【作者】 曾慧卉；

【导师】 陈平；

【作者基本信息】 中南大学 ， 内科学， 2012， 博士

【摘要】 第一部分COPD患者肺组织中Bcl-2基因启动子区异常甲基化及其与肺血管内皮细胞凋亡的关系目的：检测比较非吸烟非COPD患者、吸烟非COPD患者及吸姻并COPD患者肺组织样本间Bcl-2基因甲基化位点、Bcl-2表达、肺血管内皮细胞凋亡的变化,并分析它们之间的关系,探讨Bcl-2基因启动子区异常甲基化在COPD发病机制中的可能作用。方法：收集2007年9月至2011年2月在中南大学湘雅二医院胸外科行全麻下肺叶切除术或肺段切除术的周围型肺癌31例,所有患者的病理类型均为非小细胞肺癌。根据COPD诊断标准及吸烟习惯将其分成三组：非吸烟非COPD组10例、吸烟非COPD组10例、吸烟并COPD组(稳定期)11例。TUNEL法检测肺血管内皮细胞凋亡指数,免疫组织化学法检测肺血管内皮细胞Bcl-2蛋白的表达,Western-blot及real-time RT PCR检测肺组织Bcl-2、Bax、cyt C表达,采用亚硫酸氢盐测序法(BSP)进行甲基化水平评估和测序。各组计量资料以(Mean±SD)表示,采用Anova检验比较呈正态分布的多个独立样本均数,LSD-t检验正态分布的样本均数间的多重比较；采用Kruskal-Wallis H检验比较非正态分布的多个独立样本均数,采用Nemenyi检验非正态分布的样本均数间的多重比较；部分实验指标间作单因素直线相关分析及多元线性回归分析,P<0.05为有统计学意义。结果：吸烟并COPD(稳定期)组肺血管内皮细胞凋亡指数(22.79±5.30)%较其他两组非COPD患者增高(P<0.01)。该组的肺血管内皮细胞Bcl-2表达率为(5.18±3.76)%,较其他两组非COPD患者明显下降(P<0.01)。COPD组患者肺组织Bcl-2蛋白、mRNA的相对表达量和Bcl-2启动子区甲基化水平分别为0.31±0.11、0.14×10-3.4±0.02×10-3、(12.23±4.39)%,吸烟非COPD组肺组织Bcl-2蛋白、mRNA的相对表达量、启动子区甲基化水平分别为0.634±0.13、1.58×10-3±0.26x10-3、(1.45±1.15)%,非吸烟非COPD组肺组织Bcl-2蛋白、mRNA的相对表达量和Bcl-2启动子区甲基化水平分别为0.62±±0.12、1.70×10-3±0.16×10-3、(1.64±0.16)%;COPD组患者肺组织Bc1-2相对表达量均较非COPD组明显下降,而Bcl-2启动子区甲基化水平均较非COPD组上升(均P<0.01)。Bcl-2启动子区异常甲基化,有50%的异常甲基化发生在位于-127bp的CpG中的C位点COPD组肺组织Bax蛋白和mRNA的相对表达量分别为0.89±0.04和1.38×10-3±0.21×10-3,吸烟非COPD组肺组织Bax蛋白和mRNA的相对表达量分别为0.62±0.13和0.27×10-3±0.03×10-3,非吸烟非COPD组肺组织Bax蛋白、mRNA的相对表达量分别为0.63±0.11和0.26×10-3±0.02×10-3; COPD组患者肺组织Bax相对表达量均较非COPD组明显上升(均P<0.01)。COPD组肺组织胞浆cyt C蛋白表达均较非COPD组明显上升(P<0.01),但整体cyt C mRNA各组间并无明显差异(P>0.05)。相关性分析显示肺组织Bcl-2的蛋白表达与肺血管内皮细胞凋亡呈负相关(r=-0.78,P<0.01),与吸烟指数呈正相关(r=-0.76,P<0.05)；Bcl-2基因启动子区的甲基化水平与肺血管内皮细胞中的Bcl-2蛋白表达呈负相关(r=-0.70,P<0.01,图1-27),与吸烟指数呈正相关(r=0.83,P<0.01)结论：1.COPD患者肺血管内皮细胞凋亡增加,肺组织的Bax表达上升,肺组织及肺血管内皮细胞的Bcl-2表达下降。2.COPD患者肺内Bcl-2表达下降,可能导致线粒体依赖的凋亡途径激活,出现肺血管内皮细胞凋亡,参与COPD的疾病过程。3.COPD患者肺组织Bcl-2基因启动子区发生异常甲基化,位于-127bp的CpG中的C位点为异常甲基化好发部位。4.吸烟可能通过诱发Bcl-2基因启动区异常甲基化,从而影响Bcl-2蛋白表达,进而参与COPD患者肺血管内皮细胞凋亡。5.人群间对吸烟可能诱导的Bcl-2启动区异常甲基化具有不同的易感性。第二部分肺气肿动物模型的肺组织中Bcl-2基因启动子区异常甲基化及其与肺血管内皮细胞凋亡的关系目的：检测比较香烟提取物腹腔注射构建肺气肿动物模型、对照动物模型、去甲基化干预动物模型的肺组织样本间Bcl-2基因甲基化位点、Bcl-2表达、肺血管内皮细胞凋亡的变化,并分析它们之间的关系,探讨Bcl-2基因启动子区异常甲基化在COPD发病机制中的可能作用。方法：BALB/C小鼠40只分成四组造模,每组10只。PBS组于第1、11、15、17、19、22天经腹腔注射PBS缓冲液(0.01M,PH7.2-7.4)0.3ml; CSE组于第15、17、19天经腹腔注射PBS缓冲液(0.01M,PH7.2-7.4)0.3ml,第1,11,22天经腹腔注射CSE0.3ml; CSE+AZA组于第1、11、22天经腹腔注射CSE0.3ml,第15、17、19天经腹腔注射AZA,每次注射AZA量=2.5mg/kg×小鼠体重;AZA组于第1,11,22天腹腔注射PBS缓冲液0.3ml,第15、17、19天经腹腔注射AZA,每次注射AZA量=2.5mg/kg×小鼠体重；第28天停止实验。TUNEL法检测肺血管内皮细胞的凋亡指数,免疫组织化学法检测肺血管内皮细胞Bcl-2蛋白的表达,Western-blot及real-time RT PCR检测肺组织Bcl-2、Bax、cyt C表达。各组计量资料以(Mean±SD)表示,采用Anova检验比较呈正态分布的多个独立样本均数,LSD-t检验正态分布的样本均数间的多重比较；采用Kruskal-Wallis H检验比较非正态分布的多个独立样本均数,采用Nemenyi检验非正态分布的样本均数间的多重比较;部分实验指标间作单因素直线相关分析及多元线性回归分析,P<0.05为有统计学意义。结果：CSE组肺血管内皮细胞凋亡指数(25.88±7.55)%较其他三组明显增高(P<0.01)。该组的肺血管内皮细胞Bcl-2表达率为0.35+0.11,较其他三组动物模型明显下降(P<0.01)。COPD组患者肺组织Bcl-2蛋白、mRNA的相对表达量和Bcl-2启动子区甲基化水平分别为0.49±0.10、2.46×10-3±1.02×10-3、(35.68±5.99)%,较其他三组Bcl-2表达明显下调,启动子区甲基化程度升高明显(P<0.05)。最易发生异常甲基化的CpG位点为5’起始的第二、八个CpG位点(一1884bp.一1740bp)；有26.67%的甲基化发生在这两个位点。CSE组肺组织Bax蛋白和mRNA的相对表达量分别为0.49+0.11和1.01×10-3+0.34×10-3,胞浆cyt C蛋白的相对表达量为0.49+0.11,均较正常对照组改变明显(均P<0.01)。但AZA可以逆转CSE导致的Bcl-2启动子区甲基化水平上升、Bcl-2下调、Bax表达增加、凋亡增加等变化,且CSE+AZA组Bcl-2.Bax及胞浆cyt C表达同对照组无明显统计学差异(P>0.05)。相关性分析显示肺组织Bcl-2的蛋白表达与胞浆cyt C表达负相关(r=-0.48,P<0.01),肺组织及非血管内皮细胞Bcl-2的蛋白表达与肺血管内皮细胞凋亡均呈负相关,r(肺组织)=-0.96,r(肺血管内皮细胞)=-0.97,均P<0.01；且与动物肺功能多个指标相关。Bcl-2基因启动子区的甲基化水平与肺组织及肺血管内皮细胞中的Bcl-2蛋白表达呈负相关,r(肺组织)=-0.91,r(肺血管内皮细胞)=-0.96,均P<0.01；与肺血管内皮细胞凋亡呈正相关(r=0.95,P<0.01)结论：1.CSE腹腔注射建立的肺气肿模型中肺血管内皮细胞凋亡增加,肺组织的Bax表达上升,肺组织及肺血管内皮细胞的Bcl-2表达下降。2.CSE腹腔注射建立的肺气肿模型中肺内Bcl-2表达下降,可能通过激活线粒体依赖凋亡途径,导致肺血管内皮细胞凋亡,最终参与COPD的疾病过程。3.CSE腹腔注射建立的肺气肿模型中Bcl-2基因启动子区发生异常甲基化,异常甲基化的好发位点为-1784bp、-1705bp的CpG中C位点。4.去甲基化干预,AZA腹腔注射能够逆转CSE导致的Bcl-2启动子区异常甲基化、Bcl-2蛋白表达下调及肺血管内皮细胞凋亡。5.香烟暴露引起Bcl-2启动子区发生异常甲基化,进而Bcl-2蛋白表达下降,并可能导致cyt C释放入胞浆——肺血管内皮细胞的线粒体依赖凋亡通路启动,最终出现COPD。第三部分Bcl-2基因启动子区异常甲基化在CSE诱导HUVEC凋亡中的作用目的：研究CSE诱导HUVEC凋亡的可能机制,并探索Bcl-2基因启动子区异常甲基化在凋亡过程中扮演的可能角色。方法：体外培养HUVEC,给予不同浓度(0%-20%)和不同时间(Oh-12h)的CSE干预,采用Annexin V-FITC/PI双染,流式细胞仪检测凋亡率,免疫细胞化学法、Western-blot及realtime-RT PCR检测Bcl-2表达。进一步将HUVEC分为四组：空白组,CSE(5%)组,AZA+CSE组,AZA组,分别给予不同的干预。检测各组细胞的凋亡率；免疫细胞化学法、Western-blot及realtime-RT PCR检测Bcl-2表达,BSP法检测各组Bcl-2基因启动子区甲基化状态及水平。采用Anova检验比较呈正态分布的多个独立样本均数,LSD-t检验正态分布的样本均数间的多重比较；采用Kruskal-Wallis H检验比较非正态分布的多个独立样本均数,采用Nemenyi检验非正态分布的样本均数间的多重比较；部分实验指标间作单因素直线相关分析及多元线性回归分析,P<0.05为有统计学意义。结果：CSE孵育2h后,HUVEC凋亡呈时间依赖模式。5%CSE干预2h后的凋亡率达到(3.13+0.68)%。此后随着CSE浓度继续增加,凋亡率也增加,但细胞坏死也逐渐明显。10%CSE干预12h后HUVEC凋亡达到最高峰(46.13+5.07)%。与凋亡相反,随着CSE干预浓度的增加,Bcl-2蛋白表达逐渐降低。5%CSE干预12h后HUVEC凋亡率为(23.77±3.40)%,较0%及2.5%CSE组明显上升；而Bcl-2的蛋白相对表达量为0.16+0.05,较0%及2.5%CSE组明显下降(P<0.01)。去甲基化试剂AZA干预能够改善受损的Bcl-2表达,并降低细胞凋亡率(P<0.01)。BSP进一步发现CSE组Bcl-2基因启动区发生异常甲基化,甲基化水平为(14.55+3.15)%,而AZA干预能够逆转或预防Bcl-2基因启动子区的异常甲基化(P<0.01)。最易发生异常甲基化的位点为5’起始的第九个CpG (+5bp)的C位点；有33.33%的甲基化发生在该位点。相关性分析则发现Bcl-2蛋白表达同胞浆cyt C蛋白表达呈负相关,与Bcl-2基因启动子区甲基化水平也呈负相关。结论：(1)CSE诱导的HUVEC凋亡可能通过线粒体依赖的凋亡途径启动。(2)CSE可能通过诱导Bcl-2甲基化而下调Bcl-2表达,从而下降线粒体膜稳定性,导致cyt C线粒体释放入胞浆,激发凋亡,最终HUVEC凋亡增加。(3)香烟暴露是多种疾病的危险因素,同时内皮细胞凋亡也参与多种疾病的发病机制,脐静脉内皮细胞能够良好模拟人内皮细胞,提示CSE干预诱导的内皮细胞自身Bcl-2基因启动子区甲基化可能参与多种内皮细胞相关疾病,如COPD、冠心病、类风湿性关节炎等。更多还原

【Abstract】 The First Part The relationship between deregulation of Bcl-2promoter methylation and pulmonary endothelia apoptosis in chronic obstructive pulmonary diseaseObjective:Observe and analyze the Bcl-2methylaiton loci, Bcl-2expression level, and endothelia apoptosis status in COPD patients, asympotomous smokers and non-smokers, to investigate the possible role of deregulated Bcl-2promoter methylation in COPD pathogenesis.Method:31cases of peripheral lung cancer patients were enrolled, who were defined pathologic character as NSLC, devied into3groups as follow,11cases of COPD patients,10cases of asympotomous smokers and non-smokers. TUNEL assay was used to assess apoptosis status, immunohistochemistry to detect the Bcl-2expression in pulmonary endothelia, Western-blot and realtime-RT PCR to measure the Bcl-2, expression in lung tissue. BSP was used to assess the methylation satatus and sequence the methylation. Analyze the correlationship with regression test. Data was presented with Mean±SD, Anova and LSD-t analized the normality data, and Kruskal-Wallis H and Nemenyi analized the non-normality data. P<0.05brings the statistics significant.Result:Apoptosis index of endothelia in COPD group was(22.79±5.30)%, which was higher than the other two non-COPD groups (P<0.01). The positive rate of Bcl-2expression in endothelia from COPD group was (5.18±3.76)%, obviously lesser than the two non-COPD groups (P<0.01). Bcl-2expression in lung tissure from COPD patients was0.31±0.11(Western-blot),1.40×10-4±0.16×10-4(realtime-RT PCR) P<0.01, which was lower than the two non-COPD groups (P<0.01). Moreover the methylation lever of Bcl-2promoter in COPD group was (12.23±4.39)%, which was higer than other groups. In addition the deregulated methylation of Bcl-2has relationship with endothelia apoptosis, Bcl-2expression and smoker index.Conclusions:1. Increased endothelia apoptosis and Bax expression were found in COPD patients, with decreased Bcl-2expression.2. The decreased Bcl-2expression might stimulate the mitochondria depend apoptosis pathway, and lead to endothelia apoptosis and participate the COPD pathogenesis.3. There are abnormal methylation of Bcl-2promoter in COPD, and the-127bp C was the most common methylation loci.4. Smoke might induce bcl-2promoter abnormal methylaiton, and lead to deregulate Bcl-2expression, which might play a role in COPD pathogenesis.5. Different clones of people might show different suscepticity of abnormal methylation in Bcl-3promotor. The Second Part The relationship between deregulation of Bcl-2promoter methylation and pulmonary endothelia apoptosis in emphysematous modelObjective:Observe and analyze the Bcl-2methylaiton loci, Bcl-2expression level, and endothelia apoptosis status in CSE-induced emphysematous models, control models and demethylation models, to investigate the possible role of deregulated Bcl-2promoter methylation in COPD pathogenesis.Method:Forty BALB/C mice were devied into4groups as follow, PBS group was intraperitoneally injected with PBS in the1st,11th,15th,17th,19th,22nd days. CSE group was intraperitoneally injected as PBS group, except in the1st,11th,22nd day changed to CSE from PBS. CSE+AZA group was intraperitoneally injected as the CSE group, except changed PBS to AZA in the15th,17th,19th days. AZA group also was injected as the same, just changed the CSE to PBS in the15th,17th,19th days. TUNEL assay was used to assess apoptosis status, immunohistochemistry to detect the Bcl-2expression in pulmonary endothelia, Western-blot and realtime-RT PCR to measure the Bcl-2, expression in lung tissue. BSP was used to assess the methylation satatus and sequence the methylation. Analyze the correlationship with regression test. Data was presented with Mean±SD, Anova and LSD-t analized the normality data, and Kruskal-Wallis H and Nemenyi analized the non-normality data. P<0.05brings the statistics significant.Result:Apoptosis index of endothelia in CSE group was(25.88+7.55)%, which was higher than the other three groups (P<0.01). The positive rate of Bcl-2expression in endothelia from CSE group was0.35±0.11)%, obviously lesser than the other three groups (P<0.01). Bcl-2expression in lung tissure from COPD patients was0.49±0.10(Western-blot),2.46x10-3±0.16×10-3(realtime-RT PCR), P<0.01, which was lower than the other three groups (P<0.01). The methylation lever of Bcl-2promoter in CSE group was (35.68±5.99)%, which was higer than other groups. The most common methylated loci were-1884bp and-1740bp, almost26.67%of methylation were occurred in the two loci. The lung tissue from CSE group also present higher level of Bax and plasmic cyt C expression than control group (P<0.01). Moreover, the demethylation reagent, AZA, could improve the lung function and apoptosis with increased Bcl-2and decreased Bax. In addition, the deregulated methylation of Bcl-2has relationship with endothelia apoptosis and Bcl-2expression. On the other hand, Bcl-2protein expression also has a negative relationship with plasmic cyt C, the sign of mitochondrial-depend apoptosis pathway.Conclusions:1. Increased endothelia apoptosis and Bax expression with decreased Bcl-2expression were found in CSE-induced emphysema models.2. The decreased Bcl-2expression, induced by CSE injection, might stimulate the mitochondria-depend apoptosis pathway, and lead to endothelia apoptosis and participate the COPD pathogenesis.3. There are abnormal methylation of Bcl-2promoter in CSE-induced emphysematous models, and the-1884bp and-1740bp were the most common methylation loci.4. Demethylation treatment, AZA injection, could prevent the abnormal methylation of Bcl-2promoter, decreased expression of Bcl-2, and increased apoptosis of endothelia.5. Smoke induce bcl-2promoter abnormal methylaiton, and lead to deregulate Bcl-2expression, which might stimulate the cyt C released by mitochondrial, then enter the mitochondrial-depend apoptosis pathway, and play a role in COPD pathogenesis. The Third Part The role of deregulated Bcl-2promoter methylation in CSE-induced HUVEC apoptosisObjective:To investigate the possible mechanism in CSE-induced HUVEC apoptosis, and discuss the effect of deregulated Bcl-2promoter methylation in this apoptosis programme.Method:Treat HUVEC with different concentrates (0-20%) of CSE during different time (0-12h). Detect the apoptosis rate by flow cytometry with double die (Annexin V-FITC/PI). Immunocytochemistry、 Western-blot and realtime-RT PCR were used to assess Bcl-2expression after different concentrate of CSE treatment. Furthermore, divided cells into four groups:control group, CSE(5%)group, CSE+AZA group, AZA group, give each group treatments respectively. Measure methylation of Bcl-2promoter, Bcl-2expression, Bax expression, plasmic cyt C and apoptosis rate with BSP, immunocytochemistry, Western-blot, realtime-RT PCR and flow cytometry respectively. Data was presented with Mean±SD, Anova and LSD-t analized the normality data, and Kruskal-Wallis H and Nemenyi analized the non-normality data. P<0.05brings the statistics significant.Result:After the first2h treatment, the CSE-induced HUVEC apoptosis was increased depend time and concentrate. However, if the concentrate was too high, more than10%, the necrosis cells would be the most dead cell, rather than apoptosis. The highest apoptosis rate was46.13±5.07%after10%CSE treat last for12h. After12h of5%CSE treatment, the Bcl-2protein relative expression was0.16±0.05, obviously lower than0.0%and2.5%CSE group (P<0.01). AZA could improve the Bcl-2expression and apoptosis(P<0.01). BSP found that CSE induced abnormal methylation of Bcl-2promoter in HUVEC, though AZA could prevent this deregulation. The most common methylaltion loci was the+5bp C, almost33.33%methylation occurred in this loci. The regression analysis found that Bcl-2expression had positive relationship with plasmic cyt C, which showed a positive relationship with apoptosis, and a negative relationship with methylation level.Conclusion:1. CSE-induced HUVEC apoptosis might depend on the mitochondrial apoptosis pathway.2. CSE induce abnormal methylation of Bcl-2promoter, which might decrease the expression of Bcl-2, destruct the mitochondrial membrane, lead cyt C release to cell plasma, stimulate the mitochondrial-depend apoptosis pathway. At last, increase the apoptosis.3. Cigarette exposure was a risk factor for many diseases, and endothelia apoptosis also participates in many pathogenesis of diseases. Because the HUVEC can mimic the human endothelia, it is might be possible and inspiriting that the CSE-induced abnormal methylation of Bcl-2promoter might play a role in many endothelia relative diseases, such as COPD, coronary disease, rheumatic disease.更多还原