【作者】 彭红；

【导师】 陈平；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 目的：检测COPD患者肺组织中mtTFA. COXⅡ的表达、肺血管内皮细胞凋亡的变化,分析它们之间的关系,探讨mtTFA在COPD发病机制中的可能作用。方法：2007年9月至2008年8月在中南大学湘雅二医院胸外科行肺叶切除术或肺段切除术的周围型肺癌患者病例21例,根据COPD诊断标准将其分成两组：非COPD组10例,COPD组(稳定期)11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real-time RT-PCR检测mtTFA mRNA. COXⅡmRNA表达,免疫组织化学法和western-blot检测mtTFA蛋白的分布和表达,western-blot检测COXⅡ蛋白表达。使用SPSS 13.0统计软件包进行数据分析,正态分布的各组计量资料以(x±s)表示,两组间均数比较采用独立样本t检验,变量间相关性分析采用单因素直线相关分析(Pearson), P<0.05为差异有统计学意义。结果：COPD组吸烟指数[(33.6±12.0)包年]较非COPD组[(12.1±8.9)包年]高(P<0.01)；COPD组肺血管内皮细胞凋亡指数[(13.8±1.9)%]较非COPD组[(5.9±1.0)%]增高(P<0.01)。COPD组肺组织mtTFA mRNA. mtTFA蛋白的相对表达量分别为(0.59±0.07)和(0.32±0.07),非COPD组肺组织mtTFA mRNA. mtTFA蛋白的相对表达量分别为(0.78±0.06)和(0.55±0.09),COPD组肺组织mtTFA mRNA、mtTFA蛋白表达均较非COPD组明显下降(均P<0.01)。COPD组肺组织COX活性、COXⅡmRNA、COXⅡ蛋白的相对表达量分别为[(4.4±0.9)×10-1]U/mg、(0.72±0.12)和(0.62±0.06),非COPD组肺组织COX活性、COXⅡmRNA、COXⅡ蛋白的相对表达量分别为[(7.6±0.4)×10-1]U/mg、(0.86±0.16)和(0.89±0.13),COPD组肺组织COX活性、COXⅡmRNA和COXⅡ蛋白表达均较非COPD组明显下降(P<0.01,P<0.05,P<0.01)。相关分析显示肺血管内皮细胞凋亡指数与FEV1/FVC、FEV1%Pre呈负相关(r=-0.796,r=-0.827,均P<0.01),与吸烟指数呈正相关(r=0.703,P<0.01)；肺组织COX活性与肺血管内皮细胞凋亡指数呈负相关(r=-0.756,P<0.01)；肺组织mtTFA蛋白表达与FEV1%Pre、COXⅡ蛋白表达、COX活性均呈正相关(r=0.892,r=0.810,r=0.854,均P＜0.01),与肺血管内皮细胞凋亡指数、吸烟指数均呈负相关(r=-0.749,r=-0.763,均P<0.01)。结论：(1) COPD患者肺血管内皮细胞凋亡增加。(2) COPD患者肺组织mtTFA、COXⅡ表达下降,COX活性下降。(3)吸烟可能通过抑制mtTFA表达,从而影响COX活性而参与COPD患者肺血管内皮细胞凋亡。目的：探讨COPD患者肺组织mtTFA基因启动子区甲基化状态。方法：2007年9月至2008年8月在中南大学湘雅二医院胸外科行肺叶切除术或肺段切除术的周围型肺癌患者病例21例,根据COPD诊断标准将其分成两组：非COPD组10例,COPD组(稳定期)11例。收集患者术中肺组织标本,提取肺组织基因组DNA,亚硫酸氢钠基因组测序法(BSP)检测mtTFA基因启动子区甲基化状态。使用SPSS 13.0统计软件包进行数据分析,计量资料以(x±s)表示,两组间均数比较采用独立样本t检验。P<0.05为差异有统计学意义。结果：非COPD患者肺组织mtTFA基因启动子区不存在甲基化的CpG位点,而COPD患者肺组织mtTFA基因启动子区存在多个甲基化的CpG位点,平均甲基化率为(27.8±3.6)%。结论：COPD患者肺组织mtTFA基因启动子区存在甲基化的CpG位点。目的：研究CSE诱导HUVEC凋亡的可能机制,探讨mtTFA在其中的作用,并了解mtTFA基因甲基化是否参与其中。方法：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,分光光度法检测caspase-3的活性。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；普通RT-PCR和real-time RT-PCR检测mtTFA、COXⅡmRNA表达,western-blot检测mtTFA、COXⅡ蛋白表达。提取各组细胞基因组DNA, BSP法检测各组mtTFA基因启动子区甲基化状态。用SPSS 13.0统计软件包进行数据分析,计量数据用(x±s)表示,多组间比较采用单因素方差分析(One-Way ANOVA)。变量间相关分析采用单因素直线相关法(Pearson)。P<0.05为差异有统计学意义。结果：CSE诱导的HUVEC凋亡呈浓度依赖和时间依赖。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)%。此后随着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24 h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,同样呈浓度依赖和时间依赖。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。相关分析显示,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)。与空白对照组和其他干预组相比,CSE组HUVEC凋亡[(44.0±3.6)%]增加(P<0.01)；而COX活性[(5.94±0.36)×10-1U/mg]、COXⅡmRNA表达(0.82±0.06)、COXⅡ蛋白表达(0.73±0.05)均下降(均P<0.05)；mtTFA mRNA表达(0.40±0.02)和mtTFA蛋白表达(0.39±0.06)也下降(均P<0.01)。而AZA组、AZA+CSE组与对照组比较差异无显著性(P>0.05)。BSP发现CSE组mtTFA基因启动区有多个甲基化CpG位点,平均甲基化率为(26.5±2.4)%。而空白对照组、AZA组、AZA+CSE组mtTFA基因启动区未发现甲基化CpG位点。结论：(1)CSE诱导HUVEC凋亡呈浓度依赖和时间依赖性,该凋亡为非caspase-3-依赖,并由COX介导。(2)CSE可能通过诱导mtTFA甲基化而下调mtTFA表达,从而抑制COXⅡ表达和COX活性,导致HUVEC凋亡增加。更多还原

【Abstract】 Objective:To observe the pulmonary vascular endothelial cell apoptosis, mitochondrial transcription factor A(mtTFA) and COX subunitⅡ(COXⅡ) expression in COPD patients and analysis the relationship among them. To investigate the possible role of them in COPD pathogenesis.Methods:According to the latest COPD diagnostic criteria,21 patients underwent pneumonectomy were divided into 2 groups: non-COPD group and COPD group (stable stage). TUNEL assay was used to assess cell apoptosis of pulmonary vascular endothelial cells. COX activity was measured by spectrophotometry. Semi-quantitative and real-time reverse transcription polymerase chain reaction (RT-PCR and real-time RT-PCR) were used to measure mtTFA mRNA and COXⅡmRNA expression. Moreover, immunohistochemistry and western-blot were used to detect mtTFA protein expression respectively. Western-blot was used to measure COXⅡprotein. Data were presented as Means±SD. Statistical analysis were performed using SPSS version 13.0 and differences were considered significant if P<0.05.Results:The smoking index of COPD group[(33.6±12.0)pack year] was much higher than that in non-COPD group[(12.1±8.9)pack year]. Difference between the two groups was of great significance (P<0.01). The apoptotic index of pulmonary vascular endothelial cells in COPD group[(13.8±1.9)%] was much higher than that in non-COPD group[(5.9±1.0)%]. Difference between the two groups was of great significance (P<0.01). mtTFA mRNA and mtTFA protein were lower in COPD group[(0.59±0.07) and (0.32±0.07)] than those in non-COPD group[(0.78±0.06) and (0.55±0.09)](all P<0.01). COX activity, COXⅡmRNA and COXⅡprotein in COPD group [(4.4±0.9)×10-1U/mg、(0.72±0.12) and (0.62±0.06)]were lower than those in non-COPD group[(7.6±0.4)×10-1U/mg、(0.86±0.16) and (0.89±0.13)] (P<0.01, P <0.05, P<0.01). The correlation analysis showed that the apoptotic index was negatively correlated with FEV1/FVC and FEV1%pre (r=-0.796, r=-0.827, all P<0.01), but positively correlated with smoke index (r=0.703, P<0.01). COX activity was negatively correlated with apoptotic index (r =-0.756, P<0.01). mtTFA potein was positively correlated with FEV1% pre, COXⅡprotein and COX activity(r=0.892, r=0.810, r=0.854, all P<0.01), but negatively correlated with apoptotic index and smoke index (r=-0.749, r=-0.763, all P<0.01).Conclusions:(1) Pulmonary vascular endothelial cell apoptosis is increased in COPD patients. (2) mtTFA expression, COXⅡexpression and COX activity are inhibited in COPD patients. (3)Cigarette smoking may be related with pulmonary vascular endothelial cell apoptosis in COPD through inhibiting mtTFA and COX II expression.Objective:To investigate whether there is aberrant mtTFA methylation in the lung tissue of COPD patients.Methods:Lung tissue of 21 patients underwent pneumonectomy was collected and divided into 2 groups:non-COPD group and COPD group. Genome DNA of lung tissue was extracted and mtTFA methylation satus was detected using Bisulfite DNA sequence (BSP). Statistical analysis were performed using SPSS version 13.0 and differences were considered significant if P<0.05.Results:Several methylated CpGs were found in mtTFA of the lung tissue of COPD group but no methylated CpG was found in non-COPD group. The methylation rate of mtTFA in COPD patients is (27.8±3.6) %. Conclusions:There is aberrant mtTFA methylation in the lung tissue of COPD patients.Objective:To investigate whether mtTFA methylation is involved in CSE-induced HUVEC apoptosis.Methods: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. Then, 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. Real-time RT-PCR was used to detect mtTFA mRNA and COXⅡmRNA. Western-blot and immunocytochemistry were used to detect mtTFA protein. Western-blot was used to detect COXⅡprotein. Moreover, DNA was extracted and mtTFA methylation status was detected by BSP. Data were presented as Means±SD. Statistical analysis were performed using SPSS version 13.0 and differences were considered significant if P<0.05. Results: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% to 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). Compared with the other three groups, apoptotic index increased in CSE-treated group [(44.0±3.6)%](P<0.01). While mtTFA mRNA, mtTFA protein, COX activity, COXⅡmRNA and COXⅡprotein decreased in CSE-treated group(P<0.01, P<0.01, P<0.05, P<0.05, P<0.05). Nevertheless, no significant differences between the control group, AZA group and AZA+CSE group were found (P>0.05). After BSP, several methylated CpGs were found in CSE-treated group but no methylated CpG was found in control group, AZA group and AZA+CSE group.The methylation rate of mtTFA in CSE group is (26.5±2.4)%.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) mtTFA methylation may contribute to CSE inducing HUVEC apoptosis.更多还原