本文選題：反油酸 + 人臍靜脈內(nèi)皮細胞　； 參考：《南昌大學(xué)》2013年碩士論文

【摘要】：反式脂肪酸(trans fatty acid,TFA)是一類存在于加工油脂和含油脂類加工食品中的常見組成成分。目前已經(jīng)有很多關(guān)于TFA和心肌血管疾病關(guān)系的報道,顯示了TFA與心腦血管疾病的發(fā)生發(fā)展有著密切的關(guān)系,但對于其具體致病的作用機制尚不明確。內(nèi)皮細胞功能障礙是動脈粥樣硬化(AS)及其他很多心腦血管疾病發(fā)病的初始環(huán)節(jié),在AS發(fā)病過程中占有重要地位。內(nèi)皮細胞周期阻滯以及內(nèi)皮細胞的不正常凋亡是導(dǎo)致內(nèi)皮細胞功能障礙的重要激發(fā)因素,在AS發(fā)生發(fā)展過程中發(fā)揮了重要的推動作用。 目前世界上針對TFA對AS及其他心腦血管疾病的研究主要聚焦于內(nèi)皮細胞功能障礙和內(nèi)皮細胞過度凋亡兩個方面上,并且研究的大多是反式脂肪酸的混合物,很少有針對單個種類的反式脂肪酸對AS和心腦血管疾病的影響研究。鑒于我國居民日益增多的TFA攝入量以及我國關(guān)于TFA與人體健康研究甚少的現(xiàn)狀,研究TFA對AS的影響及其作用機制具有明顯的理論價值和社會意義。本課題針對反油酸研究其與AS及心腦血管疾病的關(guān)系,主要通過反油酸對臍靜脈內(nèi)皮細胞(HUVEC)功能障礙和內(nèi)皮細胞周期及凋亡的影響,來探討反油酸通過p53介導(dǎo)的細胞凋亡致動脈粥樣硬化的機制,為反式脂肪酸與心腦血管疾病的關(guān)系以及心血管疾病的防治提供重要理論依據(jù)和科學(xué)數(shù)據(jù)。 本研究通過ELISA法檢測了反油酸作用前后細胞培養(yǎng)液中P-選凝素、sICAM-1、TNF-α、ET-1、TXA-2、PGI2等內(nèi)皮細胞功能障礙指標(biāo)的變化,確定不同濃度的TFA對內(nèi)皮細胞功能障礙的影響；采用流式細胞儀測定TFA對內(nèi)皮細胞凋亡率的影響;通過Western-blot檢測反油酸作用前后內(nèi)皮細胞中與細胞周期和凋亡有關(guān)的CDK4和PA28γ的變化；利用RT-PCR檢測TFA作用前后細胞中p21、p53、Bcl-2、 CDK4、CyclinD1、PA28γ等周期凋亡相關(guān)蛋白mRNA表達的變化,定量檢測TFA對內(nèi)皮細胞功能障礙和細胞周期、凋亡的影響,探討反油酸通過p53介導(dǎo)的細胞凋亡致動脈粥樣硬化的機制。得到的結(jié)果如下： 1.TFA能使細胞功能障礙的生物學(xué)指標(biāo)P-選凝素、sICAM-1、ET-1、TXA-2及TNF-α的分泌升高,PGI2分泌降低。P-選凝素和sICAM-1的大量分泌會引起微血管內(nèi)皮細胞與中性粒細胞黏附,致使白細胞穿出微血管壁,引起局部組織炎癥反應(yīng),TNF-α分泌升高,易加劇炎癥反應(yīng),抗炎因子與促炎因子調(diào)節(jié)失衡,引起全身炎癥反應(yīng)綜合征,進而造成全身多器官功能障礙。ET-1升高可造成局部微循環(huán)障礙,促進血栓形成,加速了炎癥反應(yīng)和AS的進程。同時,ET-1升高可以抑制PGI2的合成和釋放,同時促進TXA-2的分泌,導(dǎo)致PGI2/TXA-2調(diào)節(jié)失衡,增加血管張力的,血液處于高凝狀態(tài),從而增加了心腦血管并發(fā)癥的發(fā)生率。各種指標(biāo)表明,TFA能夠造成內(nèi)皮細胞功能障礙。 2.TFA作用后,HUVEC細胞凋亡率增加,高濃度的反油酸對細胞凋亡影響較大,早期凋亡率和晚期凋亡率均有大幅度提高,低濃度的反油酸也使細胞凋亡率升高,但影響不大。 3. Western-blot檢測結(jié)果顯示,TFA作用后,內(nèi)皮細胞中CDK4表達明顯下降,且作用呈劑量依賴性,CDK4下降,使細胞滯留在G0/G1期,降低S和G2/M期細胞比例,阻斷了細胞周期的進行,加速細胞凋亡。PA28γ表達下降,減弱了Mdm2對p53的負反饋作用,阻礙了p53的降解,使細胞在G1停滯,DNA損傷無法修復(fù),使細胞凋亡。結(jié)果表明,TFA可以導(dǎo)致細胞周期停滯和加速細胞凋亡。 4.通過RT-PCR檢測,發(fā)現(xiàn)反油酸能夠升高細胞內(nèi)p21、p53的mRNA相對表達量,降低Bcl-2、CDK4、Cyclin1、PA28y的mRNA相對表達量,并與反油酸濃度相關(guān)。PA28γ降低,致使P53表達升高,增強了對Bcl-2的抑制,降低了Bcl-2抑制凋亡的作用,激活了Caspase9,進而激活Caspase3,出現(xiàn)細胞凋亡。p53表達升高又增加了轉(zhuǎn)錄產(chǎn)物p21的表達,使CDK4、CyclinD1下降,減弱了CDK4/CyclinD1對G1向S期轉(zhuǎn)變的調(diào)控,導(dǎo)致G1期阻滯,使細胞不能完成正常的有絲分裂,導(dǎo)致細胞凋亡。這說明反油酸即可以通過激活p53,進而激活p21,來誘導(dǎo)細胞周期阻滯導(dǎo)致細胞死亡,又可以通過激活p53,進而激活Bcl-2蛋白激活線粒體通路,最終導(dǎo)致細胞凋亡。 綜上所述,TFA能夠通過上調(diào)內(nèi)皮細胞分泌的各類炎癥因子和細胞因子造成內(nèi)皮細胞損傷導(dǎo)致內(nèi)皮細胞功能障礙,促進炎癥反應(yīng)進程,并且TFA能夠上調(diào)內(nèi)皮細胞周期及凋亡相關(guān)蛋白的mRNA表達,通過p53介導(dǎo)的內(nèi)皮細胞凋亡和細胞阻滯通路誘導(dǎo)內(nèi)皮細胞凋亡。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R151.2

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