血管周围脂肪组织在尼古丁诱导血管内皮细胞损伤中的作用

发布时间：2018-09-13 13:44

【摘要】：吸烟严重威胁人类健康,可导致动脉粥样硬化性疾病的发生,而动脉粥样硬化性疾病又是导致全球死亡的主要危险因素。吸烟使动脉粥样硬化性疾病的发生率和病死率增高2-6倍。尼古丁(Nicotine)作为烟草中的主要毒性碱成分,被认为是引起心血管损害的主要物质。以往研究多集中在吸烟对血管内皮的直接损伤进而引发动脉粥样硬化性疾病的发生,而血管周围脂肪组织在内皮细胞损伤中所起到的作用尚未得到进一步研究。近年来,随着对脂肪组织研究的深入,以及心血管疾病发病率的急剧增加,血管周围脂肪组织(Perivascular adipose tissue,PVAT)因与血管外膜间不存在任何解剖学屏障,逐渐成为学者们关注的重点。因此,本研究主要从尼古丁诱导的NF-κB通路和细胞凋亡水平,研究PVAT与血管内皮细胞之间的相关性。通过体外细胞培养,分析尼古丁刺激条件下内皮细胞、血管周围脂肪细胞及内皮-脂肪细胞共培养后,NF-κB通路相关蛋白的变化情况,以及各细胞的凋亡情况,确定尼古丁对血管内皮细胞的促凋亡作用和PVAT对其凋亡的调控作用及其作用机制。明确血管周围脂肪组织在血管内皮损伤中的作用,及其在动脉粥样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浓度的尼古丁分别处理内皮细胞和脂肪细胞,Annexin V-FITC检测细胞凋亡。此外,本研究创新性的利用transwell培养板构建内皮细胞-脂肪细胞共培养体系,10-6mol·L-1的尼古丁处理内皮-脂肪细胞共培养体系后,同样的方法检测共培养细胞凋亡。同时用qRT-PCR和western blot的方法分别检测IKKβ、NF-κB p65和IkBα的基因和蛋白表达情况,并通过免疫荧光的方法检测NF-κB p65的核转移变化。本研究结果表明,尼古丁对内皮细胞和脂肪细胞的促凋亡作用具有浓度依赖性,能够作用于NF-κB信号通路来调节细胞凋亡。其中内皮细胞中10-6 mol·L-1处理组细胞凋亡较对照组明显增加(P0.01)。相比之下脂肪细胞对尼古丁的反应较为敏感,不同作用浓度下其细胞凋亡较对照组均明显增加(P0.05)。且无论是在基因还是蛋白水平,尼古丁处理后内皮细胞和脂肪细胞中IKKβ和NF-κB p65的表达量较对照组增加(P0.01;P0.05)。而共培养后的两种细胞其细胞凋亡数目均较单纯培养组明显增加(P0.01;P0.05)。但IKKβ、NF-κB p65和IkBα的表达量的变化情况以共培养内皮细胞的变化为主。共培养内皮细胞中IKKβ、NF-κB p65的表达较单独培养内皮细胞明显增加,IkBα明显减少(P0.05)。同时,NF-κB p65的核转移也较单独培养内皮细胞明显增加。此外,NF-κB信号通路抑制剂PDTC处理内皮细胞和脂肪细胞后细胞凋亡数目明显减少(P0.05)。一定浓度的尼古丁通过激活NF-κB信号通路,促进NF-κB的磷酸化,导致内皮细胞的过度凋亡和成熟脂肪细胞过度凋亡甚至坏死。且脂肪细胞经尼古丁染毒后能够进一步激活内皮细胞中NF-κB信号通路,进而加重尼古丁对内皮细胞的毒性损伤作用,最终促进内皮细胞结构和功能的改变,加速动脉粥样硬化性疾病的发生发展。
[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.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5

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