【作者】 扈丽娟；

【导师】 胡晓芸；

【作者基本信息】 山西医科大学 ， 呼吸内科， 2011， 硕士

【摘要】 研究背景吸烟是心血管疾病发生发展的一个独立而重要的危险因素。吸烟可以损伤血管内皮细胞,导致内皮细胞功能障碍,引起体内纤溶系统的异常变化,从而增加血栓形成的风险。众所周知,血液的纤溶能力主要取决于组织型纤溶酶原激活剂(t-PA)及其快速抑制剂纤溶酶原激活物抑制物-1(PAI-1)的活性。大量文献表明,糖尿病、冠心病可伴发纤溶功能异常,而有关吸烟引起的纤溶变化研究尚少。尼古丁是烟草的主要致病成分之一,其通过损伤内皮细胞导致血液纤溶功能下降,但该作用的具体机制尚不十分清楚。蛋白激酶C(PKC)为细胞内信号转导的重要递质,广泛参与细胞的分泌、增殖、分化等功能,其作用日益受到关注。目前,尼古丁所致内皮细胞纤溶功能下降与PKC通道的关系研究较少,且结论不一。辛伐他汀为临床常用的血脂调节剂,研究表明该药还具有内皮保护、抗炎、促进纤溶等作用。辛伐他汀是否对尼古丁介导的人脐静脉内皮细胞纤溶功能紊乱具有保护作用及其可能机制目前尚无报道。目的研究尼古丁及不同浓度的PKC阻断剂Staurosporine(STS)作用于人脐静脉内皮细胞(HUVECs)后其t-PA、PAI-1蛋白及mRNA表达的变化,探讨尼古丁对HUVECs纤溶影响的可能机制;采用不同浓度的辛伐他汀预处理内皮细胞,了解该药对尼古丁介导的HUVECs表达t-PA、PAI-1蛋白及mRNA的影响。方法1.实验分组HUVECs株培养后接种于25cm×25cm的培养瓶中,随机分为①对照组:培养液中加入与尼古丁相同体积的无菌PBS液;②尼古丁组:培养液中加入终浓度为100μmol/L的尼古丁;③STS干预组:培养液中分别加入终浓度为20、50、100μmol/L的STS预处理细胞30min,再加入100μmol/L的尼古丁共同培养;④辛伐他汀干预组:培养液中分别加入终浓度为1、10、100μmol/L的辛伐他汀预处理细胞2h,再加入100μmol/L的尼古丁共同培养。2.检测方法同步培养24h后收集各组的细胞及上清液,采用酶联免疫吸附双抗体夹心法(ELISA)测定各组细胞上清液中t-PA、PAI-1蛋白浓度,逆转录-聚合酶链反应法(RT-PCR)检测各组细胞中t-PA和PAI-1 mRNA的表达水平。结果1.尼古丁组PAI-1mRNA及蛋白含量〔0.969±0.112,(66.051±2.890)ng/ml〕与对照组〔0.099±0.013,(38.660±1.749)ng/ml〕比较显著升高,差异均有统计学意义(均P﹤0.01)。尼古丁组t-PA mRNA的表达(0.099±0.011)与对照组(1.011±0.132)比较明显下降,差异有统计学意义(P﹤0.01);尼古丁组t-PA蛋白(0.725±0.211)ng/ml表达与对照组(1.082±0.131)ng/ml比较,差异无统计学意义(P>0.05)。2.STS干预结果各浓度STS干预组PAI-1mRNA及蛋白〔0.588±0.077,(61.922±5.821)ng/ml〕、〔0.530±0.070,(57.976±8.009)ng/ml〕、〔0.222±0.029,(43.291±1.878)ng/ml〕均较尼古丁组降低,差异均有统计学意义(均P﹤0.01);各STS干预组PAI-1mRNA及蛋白高于对照组,差异均有统计学意义(均P﹤0.01)。20μmol/LSTS组t-PA mRNA(0.184±0.023)与尼古丁组比较,差异无统计学意义(P>0.05),50、100μmol /L STS组t-PA mRNA(0.293±0.037,0.412±0.053)较尼古丁组升高,但仍低于对照组,差异均有统计学意义(均P﹤0.01);各组t-PA蛋白与对照组及尼古丁组比较,差异均无统计学意义(均P>0.05)。3.辛伐他汀干预结果各浓度辛伐他汀干预组PAI-1mRNA及蛋白〔0.581±0.095,(60.788±2.500)ng/ml〕、〔0.511±0.084,(54.955±3.971)ng/ml〕、〔0.0219±0.037,(41.924±4.472)ng/ml〕均较尼古丁组降低,差异均有统计学意义(均P﹤0.01),以100μmol/L辛伐他汀组降低最为显著,与对照组比较差异无统计学意义(P>0.05)。1μmol /L辛伐他汀组t-PA mRNA(0.187±0.025)与尼古丁组比较,差异无统计学意义(P>0.05);10、100μmol/L辛伐他汀组t-PA mRNA(0.278±0.038)、(0.332±0.046)较尼古丁组升高,但仍低于对照组,差异均有统计学意义(均P﹤0.01);各组t-PA蛋白与对照组及尼古丁组比较,差异均无统计学意义(均P>0.05)。结论1.尼古丁通过上调HUVECs PAI-1蛋白和mRNA的表达,对内皮细胞的纤溶活性具有抑制作用。2.尼古丁介导的内皮细胞纤溶紊乱部分通过P KC通道起作用。3.辛伐他汀对尼古丁所致的内皮细胞纤溶损害具有保护作用。更多还原

【Abstract】 BackgroudCigarette smoking was considered as a dependent danger factor to the development of cardio- vascular disease .It could damage the vascular endothelial cells, lead to endothelial cells dysfunction, as well as trigger fibrinolysis imbalance, then increasing the risk of thrombosis. As we all know, fibrinolytic system relies heavily on the activity of tissue-type plasminogen activator ( t-PA )and tissue-type plasminogen inhibitor-1(PAI-1) released by vascular endothelial cells. There has many reports about the disfunction of fibrinolysis studies associated with diabetes , coronary heart disease .But studies about disfunction of fibrinolysis associated with cigarette smoking was reported few. Nicotine, one of the main harmful substances of smoking, can damage the fibrinolytic function of endothelial cells, and its related mechanism was entirely unclear .Protein kinase C (PKC) is an important biological intracellular signal transduction pathway in the body, it widespread participate the secretion ,proliferation and differentiation of cells, its get to pay close attention increasingly .Now, the studies about nicotine decrease the fibrinolytic of endothelial cells associate with PKC is few reported. Simvastatin as a represent of liqid- lowering statin drug, it has many new function ,for example ,the protection of endothelial cells, inhibit inflammation, promote fibrolysis ,etc. Simvastatin can wether or not improved fibrinolytic activity induced by nicotine and its related mechanism has not been reported.ObjectiveTo study the effect of nicotine and different concentrations of staurosporine on the expression of t-PA and PAI-1 protein and mRNA in human umbilical vein endothelial cell (HUVECs), To explore the mechanism of fibrinolytic function stimulated by nicotine . Using different concentration of simvastatin to pretreatment the HUVECs, and observed the influence of simvastatin to the expression of t-PA,PAI-1 protein and mRNA stimulated by nicotine.MethodsExperimental Group:HUVECs were cultured in 25cm2 culture flasks and randomly divided into①the control group②100μmol/L nicotine group③STS intervention group. The cells were treated with STS at concentration of 20、50、100μmol/L for 30min,and then exposed to nicotine at concentration of 100μmol/L.④Simvastatin intervention group .The cells were treated with simvastatin at concentration of 1、10、100μmol/L for 2h,and then exposed to nicotine at concentration of 100μmol/L.The cells and supernatants of each group were collected after 24 hours cultured .The expression of t-PA and PAI-1 protein were measured by ELISA and the RNA expression of t-PA and PAI-1 was determined by RT-PCR.Results1.Compared with control group〔0.099±0.013(,38.660±1.749)ng/ml〕, the PAI-1 mRNA and protein expression of nicotine group〔0.969±0.112,(66.051±2.890)ng/ml〕were increased significantly(P<0.01, respectively).Compared with control group(1.011±0.132),the t-PA mRNA(0.099±0.011)expression of nicotine group were decreased significantly(P<0.01).However, the expression of t-PA protein was no significantly different(P>0.05).2.Compared with nicotine group , the PAI-1 mRNA and protein expression of different concentrations of STS intervention group〔0.588±0.077,(61.922±5.821)ng/ml〕、〔0.530±0.070,(57.976±8.009)ng/ml〕、〔0.222±0.029,(43.291±1.878)ng/ml〕were decreased significantly ,but still higher than control group(P<0.01, respectively). Compared with nicotine group, the t-PA mRNA expression of 20μmol/L STS group(0.184±0.023)were increased not significantly(P>0.05);The t-PA mRNA expression of 50、100μmol/L STS group(0.293±0.037)、(0.412±0.053)were increased significantly ,but still lower than control group(P<0.01, respectively).The expression of t-PA protein was no significantly different (P>0.05).3.Compared with nicotine group , the PAI-1 mRNA and protein expression of different concentrations of simvastatin intervention group〔0.581±0.095,(60.788±2.500)ng/ml〕、〔0.511±0.084,(54.955±3.971)ng/ml〕、〔1.074±0.099,(16.190±2.149)ng/ml〕were decreased significantly ,and the most notably effect of simvastatin was at the concentrations of 100μmol/L ,almost the same as that in the control group(P>0.05). Compared with nicotine group, the t-PA mRNA expression of 1μmol/L simvastatin group(0.187±0.025)were increased not significant(P>0.05);The t-PA mRNA expression of 10、100μmol/L simvastatin group(0.278±0.038)、(0.332±0.046)were increased significantly ,but still lower than control group(P<0.01, respectively). The expression of t-PA protein was no significantly different (P>0.05).Conclusions1.Nicotine through increased the expression of PAI-1 protein and mRNA in HUVECs,inhibits the fibrinolytic function of endothelial cells.2.Activation the PKC can partly disorder the fibrinolytic function of endothelial cells that induced by nicotine.3.Simvastatin can improves the fibrinolytic function of endothelial cells that induced by nicotine更多还原