）,三组间无明显统计学差异（均P＞0.50）。D、E、F三组血标本中COXⅡ甲基化阳性率分别为0%（0/10）、50%（5/10）和60%（6/10）。D组与E组或F组比较,COXⅡ甲基化阳性率差异均有统计学差异（均P＜0.05）,而E组与F组比较,统计学差异不显著（P＞0.50）。结论:COPD患者存在COXⅡ甲基化,该异常甲基化可能与吸烟有关。第三章CSE诱导HUVEC凋亡中COXⅡ甲基化状态改变及5-杂氮-2’-脱氧胞苷干预的影响目的:研究CSE诱导HUVEC凋亡的机制,探讨COX在其中的作用,并了解COXⅡ甲基化是否参与其中机制。方法:第一部分,体外培养HUVEC,给予不同浓度（0%-10%）和不同时间（0h-24h）的CSE处理,TUNEL法检测细胞凋亡,分光光度计检测其COX和caspase-3的活性。第二部分,将HUVEC分为四个处理组:空白组,AZA组,CSE（2.5%）组,AZA+CSE组,分别给予不同的干预,检测各组细胞的凋亡指数和COX活性。荧光RQ-PCR和Western Blot分别检测COXⅡmRNA和COXⅡ蛋白表达。提取各组细胞DNA,MSP法检测各组COXⅡ甲基化状态。用SPSS13.0进行数据分析,计量数据用（?）±s表示,多组间比较采用单因素方差分析（One-WayANOVA）。变量间采用单因素相关分析（Pearson）法。P＜0.05为差异有统计学意义。结果:第一部分:CSE诱导的HUVEC凋亡呈浓度-和时间-依赖。1%CSE组即出现HUVEC凋亡增加[（26.0±2.1）%],2.5%CSE时凋亡指数达到（42.6±2.6）%。此后CSE浓度继续增加,虽然凋亡仍在增加,但细胞坏死也逐渐明显。10%CSE干预24h后HUVEC凋亡达到最高峰（49.5±0.7）%。2.5%CSE干预HUVEC,随着干预时间的延长凋亡逐渐增多。与凋亡相反,随着CSE干预浓度和时间的增加,COX活性下降愈明显,同样呈浓度-和时间-依赖。10%CSE干预24h后,COX活性下降约25.6%。在较低CSE干预浓度（0.5%-2.5%）时caspase-3活性随着CSE浓度增加而增加,2.5%CSE组达到最大值（255.0±24.2%）。而在较高CSE浓度（5%-10%）干预时,caspase-3活性出现回降,10%CSE组降至（68.6±3.7）%,低于空白对照组。经相关分析,HUVEC凋亡与COX活性呈负相关（r=-0.884,P＜0.01）,而与caspase-3活性无关（r=-0.236,P＞0.05）。第二部分:与对照组相比,CSE组[（44.0±3.6）%]和AZA+CSE组[（21.7±2.3）%]HUVEC凋亡增加;而COX活性[分别为（5.94±0.36）×10^-1U/mg和（6.83±0.26）×10^-1U/mg]、COXⅡmRNA表达[分别为（0.82±0.06）和（1.18±0.08）]和COXⅡ蛋白表达[分别为（0.73±0.05）和（1.10±0.12）]均下降,三组间各指标差异均有统计学意义（均P＜0.05）。而AZA组与对照组比较无显著差异。MSP检验发现CSE组和AZA+CSE组出现COXⅡ甲基化阳性表达,而对照组和AZA组甲基化阴性。结论:（1）CSE诱导HUVEC凋亡呈浓度-和时间-依赖性,该凋亡为非caspase-3-依赖,并由COX介导。（2）CSE可能通过诱导COXⅡ发生甲基化而抑制COXⅡ转录。（3）AZA能一定程度逆转CSE诱导的COXⅡ甲基化,对COX和HUVEC有保护作用。更多还原

【Abstract】 Chapter 1.Pulmonary vascular endothelial apoptosis and COX expression in COPDObjective:To observe the pulmonary vascular endothelial cell apoptosis,COX activity and COXⅡexpression in COPD patients,and to investigate the possible mechanism of COPD pathogenesis.Methods:According to the latest COPD diagnostic criteria and WHO standards for smoking-related investigation,20 patients underwent pneumonectomy were divided into 3 groups:group A,non-smokers without COPD;group B,smokers without COPD;and group C,smokers with COPD. TUNEL assay was used to assess cell apoptosis of pulmonary vascular endothelial cells.COX activity was measured by spectrophotometry. Semi-quantitative and fluorescence quantitative reverse transcription polymerase chain reaction（RT-PCR and RQ-PCR） were used to measure COX subunitⅡ（COXⅡ） mRNA expression.Moreover, immunohistochemistry and Western Blot were used to detect COXⅡprotein expression respectively.Data were presented as Means±SD.Statistical analyses were performed using SPSS version 13.0 and differences were considered significant if P＜0.05.Results:The apoptotic index of pulmonary vascular endothelial cells in group C[（13.8±1.8）%]was much higher than that in group B[（9.6±0.8）%] and group A[（5.9±1.0）%],differences between every two groups were of great significance（all P＜0.05）.COX activity,COXⅡmRNAΔΔCt value and COXⅡprotein OD value in group A were（7.6±0.4）×10^-1U/mg, （0.86±0.19） and（0.99±0.14） respectively.And those in group B were （6.0±0.6）×10^-1U/mg,（0.81±0.15） and（0.81±0.09）;in group C were （4.4±0.7）×10^-1U/mg,（0.76±0.17） and（0.60±0.07）.Each variant decreased in turn and differences between every two groups were siganificant（all P＜0.05）.The correlation analyses showed that the apoptotic index was negatively correlated with FEV₁/Pre（r=-0.846,P＜0.01）,but positively correlated with smoke index（r=0.794,P＜0.01）,while COX activity was positively correlated with FEV₁/Pre（r=0.812,P＜0.01）,but negatively correlated with smoke index（r=-0.783,P＜0.01）.Conclusions:（1） Pulmonary vascular endothelial cell apoptosis is increased in COPD patients.（2） COXⅡexpression and COX activity are inhibited in COPD patients.（3） Cigarette smoking may be related with pulmonary vascular endothelial cell apoptosis and inhibition COXⅡexpression and COX activity.（4） COX mediates pulmonary vascular endothelial cell apoptosis in COPD. Chapter 2.The methylation status of COXⅡin COPD and its association with COPDObjective:To investigate whether there is COXⅡmethylation in COPD and the relationship between COXⅡmethylation and COPD.Methods:Lung tissues of 20 patients underwent pneumonectomy were collected and devided into 3 groups:group A,non-smokers without COPD; group B,smokers without COPD;and group C,smokers with COPD.Blood samples of another 10 in patients with COPD and 20 healthy controls were collected and divided into 3 other groups:group D,non-smokers without COPD;group E,smokers without COPD;and group F smokers with COPD. Extracted genome DNA in either lung tissues or peripheral bloods,and detected the COXⅡmethylation in them using methylation-specific PCR. Quantitative data were presented as Means±SD,and analyzed using One-Way ANOVA.While methylated frequencies of COXⅡwere presented as percentage and analyzed using Fisher’s exact test.Statistical analyses were performed using SPSS version 13.0 and differences were considered significant if P＜0.05.Results:The methylation frequencies of COXⅡin lung tissues were 71.4%（5/7） in group A,71.4%（5/7） in group B and 66.7%（4/6） in group C. Through Fisher exact test,no difference among them was found（P＞0.05）. while in blood samples,methylated frequencies of COXⅡwere 0%（0/10） in group D,50%（5/10） in group E and 60%（6/10） in group F.Differences between group D and group E or group F were statistic significant（all P＜0.05）,but no difference was found between group E and group F（P＞0.50）.Conclusions:There is COXⅡmethylation in COPD patients,and this aberrant methylation may be associated with cigarette smoke.Chapter 3.Methylation status of COXⅡin CSE-induced HUVEC apoptosis and the effect of 5-aza-2’-deoxycytidine interventionObjective:To investigate whether COX activity and COXⅡmethylation are involved in CSE-induced HUVEC apoptosis.Methods:In part one,cultured HUVECs were exposed to CSE at various concentrations and for different durations.TUNEL assay was used to detect apoptosis.COX activity and caspase-3 activity were measured by spectrophotometry.In part two,HUVECs were divided into 4 groups: control group;AZA group;CSE group and AZA+CSE group.Apoptotic index and COX activity were assessed in each group.Fluorescence RQ-PCR and Western Blot were used to detect COXⅡmRNA and COXⅡprotein, respectively.Moreover,DNA was extracted and COXⅡmethylation was detected by MSP.Data were presented as Means±SD.Statistical analyses were performed using SPSS version 13.0 and differences were considered significant if P＜0.05.Results:Part one,CSE-induced HUVEC apoptosis increased in a dose-and time-dependent manner with obvious cell death in 5%and 10% CSE-treated groups.COX activity decreased paralleled to CSE concentration and duration.Caspase-3 activity increased with CSE at a lower concentration of 0.5%-2.5%,but suddenly decreased after 5%and 10%CSE-treatment.This CSE-induced apoptosis in HUVEC was found negatively correlated with COX activity（r=-0.884,P＜0.01）,but independent of caspase-3 activity（r=-0.236,P＞0.05）.Part two,compared with control group,apoptotic index increased in CSE-treated group [（44.0±3.6）%]and AZA+CSE-treated group[（21.7±2.3）%].While COX activity,COXⅡmRNA and COXⅡprotein expression decreased in these two groups[（5.94±0.36）×10^-1U/mg,（0.82±0.06） and（0.73±0.05） in CSE-treated group;（6.83±0.26）×10^-1U/mg,（1.18±0.08） and（1.10±0.12） in AZA+CSE-treated group].Differences of variants between every two of these three groups were significant（all P＜0.05）.Nevertheless,no significant difference between control group and AZA group was found（P＞0.05）.After MSP,positive COXⅡmethylation（semi-methylation） in CSE group and AZA+CSE group was found.Conclusions:（1） CSE induces HUVEC apoptosis in a dose- and time-dependent manner.This apoptosis is independent of caspase-3 activation and is mediated by COX.（2） CSE inhibits COXⅡtranscription via inducting methylation of COXⅡ.（3） AZA demethylation may partially reverse CSE -induced COXⅡmethylation,and serve as a protective factor of COX and HUVEC.更多还原