细胞色素C氧化酶在慢性阻塞性肺疾病中的表达及其与肺血管内皮细胞凋亡的关系

Cytochrome C oxidase expression and endothelial cell apoptosis in lungs of patients with chronic obstructive pulmonary disease

目的观察COPD患者肺组织中细胞色素C氧化酶（COX）和血管内皮细胞凋亡的变化，探讨COPD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行肺切除术的周围型肺癌患者20例，参照COPD的诊断标准和患者的吸烟状况分为对照组（7例）、吸烟非COPD组（7例）和吸烟并COPD组（6例），取远离病变的肺组织。采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法检测肺血管内皮细胞凋亡，采用分光光度法、半定量逆转录PCR法和Western blot法分别检测肺组织中COX活性、COXⅡ亚基（COXⅡ）mRNA和蛋白表达，免疫组织化学法检测肺血管内皮细胞COXⅡ蛋白的分布和表达。多组间比较采用单因素方差分析，用LSD—t检验进行两两比较。结果吸烟并COPD组肺血管内皮细胞凋亡指数为（13．8±1．9）％，明显高于吸烟非COPD组[（9．6±0．8）％]和对照组[（5．9±1．0）％]；吸烟并COPD组肺组织中COX活性为（4．4±0．7）×10^5U／kg，COXⅡ mRNA相对表达量为0．76±0．17，肺血管内皮细胞中COXⅡ蛋白相对表达量为14．5±1．6，明显低于吸烟非COPD组[（6．0±0．6）×10^5U／kg、0．81±0．15和18．6±2．1]和对照组[（7．6±0．4）×10^5U／kg、0．86±0．20和23．8±3．4]，差异均有统计学意义（t值为-13．66～13．27，均P〈0．05）；COX活性与FEV，占预计值％和FEV1／FVC呈显著正相关（r值分别为0．84和0．91，均P〈0．01），与吸烟指数呈显著负相关（r=-0．78，P〈0．01）；肺血管内皮细胞凋亡与COXⅡ蛋白表达呈显著负相关（r=-0．75，P〈0．01）。结论COPD患者的肺血管内皮细胞凋亡增加，COX表达和活性下降，且均与吸烟有相关性。COX可能参与介导COPD肺血管内皮细胞凋亡过程。

Objective To observe the expression and activity of cytochrome C oxidase and pulmonary endothelial apoptosis in lungs of COPD patients, and therefore to investigate the pathogenesis of COPD. Methods According to COPD diagnostic criteria and patients＇ smoking status, 20 patients who had undergone lung resections were divided into 3 groups： group A, non-smokers without COPD （7 patients） ; group B, smokers without COPD （7 patients）; and group C, smokers with COPD （6 patients）. Their normal lung tissues were used in the study. TUNEL assay was used to assess pulmonary vascular endothelial apoptosis. Cytochrome C oxidase （COX） activity was measured by spectrophotometry. Semi-quantitative polymerase chain reaction and Western blot were used to measure COX subunit Ⅱ （ COX Ⅱ） mRNA and protein expression respectively. Moreover, immunohistochemistry was used to detect COX Ⅱprotein expression in pulmonary endothelial cells. One-way ANOVA and LSD-t test were used for statistics. Results The apoptotic index of pulmonary vascular endothelial cells in group C [ （ 13. 9 ± 1.9） % ] was significantly higher than that in group B [ （9. 6 ± 0. 8 ） % ] and group A [ （5. 9 ± 1.0） % ]. While COX activity and COX Ⅱ mRNA expression of lung tissues and COX Ⅱprotein expression of pulmonary endothelial ceils in group C [ （4.4 ±0.7）×10^5 U/kg, （0.76 ±0. 17） and （ 14.5 ± 1.6）, respectively] were significantly lower than those in group A [ （7.6 ± 0. 4） ×10^5U/kg, 0. 86 ± 0. 20 and 23.8 ± 3. 4, respectively ] and groupB [（6.0±-0.6） xl05 U/kg, 0.81±0.15 and 18.6±2.1, respectively] （t= -13.66-13.27, all P 〈0.05）. The correlation analyses showed that COX activity was correlated positively with FEV1% pred and FEV1/FVC （ r = 0. 84, 0. 91, all P 〈 0.01 ）, but negatively with smoking index （ r = - 0. 78, P 〈 0. 01 ）. The apoptotic index of pulmonary vascular endothelial cells was negatively correlated with COX Ⅱ expression （ r = - 0. 75, P 〈 0. 01 ）. Conclusions Increased pulmonary vascular endothelial apoptosis and decreased COX expression and activity, which were often associated with cigarette smoking, were present in COPD patients. COX might mediate the pulmonary vascular endothelial apoptosis in COPD.