反油酸诱导内皮细胞凋亡作用机制的研究

发布时间：2018-06-18 21:03

本文选题：反油酸 + 人脐静脉内皮细胞　；参考：《南昌大学》2013年硕士论文

【摘要】：反式脂肪酸(trans fatty acid,TFA)是一类存在于加工油脂和含油脂类加工食品中的常见组成成分。目前已经有很多关于TFA和心肌血管疾病关系的报道,显示了TFA与心脑血管疾病的发生发展有着密切的关系,但对于其具体致病的作用机制尚不明确。内皮细胞功能障碍是动脉粥样硬化(AS)及其他很多心脑血管疾病发病的初始环节,在AS发病过程中占有重要地位。内皮细胞周期阻滞以及内皮细胞的不正常凋亡是导致内皮细胞功能障碍的重要激发因素,在AS发生发展过程中发挥了重要的推动作用。目前世界上针对TFA对AS及其他心脑血管疾病的研究主要聚焦于内皮细胞功能障碍和内皮细胞过度凋亡两个方面上,并且研究的大多是反式脂肪酸的混合物,很少有针对单个种类的反式脂肪酸对AS和心脑血管疾病的影响研究。鉴于我国居民日益增多的TFA摄入量以及我国关于TFA与人体健康研究甚少的现状,研究TFA对AS的影响及其作用机制具有明显的理论价值和社会意义。本课题针对反油酸研究其与AS及心脑血管疾病的关系,主要通过反油酸对脐静脉内皮细胞(HUVEC)功能障碍和内皮细胞周期及凋亡的影响,来探讨反油酸通过p53介导的细胞凋亡致动脉粥样硬化的机制,为反式脂肪酸与心脑血管疾病的关系以及心血管疾病的防治提供重要理论依据和科学数据。本研究通过ELISA法检测了反油酸作用前后细胞培养液中P-选凝素、sICAM-1、TNF-α、ET-1、TXA-2、PGI2等内皮细胞功能障碍指标的变化,确定不同浓度的TFA对内皮细胞功能障碍的影响；采用流式细胞仪测定TFA对内皮细胞凋亡率的影响;通过Western-blot检测反油酸作用前后内皮细胞中与细胞周期和凋亡有关的CDK4和PA28γ的变化；利用RT-PCR检测TFA作用前后细胞中p21、p53、Bcl-2、 CDK4、CyclinD1、PA28γ等周期凋亡相关蛋白mRNA表达的变化,定量检测TFA对内皮细胞功能障碍和细胞周期、凋亡的影响,探讨反油酸通过p53介导的细胞凋亡致动脉粥样硬化的机制。得到的结果如下： 1.TFA能使细胞功能障碍的生物学指标P-选凝素、sICAM-1、ET-1、TXA-2及TNF-α的分泌升高,PGI2分泌降低。P-选凝素和sICAM-1的大量分泌会引起微血管内皮细胞与中性粒细胞黏附,致使白细胞穿出微血管壁,引起局部组织炎症反应,TNF-α分泌升高,易加剧炎症反应,抗炎因子与促炎因子调节失衡,引起全身炎症反应综合征,进而造成全身多器官功能障碍。ET-1升高可造成局部微循环障碍,促进血栓形成,加速了炎症反应和AS的进程。同时,ET-1升高可以抑制PGI2的合成和释放,同时促进TXA-2的分泌,导致PGI2/TXA-2调节失衡,增加血管张力的,血液处于高凝状态,从而增加了心脑血管并发症的发生率。各种指标表明,TFA能够造成内皮细胞功能障碍。 2.TFA作用后,HUVEC细胞凋亡率增加,高浓度的反油酸对细胞凋亡影响较大,早期凋亡率和晚期凋亡率均有大幅度提高,低浓度的反油酸也使细胞凋亡率升高,但影响不大。 3. Western-blot检测结果显示,TFA作用后,内皮细胞中CDK4表达明显下降,且作用呈剂量依赖性,CDK4下降,使细胞滞留在G0/G1期,降低S和G2/M期细胞比例,阻断了细胞周期的进行,加速细胞凋亡。PA28γ表达下降,减弱了Mdm2对p53的负反馈作用,阻碍了p53的降解,使细胞在G1停滞,DNA损伤无法修复,使细胞凋亡。结果表明,TFA可以导致细胞周期停滞和加速细胞凋亡。 4.通过RT-PCR检测,发现反油酸能够升高细胞内p21、p53的mRNA相对表达量,降低Bcl-2、CDK4、Cyclin1、PA28y的mRNA相对表达量,并与反油酸浓度相关。PA28γ降低,致使P53表达升高,增强了对Bcl-2的抑制,降低了Bcl-2抑制凋亡的作用,激活了Caspase9,进而激活Caspase3,出现细胞凋亡。p53表达升高又增加了转录产物p21的表达,使CDK4、CyclinD1下降,减弱了CDK4/CyclinD1对G1向S期转变的调控,导致G1期阻滞,使细胞不能完成正常的有丝分裂,导致细胞凋亡。这说明反油酸即可以通过激活p53,进而激活p21,来诱导细胞周期阻滞导致细胞死亡,又可以通过激活p53,进而激活Bcl-2蛋白激活线粒体通路,最终导致细胞凋亡。综上所述,TFA能够通过上调内皮细胞分泌的各类炎症因子和细胞因子造成内皮细胞损伤导致内皮细胞功能障碍,促进炎症反应进程,并且TFA能够上调内皮细胞周期及凋亡相关蛋白的mRNA表达,通过p53介导的内皮细胞凋亡和细胞阻滞通路诱导内皮细胞凋亡。
[Abstract]:Trans fatty acid (TFA) is a common component of processed oils and oil containing processed foods. There are many reports about the relationship between TFA and cardiac vascular diseases. It shows that there is a close relationship between TFA and the development of cardiovascular and cerebrovascular diseases, but the mechanism of its specific pathogenesis is still still available. Endothelial cell dysfunction is the initial link in the pathogenesis of atherosclerosis (AS) and many other cardiovascular and cerebrovascular diseases. It plays an important role in the pathogenesis of AS. Endothelial cell cycle arrest and abnormal apoptosis of endothelial cells are important triggers for endothelial cell dysfunction, which are developed during the development of AS. It plays an important role.
At present, the study of TFA on AS and other cardiovascular and cerebrovascular diseases mainly focuses on two aspects of endothelial dysfunction and endothelial cell apoptosis, and most of them are a mixture of trans fatty acids, and few of them have been used to study the effects of single type of trans fatty acids on AS and cardiovascular diseases. The increasing number of TFA intake and the lack of research on TFA and human health in our country, the study of the effect of TFA on AS and its mechanism has obvious theoretical value and social significance. This topic aims at the study of the relationship between the anti oleic acid and AS and the cardiovascular and cerebrovascular diseases, mainly through the function of the anti oleic acid on the umbilical vein endothelial cells (HUVEC) work. The effect of barrier and endothelial cell cycle and apoptosis to explore the mechanism of atherosclerosis induced by p53 mediated apoptosis by oleic acid, and provide important theoretical basis and scientific data for the relationship between trans fatty acid and cardiovascular and cerebrovascular diseases and the prevention and treatment of cardiovascular diseases.
In this study, the changes of endothelial cell dysfunction, such as P-, sICAM-1, TNF- a, ET-1, TXA-2 and PGI2, were detected by ELISA method, and the effect of TFA on endothelial cell dysfunction was determined by different concentrations of TFA, and the effect of TFA on the apoptosis rate of endothelial cells was determined by flow cytometry; and Western-bl was used to determine the effect of TFA on the apoptosis rate of endothelial cells. Ot detection of the changes of CDK4 and PA28 gamma related to cell cycle and apoptosis in endothelial cells before and after the action of oleic acid; the changes in the expression of p21, p53, Bcl-2, CDK4, CyclinD1, PA28 gamma, and so on, and the effect of RT-PCR on endothelial cell dysfunction and cell cycle and apoptosis To explore the mechanism of oleic acid induced atherosclerosis through p53 mediated apoptosis.
1.TFA, a biological indicator of cell dysfunction, the secretion of P-, sICAM-1, ET-1, TXA-2 and TNF- alpha, the secretion of.P- and sICAM-1 by PGI2 secretion can cause adhesion between microvascular endothelial cells and neutrophils, causing leukocytes to penetrate the microvascular wall, causing local tissue inflammation, TNF- alpha secretion rising, and easy to increase. Aggravating the inflammatory response, regulating the imbalance of anti-inflammatory factors and proinflammatory factors and causing systemic inflammatory response syndrome, resulting in.ET-1 elevation of multiple organ dysfunction can cause local microcirculation disorder, promote thrombosis, accelerate the process of inflammatory response and AS. At the same time, the rise of ET-1 can inhibit the synthesis and release of PGI2 and promote TXA-2 The secretion of PGI2/TXA-2 regulates imbalance, increases vascular tension, and the blood is in a hypercoagulable state, which increases the incidence of cardiovascular and cerebrovascular complications. Various indicators suggest that TFA can cause endothelial cell dysfunction.
After the action of 2.TFA, the apoptosis rate of HUVEC cells increased, the high concentration of oleic acid had great influence on the apoptosis, the early apoptosis rate and the late apoptosis rate were greatly improved, and the low concentration of oleic acid also increased the apoptosis rate, but the effect was not significant.
The results of 3. Western-blot detection showed that the expression of CDK4 in endothelial cells decreased significantly after TFA action, and the effect was dose-dependent and CDK4 decreased. The cells remained in G0/G1 stage, reduced the proportion of S and G2/M cells, blocked the cell cycle, accelerated the decrease of.PA28 Y expression of apoptosis, weakened the negative feedback effect of Mdm2 to p53, hindered P5. The degradation of G1 caused the cell to stop at DNA, and the TFA damage could not be repaired. The result showed that TFA could lead to cell cycle arrest and accelerate cell apoptosis.
4. by RT-PCR detection, it was found that oleic acid could increase the relative expression of p21 and p53 in cells, reduce the mRNA relative expression of Bcl-2, CDK4, Cyclin1, PA28y, and reduce the expression of.PA28 gamma related to the concentration of anti oleic acid, which resulted in the increase of P53 expression, enhanced the inhibition of Bcl-2 and reduced the effect of inhibiting apoptosis. Pase3, the increased expression of apoptosis.P53 and the expression of p21 of the transcriptional product increased the CDK4, CyclinD1, weakened the CDK4/CyclinD1 regulation of the G1 to S phase transition, resulting in the G1 phase block, causing the cells to fail to complete normal mitosis and lead to cell apoptosis. This indicates that anti oleic acid can be induced by activating p53 and then activating p21 to induce induction. Cell cycle arrest leads to cell death, and by activating p53, it activates the Bcl-2 protein and activates the mitochondrial pathway, which ultimately leads to apoptosis.
To sum up, TFA can cause endothelial cell dysfunction to cause endothelial cell dysfunction by up-regulating various inflammatory factors and cytokines secreted by endothelial cells, promoting the process of inflammatory response, and TFA can up-regulate the mRNA expression of endothelial cell cycle and apoptosis related proteins, through p53 mediated endothelial cell apoptosis and cell block pathway Induce apoptosis of endothelial cells.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R151.2

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