吸烟对人体微血管结构的损伤及其机制探讨

发布时间：2018-03-02 07:27

本文关键词： 循环内皮细胞内皮通透性紧密连接香烟提取物 15-脱氧前列腺素J_2　出处：《北京协和医学院》2011年博士论文　论文类型：学位论文

【摘要】：目的第一部分：探讨吸烟和被动吸烟者体内循环内皮细胞(CECs)的变化,考察炎症在微血管损伤中的作用。第二部分：采用香烟提取物(CSE)在体外干预内皮细胞,研究香烟烟雾损伤人微血管的分子机制。方法第一部分：募集24对健康夫妇作为志愿者检测外周血CECs,其中的12对夫妇由主动吸烟的丈夫和不吸烟的妻子组成,后者被认为是被动吸烟者。另外的12对均不吸烟的夫妇为年龄匹配的对照组。用流式细胞仪检测CD45lowCD133-CD146+的CECs。用ELISA法检测血浆中肿瘤坏死因子-α(TNF-α)、白介素-6 (IL-6)、C-反应蛋白(CRP)、假性血友病因子(vWF)、可溶性E-selectin (sE-selectin)、可溶性细胞间粘附分子-1(sICAM-1)和可溶性血栓调节蛋白(sTM)。第二部分：MTT法检测不同浓度CSE对HUVECs增殖的影响。Transwell系统检测HUVECs单层通透性。“划痕法”检测HUVECs的迁移。毛细管样成管实验检测HUVECs的血管新生。免疫荧光染色occludin、claudin-5和ZO-1蛋白观察内皮屏障的改变。Western blot检测occludi、claudin-5、ZO-1和NF-κB p65蛋白表达。结果第一部分：CECs水平在男性主动吸烟者显著高于男性不吸烟的对照组(365-3528个/mL、中位数1078个/mL versus143-1827个/mL、中位数536个/mL,p0.05),在女性被动吸烟者显著高于女性不吸烟的对照组(261-3673个／mL、中位数1597个/mL versus 69-1834个/mL、中位数455个/mL,p0.01)。血浆TNF-α、IL-6、CRP、vWF、sE-selectin和sTM水平在男性主动吸烟者显著高于男性不吸烟的对照组(p0.05或p0.01),而在女性被动吸烟者显著高于女性不吸烟的对照组(p0.05或p0.01)。与男性不吸烟的对照组相比,血浆sICAM-1水平在男性吸烟者中显著升高(p0.05)。第二部分：10%CSE不会对HUVECs (?)舌性造成明显影响。与对照组相比,CSE干预可显著增加内皮间通透性。以15d-PGJ2(10μM)预处理HUVEC单层可有效抑制CSE诱导的通透性增加。与对照组相比,CSE显著抑制HUVECs迁移,使单个视野划痕内细胞数由629±28降低至364±17(P0.01),15d-PGJ2干预可使细胞数回升至546±20(P0.01)。CSE抑制HUVECs血管新生,15d-PGJ2可逆转CSE对HUVECs血管新生的抑制。免疫荧光和Western blot分析结果显示15d-PGJ2可减轻CSE介导的内皮紧密连接蛋白occludin、claudin-5和ZO-1表达的下调。15d-PGJ2可降低CSE诱导的NF-κB激活。结论第一部分：主动和被动吸烟与CECs升高及炎症诱导内皮损伤的标志物水平上调有关；主动和被动吸烟者体内存在的低度全身性炎症可能是CECs升高的最关键因素。第二部分：15d-PGJ2通过NF-κB信号转导通路发挥其抗炎效应而减弱CSE介导的内皮损伤与功能障碍。
[Abstract]:Objective to investigate the changes of circulating endothelial cells (CECs) in smoking and passive smokers and to investigate the role of inflammation in microvascular injury. To study the molecular mechanism of cigarette smoke damaging human microvessels. Methods the first part: 24 healthy couples were recruited as volunteers to detect CECs in peripheral blood. Twelve of them were composed of active smoking husbands and non-smoking wives. The latter was considered to be a passive smoker. The other 12 couples who did not smoke were age-matched controls. CECs of CD45lowCD133-CD146 were detected by flow cytometry. Plasma TNF- 伪 TNF- 伪 and IL-6TNF- 伪 were detected by ELISA assay. Pseudophilic factor, soluble E-selectin sE-selectinine, soluble intercellular adhesion molecule-1 ICAM-1) and soluble thrombomodulin monolayer were used to detect the effect of different concentrations of CSE on the proliferation of HUVECs. Transwell system was used to detect the permeability of HUVECs monolayer. "Detection of HUVECs migration. Capillary tube formation assay was used to detect the angiogenesis of HUVECs. The changes of endothelial barrier were observed by immunofluorescence staining. Western blot was used to detect the expression of HUVECs claudin-5 ZO-1 and NF- 魏 B p65 protein. Results part one: CECs levels in male active smokers were significantly higher than those in non-smoking male controls (365-3528 / mL, median 1078 / mL versus143-1827 / mL / mL, median 536 / mLp0.05), and in female passive smokers were significantly higher than those in non-smoking female controls. 261-3673 / mL, median 1597 / mL versus 69-1834 / mLand median 455 / mLp0.01mL.Plasma TNF- 伪 -IL-6, CRPPvWFFsE-selectin and sTM levels in active male smokers were significantly higher than those in non-smoking male controls (p0.05 or p0.01g), while those in passive female smokers were significantly higher than those in non-smoking female controls (p0.05 or p0.01g). The exposure group (p0.05 or p0.01g) was compared with the control group (non-smoking male). Plasma sICAM-1 levels are significantly higher in male smokers than in male smokers. Compared with the control group, HUVEC monolayer pretreated with 15d-PGJ210 渭 M could effectively inhibit the increase of permeability induced by CSE. By reducing the number of cells in single field scratch from 629 卤28 to 364 卤17 P0.01P0.01D PGJ2, the number of cells increased to 546 卤20 P0.01G 路CSE. CSE inhibited the inhibition of HUVECs angiogenesis by CSE from 15d-PGJ2. Immunofluorescence and Western blot analysis showed that 15d-PGJ2 could alleviate CSE mediated angiogenesis. The down-regulation of claudin-5 and ZO-1 expression of Occludin-claudin-5 and ZO-1 could decrease the activation of NF- 魏 B induced by CSE. Conclusion part one: active and passive smoking is related to the increase of CECs and the up-regulation of markers of inflammation induced endothelial injury. Low degree systemic inflammation in active and passive smokers may be the most important factor in the elevation of CECs. Part two: 15d-PGJ2 attenuates endothelial damage and dysfunction mediated by CSE through NF- 魏 B signal transduction pathway.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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