【摘要】：吸煙嚴(yán)重威脅人類健康,可導(dǎo)致動(dòng)脈粥樣硬化性疾病的發(fā)生,而動(dòng)脈粥樣硬化性疾病又是導(dǎo)致全球死亡的主要危險(xiǎn)因素。吸煙使動(dòng)脈粥樣硬化性疾病的發(fā)生率和病死率增高2-6倍。尼古丁(Nicotine)作為煙草中的主要毒性堿成分,被認(rèn)為是引起心血管損害的主要物質(zhì)。以往研究多集中在吸煙對(duì)血管內(nèi)皮的直接損傷進(jìn)而引發(fā)動(dòng)脈粥樣硬化性疾病的發(fā)生,而血管周圍脂肪組織在內(nèi)皮細(xì)胞損傷中所起到的作用尚未得到進(jìn)一步研究。近年來,隨著對(duì)脂肪組織研究的深入,以及心血管疾病發(fā)病率的急劇增加,血管周圍脂肪組織(Perivascular adipose tissue,PVAT)因與血管外膜間不存在任何解剖學(xué)屏障,逐漸成為學(xué)者們關(guān)注的重點(diǎn)。因此,本研究主要從尼古丁誘導(dǎo)的NF-κB通路和細(xì)胞凋亡水平,研究PVAT與血管內(nèi)皮細(xì)胞之間的相關(guān)性。通過體外細(xì)胞培養(yǎng),分析尼古丁刺激條件下內(nèi)皮細(xì)胞、血管周圍脂肪細(xì)胞及內(nèi)皮-脂肪細(xì)胞共培養(yǎng)后,NF-κB通路相關(guān)蛋白的變化情況,以及各細(xì)胞的凋亡情況,確定尼古丁對(duì)血管內(nèi)皮細(xì)胞的促凋亡作用和PVAT對(duì)其凋亡的調(diào)控作用及其作用機(jī)制。明確血管周圍脂肪組織在血管內(nèi)皮損傷中的作用,及其在動(dòng)脈粥樣硬化性疾病中的作用。用10-6 mol·L-1,10-7 mol·L-1和10-8 mol·L-1濃度的尼古丁分別處理內(nèi)皮細(xì)胞和脂肪細(xì)胞,Annexin V-FITC檢測(cè)細(xì)胞凋亡。此外,本研究創(chuàng)新性的利用transwell培養(yǎng)板構(gòu)建內(nèi)皮細(xì)胞-脂肪細(xì)胞共培養(yǎng)體系,10-6mol·L-1的尼古丁處理內(nèi)皮-脂肪細(xì)胞共培養(yǎng)體系后,同樣的方法檢測(cè)共培養(yǎng)細(xì)胞凋亡。同時(shí)用qRT-PCR和western blot的方法分別檢測(cè)IKKβ、NF-κB p65和IkBα的基因和蛋白表達(dá)情況,并通過免疫熒光的方法檢測(cè)NF-κB p65的核轉(zhuǎn)移變化。本研究結(jié)果表明,尼古丁對(duì)內(nèi)皮細(xì)胞和脂肪細(xì)胞的促凋亡作用具有濃度依賴性,能夠作用于NF-κB信號(hào)通路來調(diào)節(jié)細(xì)胞凋亡。其中內(nèi)皮細(xì)胞中10-6 mol·L-1處理組細(xì)胞凋亡較對(duì)照組明顯增加(P0.01)。相比之下脂肪細(xì)胞對(duì)尼古丁的反應(yīng)較為敏感,不同作用濃度下其細(xì)胞凋亡較對(duì)照組均明顯增加(P0.05)。且無論是在基因還是蛋白水平,尼古丁處理后內(nèi)皮細(xì)胞和脂肪細(xì)胞中IKKβ和NF-κB p65的表達(dá)量較對(duì)照組增加(P0.01;P0.05)。而共培養(yǎng)后的兩種細(xì)胞其細(xì)胞凋亡數(shù)目均較單純培養(yǎng)組明顯增加(P0.01;P0.05)。但I(xiàn)KKβ、NF-κB p65和IkBα的表達(dá)量的變化情況以共培養(yǎng)內(nèi)皮細(xì)胞的變化為主。共培養(yǎng)內(nèi)皮細(xì)胞中IKKβ、NF-κB p65的表達(dá)較單獨(dú)培養(yǎng)內(nèi)皮細(xì)胞明顯增加,IkBα明顯減少(P0.05)。同時(shí),NF-κB p65的核轉(zhuǎn)移也較單獨(dú)培養(yǎng)內(nèi)皮細(xì)胞明顯增加。此外,NF-κB信號(hào)通路抑制劑PDTC處理內(nèi)皮細(xì)胞和脂肪細(xì)胞后細(xì)胞凋亡數(shù)目明顯減少(P0.05)。一定濃度的尼古丁通過激活NF-κB信號(hào)通路,促進(jìn)NF-κB的磷酸化,導(dǎo)致內(nèi)皮細(xì)胞的過度凋亡和成熟脂肪細(xì)胞過度凋亡甚至壞死。且脂肪細(xì)胞經(jīng)尼古丁染毒后能夠進(jìn)一步激活內(nèi)皮細(xì)胞中NF-κB信號(hào)通路,進(jìn)而加重尼古丁對(duì)內(nèi)皮細(xì)胞的毒性損傷作用,最終促進(jìn)內(nèi)皮細(xì)胞結(jié)構(gòu)和功能的改變,加速動(dòng)脈粥樣硬化性疾病的發(fā)生發(fā)展。
[Abstract]:Smoking is a serious threat to human health and can lead to the occurrence of atherosclerotic diseases, and atherosclerotic diseases are the main risk factors leading to global death. Smoking increases the incidence and mortality of atherosclerotic diseases by 2-6 times. Nicotine, as the main toxic alkaloid in tobacco, is considered to be the main toxic alkaloid. Previous studies have focused on the direct damage of smoking to vascular endothelium and atherosclerosis, but the role of perivascular adipose tissue in endothelial cell injury has not been further studied. The incidence of vascular diseases has increased dramatically. There is no anatomical barrier between perivascular adipose tissue (PVAT) and adventitia, which has gradually become the focus of attention. The changes of NF-kappa B pathway related proteins and apoptosis of endothelial cells, perivascular adipocytes and endothelial-adipocytes co-cultured with nicotine in vitro were analyzed to determine the effect of nicotine on promoting apoptosis of vascular endothelial cells and the regulation of PVAT on apoptosis. To clarify the role of perivascular adipose tissue in vascular endothelial injury and its role in atherosclerotic diseases, we treated endothelial cells and adipocytes with nicotine at concentrations of 10-6 mol. L-1, 10-7 mol. L-1 and 10-8 mol. L-1 respectively, and detected apoptosis by Annexin V-FITC. The co-culture system of endothelial cells and adipocytes was constructed by Transwell plate, and the co-culture system of endothelial cells and adipocytes was treated with nicotine of 10-6 mol L-1. The apoptosis of co-cultured cells was detected by the same method. The gene and protein expression of IKK beta, NF-kappa B p65 and IkB alpha were detected by qRT-PCR and Western blot, respectively. The results showed that nicotine could induce apoptosis of endothelial cells and adipocytes in a concentration-dependent manner, and regulate apoptosis through the NF-kappa B signaling pathway. Compared with the control group, the response of adipocytes to nicotine was more sensitive, and the apoptosis of adipocytes at different concentrations was significantly increased (P 0.05). Moreover, the expression of IKK beta and NF-kappa B p65 in endothelial cells and adipocytes after nicotine treatment was higher than that in control group (P 0.01; P 0.05). The number of apoptotic cells in co-cultured endothelial cells was significantly higher than that in cultured endothelial cells (P 0.01; P 0.05). However, the expression of IKK-beta, NF-kappa B p65 and IkB alpha in co-cultured endothelial cells was mainly changed. The expression of IKK-beta, NF-kappa B p65 in co-cultured endothelial cells was significantly higher than that in cultured endothelial cells alone, and IkB alpha was significantly decreased (P 0.05). In addition, PDTC, an inhibitor of NF-kappa B signaling pathway, significantly decreased the number of apoptosis in endothelial cells and adipocytes (P 0.05). Nicotine at a certain concentration promoted the phosphorylation of NF-kappa B by activating the NF-kappa B signaling pathway, leading to excessive apoptosis and mature lipids in endothelial cells. Fatty cells apoptosis and even necrosis are excessive, and after nicotine exposure, adipocytes can further activate the NF-kappa B signaling pathway in endothelial cells, thereby aggravating the toxic effects of nicotine on endothelial cells, and ultimately promote the structural and functional changes of endothelial cells, and accelerate the occurrence and development of atherosclerotic diseases.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R543.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉新農(nóng);劉秀;李天佳;王占啟;倪冷;劉暴;劉昌偉;;誘導(dǎo)劑作用時(shí)間對(duì)3T3-L1前脂肪細(xì)胞系分化的影響[J];中國(guó)醫(yī)學(xué)科學(xué)院學(xué)報(bào);2016年03期

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本文編號(hào)：2241344