【作者】 杜艳；

【导师】 胡晓芸；

【作者基本信息】 山西医科大学 ， 呼吸内科， 2010， 硕士

【摘要】 研究背景流行病学资料提示,吸烟与动脉或静脉血栓形成关系密切。吸烟可以损伤血管内皮细胞,导致血管内皮细胞功能障碍,引发纤溶失衡,从而增加血栓形成的风险。组织型纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制物-1(PAI-1)为纤溶系统的主要成分,主要由血管内皮细胞合成和分泌,二者的动态平衡决定着纤溶系统的功能。有关吸烟对内皮细胞凝血-纤溶系统功能影响的研究多集中在凝血方面,吸烟对纤溶系统的影响研究较少,其作用机制尚不完全清楚。蛋白激酶C (PKC)为细胞内信号转导的重要通道,在血管内皮功能紊乱中发挥关键作用。尼古丁作为香烟烟雾中的主要有害物质之一,可损伤内皮细胞,导致纤溶系统功能下降,但这一作用是否通过PKC信号途径发挥作用未见报道。目的研究不同实验条件下尼古丁对人脐静脉内皮细胞(HUVECs) t-PA及PAI-1表达的影响,选择尼古丁作用的最佳时间和浓度。进一步采用PKC抑制剂星型胞菌素Staurosporine(STS)作为干预药物,观察其对尼古丁诱导的内皮细胞t-PA、PAI-1蛋白和mRNA表达的影响。从体外实验探讨尼古丁对内皮细胞纤溶活性的影响,以及PKC途径在该变化中的作用。方法采用HUVECs细胞株进行以下实验。(1)尼古丁对血管内皮细胞纤溶活性影响的时间及浓度依赖性：HUVECs培养后接种于24孔培养板中,随机分为对照组及实验组,进行以下研究。①尼古丁作用的浓度依赖性：分别用0、0.1、1、10及100μmol/L的尼古丁孵育细胞,12h后收集各组上清液；②尼古丁作用的时间依赖性：用100μmol/L的尼古丁与细胞分别孵育0、4、6、8、12及24 h,收集各组上清液。采用酶联免疫吸附双抗体夹心分析法(ELISA)测定各组t-PA和PAI-1的抗原浓度。(2)PKC抑制剂(STS)干预实验：HUVECs培养后接种于25cm X 25cm的培养瓶中,随机分为对照组、尼古丁组、STS组及尼古丁+STS组。对照组培养液中不加任何干预；尼古丁组于培养液中加入终浓度为100μmol/L的尼古丁；STS组于培养液中加入终浓度为100nmol/L的STS；尼古丁+STS组于培养液中加入100nmol/L STS预处理细胞30min,再加入100μmol/L的尼古丁共同培养。同步培养12h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分析法(ELISA)测定HUVECs培养液中t-PA和PAI-1的抗原浓度,逆转录-聚合酶链反应(RT-PCR)法检测各组细胞t-PA和PAI-1 mRNA的表达水平。结果(1)尼古丁对HUVECs纤溶平衡的影响：HUVECs与不同浓度尼古丁孵育12h后,100μmol/L尼古丁组PAI-1蛋白表达较对照组明显增加,差异有统计学意义(P<0.01)；0.1μmol/L、1μmol/L及10μmol/L尼古丁组PAI-1蛋白表达与对照组比较,差异均无统计学意义(均P>0.05)；各浓度尼古丁组t-PA蛋白表达与对照组比较,差异均无统计学意义(均P>0.05)。HUVECs与100μmol/L的尼古丁分别孵育0、4、6、8、12及24 h后,各尼古丁组PAI-1蛋白表达均较对照组明显升高,差异有统计学意义(P<0.05),且其升高呈时间依赖性；各尼古丁组t-PA蛋白表达与对照组比较,差异均无统计学意义(均P>0.05)。(2)STS在尼古丁介导HUVECs纤溶失衡中的作用：①尼古丁组PAI-1 mRNA及蛋白含量显著升高(1.321±0.198, (21.084±0.829) ng/ml),与对照组(0.729±0.100,(13.388±0.927) ng/ml)比较,差异均有统计学意义(均P<0.01)；②尼古丁+STS组PAI-1 mRNA及蛋白含量(1.074±0.099, (16.190±2.149) ng/ml)较尼古丁组降低,但仍高于对照组,差异均有统计学意义(均P<0.01)；③尼古丁组t-PA mRNA的表达明显下降(0.193±0.073),与对照组(0.782±0.152)比较,差异有统计学意义(P<0.01)；t-PA蛋白((0.387±0.089)ng/ml)表达较对照组((0.461±0.090) ng/ml)有所下降,但差异无统计学意义(P>0.05)；④尼古丁+STS组t-PA mRNA (0.394±0.076)的表达较尼古丁组升高,但仍低于对照组,差异均有统计学意义(均P<0.01)；t-PA蛋白表达无明显变化(P>0.05)。结论(1)尼古丁可上调HUVECs PAI-1 mRNA的表达,引起HUVECs PAI-1蛋白释放增高,对内皮细胞纤溶功能具有抑制作用。(2)尼古丁对HUVECs t-PA蛋白的影响不大,主要通过增加PAI-1含量,导致t-PA／PAI-1失衡而影响血管内皮细胞的纤溶活性。(3)PKC通道在尼古丁诱导的血管内皮细胞纤溶紊乱中发挥部分作用。更多还原

【Abstract】 BackgroudEpidemiological surveys have shown that cigarette smoking has an influence on both arterial-type and venous-type thrombosis. It could damage the vascular endothelial cells, lead to vascular endothelial cells dysfunction, as well as trigger fibrinolysis imbalance, then increasing the risk of thrombosis. As the major anticoagulation system in the body, 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. A majority of experimental studies associated with smoking and coagulation-fibrinolysis of endothelial cells focus on the coagulation system. But studies about cigarette smoking on fibrinolytic activity of vascular endothelial cells are rare, and its related mechanism is entirely unclear.Protein kinase C (PKC) is an important biological intracellular signal transduction pathway in the body. Staurosporine (STS), a specific inhibitor, can block PKC expression. Nicotine, one of the main harmful substances of smoking, can damage the fibrinolytic function of endothelial cells. But the mechanism of nicotine on the fibrinolytic damage of endothelial cells is unclear. Whether the PKC signaling pathway plays a role in fibrinolytic dysfunction caused by nicotine needs to be investigated.ObjectiveTo study the effect of nicotine on the expression of secreting t-PA and PAI-1 in Human Umbilical Vein Endothelial Cells (HUVECs) on the different experimental conditions in order to select the best time and concentration of nicotine. STS was used to investigate the possible role of PKC in the process of t-PA or PAI-1 protein and mRNA expression in HUVECs induced by nicotine.Methods(1) HUVECs were cultured in 24-well tissue-culture plates and randomly divided into the control group and nicotine treated group.①Concentration-dependent effect of nicotine on the expression of t-PA and PAI-1:HUVECs were incubated with 0,0.1,1,10,100μmol/L nicotine for 12 hours respectively.②Time-dependent effect of nicotine on the expression of t-PA and PAI-1:The cells were exposured to 100μmol/L nicotine for 0,4,6,8,12,24 hours respectively. The cells of control groups were exposured to the same volume of PBS instead of nicotine at each time points. The supematants were collected respectively. The expression of t-PA and PAI-1 were determined by the enzyme linked immunosorbent assay (ELISA).(2) STS intervention trials. HUVECs were cultured in 25 cm×25cm culture flasks and randomly divided into the control group, 100μmol/L nicotine group, 100nmol/L STS group,and nicotine+STS group. The cells and supernatants of each group were collected after 12 hours cultured.The expression of t-PA and PAI-1 protein were measured by ELISA and the expression of t-PA and PAI-1 mRNA were determined by RT-PCR.Results(1) Compared with control group,the expression levels of PAI-1 protein of 100μmol/L nicotine treated group were increased significantly (P<0.01).However, the PAI-1 protein expression of other concentrations nicotine group had no significant difference (P>0.05, respectively).The stimulated with 100μmol/L nicotine for 0,4,6,8,12,24 hours, the levels of PAI-1 protein increased over time and reached the peak at 12 hours, which were significantly higher than those of control groups (P＜0.05, respectively).But the expression levels of t-PA protein had no significant levels of t-PA protein of each group had no significant difference (P>0.05, respectively).After difference than those of control groups (P>0.05, respectively).(2) Compared with control group (0.729±0.100, (13.388±0.927) ng/ml), the PAI-1 mRNA and protein expression of nicotine group (1.321±0.198, (21.084±0.829) ng/ml) were increased significantly (P＜0.01). Compared with nicotine group, the PAI-1 mRNA and protein expression of nicotine+STS group (1.074±0.099, (16.190±2.149) ng/ml) were decreased significantly, but still higher than control group (P＜0.01, respectively).Compared with control group (0.782±0.152), the t-PA mRNA (0.193±0.073) expression of nicotine group were decreased significantly (P＜0.01), However, the decrease of t-PA protein expression was no significant (P＞0.05). Compared with nicotine group, the t-PA mRNA expression of nicotine+STS group(0.394±0.076) were increased significantly, but still lower than control group (P＜0.01, respectively), but no significant on the t-PA protein expression (P>0.05).Conclusions(1) Nicotine can increased the expression of PAI-1 mRNA and protein in HUVECs, then inhibits the fibrinolytic function of endothelial cells.(2) Nicotine had no significant effect on the expression of t-PA protein.But increasing expression of PAI-1 can disturb the balance of t-PA/PAI-1 and affecting the fibrinolytic activity of endothelial cells.(3) PKC-dependent pathway may play a partial role on the fibrinolytic disorders of endothelial cells that induced by nicotine.更多还原