【作者】 张文曦；

【导师】 奚肇庆；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 目的:慢性阻塞性肺病（COPD）肺动脉高压（PAH）的发病机制为,肺血管内皮功能失调、低氧性肺血管收缩、肺血管重构,而内皮细胞损伤是PAH的起始环节。复方薤白胶囊治疗COPD并PAH疗效显著,并于2004年取得国家发明专利权,专利号ZL02160139.9。本课题研究复方薤白提取液对过氧化氢（H₂O₂）诱导的凋亡内皮细胞的保护作用,及对凋亡相关基因及信号转导通路的影响,以探讨其抗凋亡机制。方法:体外培养人脐静脉内皮细胞（HUVEC）,以过氧化氢（H₂O₂）行内皮细胞凋亡造模,以MTT法筛选H₂O₂造模浓度及实验时间。设立5个实验组:正常组（HUVEC）、对照组（HUVEC+H₂O₂）、中药低、中、高剂量组（HUVEC+H₂O₂+复方薤白胶囊提取液3μg/ml、30μg/ml、300μg/ml）。分别以MTT法检测各组细胞增殖情况、光镜下形态学及Hoechst染色观察细胞凋亡、流式细胞检测细胞凋亡率、以Western Blot检测抗凋亡基因Bcl-2蛋白表达变化、罗丹明123（Rh123）染色检测线粒体膜电位、免疫组织化学法检测信号转导通路Caspase-3、NF—KB的表达。结果:1.造模结果:200μmol/l的H₂O₂为最佳造模浓度,该浓度作用HUVEC 2小时造模成功。造模后24小时作为最佳实验研究时间。2.MTT法检测细胞增殖结果显示:对照组HUVEC吸光度（OD值）明显降低,细胞增殖率仅9.29%（P＜0.01）。经不同浓度复方薤白胶囊提取液处理后,HUVEC的OD值、增殖率逐渐提高,分别为14.77%、31.98%、41.45%,以中、高剂量组增殖最为明显,与对照组比P＜0.01,具有明显的统计学意义。3.Hoechst染色观察细胞凋亡结果示:正常组凋亡率为2.0%,对照组见大量凋亡细胞,凋亡率为32.17%（P＜0.01）。中药低、中、高浓度组的凋亡率分别为28.33%、20.17%、13.33%。其中以中、高剂量组与对照组比P＜0.01,具有明显的统计学意义。4.流式细胞检测细胞凋亡率结果示:正常组细胞凋亡率为0.37%。对照组达14.39%（P＜0.01）。低、中、高中药组HUVEC细胞凋亡率分别为9.10%、7.91%、6.48%。与对照组比P＜0.01,具有明显的统计学意义。5.Western Blot检测Bcl-2结果示:正常组为0.673,对照组为0.438,低、中、高中药组分别为0.572、0.613、0.628,各中药组的Bcl-2基因表达量较对照组明显增高。6.罗丹明123（Rh123）染色检测线粒体膜电位结果显示:正常组细胞发出明显绿色荧光,而对照组荧光强度微弱。而经复方薤白胶囊提取液处理后,各中药组随药物浓度的增加,绿色荧光逐渐增多,其中以中、高剂量组为明显。7.Caspase-3免疫组织化学结果示:对照组的胞浆中见明显紫红色颗粒,各中药组胞浆中紫红色颗粒逐渐减少,且与药物浓度呈正相关。正常组阳性细胞为1.83%,对照组高达20.67%（P＜0.01）,低、中、高中药组的阳性细胞数分别为19.33%、14.33%、5.33%,其中以中、高剂量组与对照组相比,P＜0.01,具有明显的统计学意义。8.NF—KB免疫组织化学结果示:对照组的胞浆中见明显紫红色颗粒,各中药组胞浆中紫红色颗粒逐渐减少,且与药物浓度呈正相关。正常组阳性细胞为3.33%,对照组达18.5%,P＜0.01,低、中、高中药组分别为16.67%、12.33%、6.67%,其中以中、高剂量组与对照组相比,P＜0.01,具有明显的统计学意义。结论:复方薤白胶囊对氧化损伤所致凋亡的内皮细胞具有保护作用,能抗内皮细胞凋亡。其抗凋亡的机理为提高抗凋亡基因Bcl-2水平的表达。而该基因表达的上调,与该药改变了内皮细胞的线粒体膜的通透性,增强线粒体跨膜电位的表达,下调了Caspase-3及NF-κB的信号转导通路有关。由此我们认为,复方薤白胶囊治疗COPD、PAH的作用机制是,通过多个作用靶点保护内皮细胞,起到抗凋亡作用。更多还原

【Abstract】 Objective:The pathogenesis of chronic obstructive pulmonary disease（COPD） and pulmonary artery hypertension（PAH） are pulmonary endothelial dysfunction,hypoxic pulmonary vasoconstriction,vascular remodelling.Recent investigation proves that the damage of endothelial cell is the initiation of PAH.Compound Macrostem Onion Capsule （CMOC）,which got patent right in 2004,NO.ZL02160139.9,is an effective formula for patients with COPD and PAH.This project study was designed to assess the protective effect of CMOC on hydrogen dioxided（H₂O₂） induced apoptosis in human umbilical vascular endothelial cell（HUVEC）,apoptosis-related genes expression and signal transduction factors, to reveal the mechanism of anti-apoptosis of CMOC on Endothelial Cell.Methods:To establish the apoptotic models of HUVEC in vitro,using MTT assay to decide the best dose and the best time of H₂O₂.HUVEC were divided into five experimental groups:normal group,control group,CMOC group with three concentrations of 3μg/ml, 30μg/ml and 300μg/ml respectively MTT assay was used to measure the proliferation of the HUVEC.The apoptotic characteristics were detected by means of flow cytometry and Hoechst stain.Genes bcl-2 were detected by Western blotting.Using Rh123 dying to detect the expression of mitochondrial membrane potential.Using immunohistochemistry staining to detect the expression of caspase-3 and NF—KB.Results:1.The model of apoptotic were established by H₂O₂.The result showed that the best concentration of H₂O₂ was 200μmol/l,and the best duration to establish apoptotic model was 24 hours.2.MTT assay result:Cell optical density were lower than normal group and the proliferation rate of the cells was 9.29%in the control group（P＜0.01）.When treated by CMOC,the OD value were increased,and the proliferation rate of the cells were14.77%, 31.98%and 41.45%in CMOC groups respectively which increased with the increase of concentration.There were apparent statistical significance in the middle and high concentration groups,as compared with the control group,P＜0.01.3.Hoechst dying result:Apoptosis cell were seldom found in the normal group the apoptosis rate was 2.0%.In the control group a mass of apoptosis were detected and the apoptosis rate was 32.17%.After treated with CMOC,apoptosis cells were reduced in number.The apoptosis rate were 28.33%,20.17%,13.33%in CMOC groups respectively,in direct proportion to concentration.There were apparent statistical significance in the middle and high concentration groups,as compared with the control group,P＜0.01.4.Flow cytometry assay result:The cellular apoptosis rate were 0.37%in the normal group,14.38%in the control group,9.1%,7.9%and 6.48%in CMOC groups respectively which were proportional to the increase of concentration.There were apparent statistical significance compared with the control group,P＜0.01.5.Western blot result:The value of Bcl-2 expression were 0.673 in normal group, 0.438 in control group,0.572,0.613 and 0.628 in CMOC groups respectively,in direct proportion to concentration.It shows that the anti-apoptosis gene expression was increased after CMOC treatment.6.Mitochondrial membrane potential result:In the normal group Rh123 dying emitted apparent green florescent light,while in the control group the florescent light were feeble. After treated with CMOC,the florescent light increased in intensity and were positively correlated with concentration,especially in the middle and high concentration groups.7.Caspase-3 immunohistochemistry result:The prunosus particle in the control group were the most apparent,after treated with CMOC,the prunosus particle reduced in number. The positive cells were 1.83%in normal group,20.67%in control group,19.33%,14.33% and 5.33%in CMOC groups respectively,in direct proportion to concentration.The most apparent statistical significance were found in the middle and high concentration groups,as compared with the control group,P＜0.01.8.NF—KB immunohistochemistry result:The prunosus particle in the control group were the most apparent,but after treated with CMOC,the prunosus particle reduced in number.The positive cells were 3.33%in normal group,18.5%in control group, 16.67%,12.33%and 6.67%in CMOC groups respectively,which were again in direct proportion to concentration.The most apparent statistical significance were found in the middle and high concentration groups,as compared with the control group,P＜0.01.Conclusions:These results indicate that Compound Macrostem Onion Capsule（CMOC） has the protective effect on the H₂O₂ induced cellular apoptosis in HUVEC,and has the function of anti-apoptosis.Its mechanism lies in its effect in increasing the expression of Bcl-2.The expression of this gene may change the permeability of mitochondrial,enhance the expression of membrane potential,reduced Caspae-3 and NF-KB signal pathway,and suppress caspase-3 induced cell apoptosis.Thus we think CMOC can protect HUVEC via many targets and can prevent apoptosis.更多还原