本文關(guān)鍵詞： 循環(huán)內(nèi)皮細(xì)胞 內(nèi)皮通透性 緊密連接 香煙提取物 15-脫氧前列腺素J_2　出處：《北京協(xié)和醫(yī)學(xué)院》2011年博士論文　論文類型：學(xué)位論文

【摘要】：目的第一部分：探討吸煙和被動(dòng)吸煙者體內(nèi)循環(huán)內(nèi)皮細(xì)胞(CECs)的變化,考察炎癥在微血管損傷中的作用。第二部分：采用香煙提取物(CSE)在體外干預(yù)內(nèi)皮細(xì)胞,研究香煙煙霧損傷人微血管的分子機(jī)制。 方法第一部分：募集24對(duì)健康夫婦作為志愿者檢測(cè)外周血CECs,其中的12對(duì)夫婦由主動(dòng)吸煙的丈夫和不吸煙的妻子組成,后者被認(rèn)為是被動(dòng)吸煙者。另外的12對(duì)均不吸煙的夫婦為年齡匹配的對(duì)照組。用流式細(xì)胞儀檢測(cè)CD45lowCD133-CD146+的CECs。用ELISA法檢測(cè)血漿中腫瘤壞死因子-α(TNF-α)、白介素-6 (IL-6)、C-反應(yīng)蛋白(CRP)、假性血友病因子(vWF)、可溶性E-selectin (sE-selectin)、可溶性細(xì)胞間粘附分子-1(sICAM-1)和可溶性血栓調(diào)節(jié)蛋白(sTM)。第二部分：MTT法檢測(cè)不同濃度CSE對(duì)HUVECs增殖的影響。Transwell系統(tǒng)檢測(cè)HUVECs單層通透性�！皠澓鄯ā睓z測(cè)HUVECs的遷移。毛細(xì)管樣成管實(shí)驗(yàn)檢測(cè)HUVECs的血管新生。免疫熒光染色occludin、claudin-5和ZO-1蛋白觀察內(nèi)皮屏障的改變。Western blot檢測(cè)occludi、claudin-5、ZO-1和NF-κB p65蛋白表達(dá)。 結(jié)果第一部分：CECs水平在男性主動(dòng)吸煙者顯著高于男性不吸煙的對(duì)照組(365-3528個(gè)/mL、中位數(shù)1078個(gè)/mL versus143-1827個(gè)/mL、中位數(shù)536個(gè)/mL,p0.05),在女性被動(dòng)吸煙者顯著高于女性不吸煙的對(duì)照組(261-3673個(gè)／mL、中位數(shù)1597個(gè)/mL versus 69-1834個(gè)/mL、中位數(shù)455個(gè)/mL,p0.01)。血漿TNF-α、IL-6、CRP、vWF、sE-selectin和sTM水平在男性主動(dòng)吸煙者顯著高于男性不吸煙的對(duì)照組(p0.05或p0.01),而在女性被動(dòng)吸煙者顯著高于女性不吸煙的對(duì)照組(p0.05或p0.01)。與男性不吸煙的對(duì)照組相比,血漿sICAM-1水平在男性吸煙者中顯著升高(p0.05)。第二部分：10%CSE不會(huì)對(duì)HUVECs (?)舌性造成明顯影響。與對(duì)照組相比,CSE干預(yù)可顯著增加內(nèi)皮間通透性。以15d-PGJ2(10μM)預(yù)處理HUVEC單層可有效抑制CSE誘導(dǎo)的通透性增加。與對(duì)照組相比,CSE顯著抑制HUVECs遷移,使單個(gè)視野劃痕內(nèi)細(xì)胞數(shù)由629±28降低至364±17(P0.01),15d-PGJ2干預(yù)可使細(xì)胞數(shù)回升至546±20(P0.01)。CSE抑制HUVECs血管新生,15d-PGJ2可逆轉(zhuǎn)CSE對(duì)HUVECs血管新生的抑制。免疫熒光和Western blot分析結(jié)果顯示15d-PGJ2可減輕CSE介導(dǎo)的內(nèi)皮緊密連接蛋白o(hù)ccludin、claudin-5和ZO-1表達(dá)的下調(diào)。15d-PGJ2可降低CSE誘導(dǎo)的NF-κB激活。結(jié)論第一部分：主動(dòng)和被動(dòng)吸煙與CECs升高及炎癥誘導(dǎo)內(nèi)皮損傷的標(biāo)志物水平上調(diào)有關(guān)；主動(dòng)和被動(dòng)吸煙者體內(nèi)存在的低度全身性炎癥可能是CECs升高的最關(guān)鍵因素。第二部分：15d-PGJ2通過NF-κB信號(hào)轉(zhuǎn)導(dǎo)通路發(fā)揮其抗炎效應(yīng)而減弱CSE介導(dǎo)的內(nèi)皮損傷與功能障礙。
[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- 偽 TNF- 偽 and IL-6TNF- 偽 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- 偽 -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 鹵28 to 364 鹵17 P0.01P0.01D PGJ2, the number of cells increased to 546 鹵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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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