本文選題：白藜蘆醇　切入點(diǎn)：人臍靜脈內(nèi)皮細(xì)胞　出處：《河北醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:糖尿病是一種代謝紊亂性疾病,可以導(dǎo)致糖尿病特有的微血管并發(fā)癥(視網(wǎng)膜病變、腎病、神經(jīng)病變)和大血管并發(fā)癥(心血管疾病和中風(fēng)),最終導(dǎo)致患者壽命的縮短。我國(guó)2010年糖尿病流行病學(xué)調(diào)查顯示,成人糖尿病的患病率為9.7%,如果將Hb A1c≥6.5%也作為糖尿病的診斷標(biāo)準(zhǔn),則糖尿病的患病率達(dá)11.6%。糖尿病是心腦血管疾病的獨(dú)立危險(xiǎn)因素,據(jù)報(bào)道糖尿病患者發(fā)生心血管疾病的風(fēng)險(xiǎn)是非糖尿病患者的2-4倍。大多數(shù)的糖尿病患者死于心腦血管疾病及糖尿病腎病,可見(jiàn)糖尿病慢性并發(fā)癥的治療對(duì)于糖尿病患者的重要性。高血糖的糖毒性參與了糖尿病慢性并發(fā)癥的發(fā)生,但是從臨床治療和一些臨床實(shí)驗(yàn)來(lái)看,通過(guò)藥物使血糖控制在理想范圍內(nèi)的糖尿病患者仍可能發(fā)生糖尿病并發(fā)癥,且這些并發(fā)癥持續(xù)進(jìn)展。為此人們進(jìn)行了一些大型臨床試驗(yàn)研究,提出高血糖代謝記憶效應(yīng),很好的解釋了這一現(xiàn)象。糖尿病患者早期高血糖狀態(tài)可對(duì)機(jī)體各個(gè)器官造成損害,即使后期血糖達(dá)到理想水平,這種損傷仍然持續(xù)存在,這種現(xiàn)象稱(chēng)為“代謝記憶”現(xiàn)象。多項(xiàng)研究表明,氧化應(yīng)激在糖尿病患者心血管病變的發(fā)病機(jī)制中起關(guān)鍵作用。機(jī)體在高血糖刺激下出現(xiàn)氧化應(yīng)激反應(yīng),從而引起糖尿病的發(fā)生、發(fā)展。當(dāng)前,已被證實(shí)有4種主要機(jī)制參與了高血糖引起的組織損傷:多元醇通路的增加、細(xì)胞內(nèi)糖基化終產(chǎn)物(AGEs)的增加、蛋白激酶C(PKC)的激活、己糖胺通路的過(guò)度激活。高血糖可以通過(guò)激活多元醇通路和己糖胺通路使機(jī)體產(chǎn)生過(guò)量的ROS,誘導(dǎo)糖基化終產(chǎn)物(AGEs)形成,并使其與特異性受體(RAGE)結(jié)合。反過(guò)來(lái),AGEs與其特異性受體(RAGE)的結(jié)合,也促進(jìn)了活性氧簇(ROS)的產(chǎn)生,進(jìn)而激活核轉(zhuǎn)錄因子NF-κB通路,使細(xì)胞長(zhǎng)期處于炎癥狀態(tài),加速血管動(dòng)脈粥樣硬化的形成。并且,細(xì)胞外基質(zhì)中的AGEs,可以使血管彈性下降、一氧化氮的活性降低、血管內(nèi)皮依賴(lài)性舒張功能減低。此外,慢性高血糖增加了循環(huán)血中細(xì)胞因子、生長(zhǎng)因子、內(nèi)皮素-I、血管緊張素II含量,這些物質(zhì)與相應(yīng)的細(xì)胞表面受體結(jié)合,激活蛋白激酶C(PKC)通路,而PKC通路的激活可誘導(dǎo)血管損傷,抑制內(nèi)皮細(xì)胞中一氧化氮合酶(e NOS)的表達(dá),抑制平滑肌細(xì)胞中一氧化氮的產(chǎn)生,提高纖溶酶原激活物(PAI-I)的表達(dá),增加氧化還原敏感的核轉(zhuǎn)錄因子NF-κB表達(dá),提高促氧化劑NADPH氧化酶的活性,進(jìn)而對(duì)血管內(nèi)皮細(xì)胞造成損傷。白藜蘆醇(Resveratrol,Res),化學(xué)名為3,5,4-三羥基-1,2-二苯乙烯,是一種天然的多酚類(lèi)化合物。白藜蘆醇在葡萄皮及紅酒中含量豐富,可以作為活性氧(ROS)的清除劑、金屬螯合金、酶調(diào)節(jié)劑。一些研究表明紅酒或白藜蘆醇可以使糖尿病大鼠的不同大腦區(qū)域的脂質(zhì)過(guò)氧化降低,同時(shí)增加抗氧化酶水平。并且最近的研究也表明,白藜蘆醇對(duì)不同的組織以及心血管疾病、炎性反應(yīng)、癌癥、糖尿病等病理疾病有抗氧化應(yīng)激保護(hù)作用。本研究旨在通過(guò)體外培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞,探討高血糖代謝記憶對(duì)于體外培養(yǎng)的血管內(nèi)皮細(xì)胞的氧化應(yīng)激作用,并且探討白藜蘆醇在高糖代謝記憶中對(duì)血管內(nèi)皮細(xì)胞的保護(hù)作用。方法:人臍靜脈內(nèi)皮細(xì)胞株(HUVECs)體外培養(yǎng)貼壁后隨機(jī)分組:正常對(duì)照組:NG,5.5mmol/L D-葡萄糖×3天;甘露醇對(duì)照組:MA,5.5mmol/L D-葡萄糖、24.5mmol/L D-甘露醇×3天;高糖持續(xù)組:HG,30mmol/L D-葡萄糖×3天;高糖記憶組:TG,30mmol/L D-葡萄糖×1天更換為5.5mmol/L D-葡萄糖×2天;高糖記憶+白藜蘆醇組:TG+100μmol/L、10μmol/L、1μmol/L、0.1μmol/L Res×1天更換為5.5mmol/L D-葡萄糖×2天。NG組、MA組、HG組、TG組、TG+Res組分別于培養(yǎng)24h、48h和72h用MTT法檢測(cè)細(xì)胞增殖情況,用wst-1法檢測(cè)細(xì)胞內(nèi)超氧化物歧化酶(SOD)的活力,用TBA法檢測(cè)細(xì)胞內(nèi)丙二醛(MDA)的含量。結(jié)果:1與正常對(duì)照組比較,高糖組明顯抑制臍靜脈內(nèi)皮細(xì)胞增殖活性(P0.01)。更換為正常糖濃度后,內(nèi)皮細(xì)胞增殖活性部分恢復(fù)(P0.05),但仍低于正常對(duì)照組(P0.01)。白藜蘆醇以劑量依賴(lài)形式增加代謝記憶組細(xì)胞增殖活性(P0.05)。甘露醇對(duì)照組對(duì)內(nèi)皮細(xì)胞的增殖也有一定的抑制作用(P0.05)。2甘露醇組細(xì)胞內(nèi)MDA含量高于正常對(duì)照組(P0.05)。與正常對(duì)照組比較,高糖組細(xì)胞內(nèi)MDA含量明顯升高(P0.01)。高糖代謝記憶組細(xì)胞內(nèi)MDA含量低于高糖組(P0.05),但高于正常對(duì)照組(P0.01)。白藜蘆醇以濃度依賴(lài)形式降低高糖記憶組內(nèi)皮細(xì)胞內(nèi)MDA含量(P0.05)。3與正常對(duì)照組相比,甘露醇組細(xì)胞內(nèi)SOD水平下降(P0.05),高糖組、高糖記憶組和白藜蘆醇干預(yù)組細(xì)胞內(nèi)SOD水平明顯下降(P0.01)。高糖持續(xù)組細(xì)胞內(nèi)SOD水平明顯降低高糖代謝記憶組(P0.01)。白藜蘆醇呈濃度依賴(lài)性恢復(fù)代謝記憶組內(nèi)皮細(xì)胞內(nèi)SOD水平(P0.05)。結(jié)論:1高糖抑制體外HUVECs增殖、誘發(fā)HUVECs氧化應(yīng)激,去除高糖刺激后,細(xì)胞增殖抑制作用和氧化應(yīng)激持續(xù)存在。提示代謝記憶效應(yīng)參與糖尿病的血管內(nèi)皮損傷機(jī)制。2白藜蘆醇劑量依賴(lài)地恢復(fù)高糖代謝記憶效應(yīng)介導(dǎo)的細(xì)胞增殖抑制及氧化應(yīng)激效應(yīng)。提示白藜蘆醇可能對(duì)代謝記憶效應(yīng)介導(dǎo)的內(nèi)皮細(xì)胞損傷具有潛在的保護(hù)作用。
[Abstract]:Objective: diabetes mellitus is a metabolic disorder, microvascular complications can lead to diabetes specific (retinopathy, nephropathy, neuropathy) and macrovascular complications (cardiovascular disease and stroke), eventually lead to shorten the patient life. 2010 survey of diabetes epidemiology in China, adult prevalence rate of diabetes was 9.7%, if the Hb A1c = 6.5% as the diagnostic standard of diabetes, the diabetes prevalence rate of 11.6%. diabetes is the independent risk factor for cardiovascular disease risk, according to reports of cardiovascular disease in patients with diabetes is 2-4 times higher than the non diabetes patients. Most of the diabetic patients died of cardiovascular and cerebrovascular diseases and diabetic nephropathy, treatment of chronic complications of diabetes mellitus for visible the importance of diabetes. High blood sugar glucose toxicity in chronic diabetic complications, but from clinical treatment Treatment and clinical trials, patients with diabetes control in the ideal range by drugs to blood glucose may still occur in diabetic complications, and the complication of continuous progress. Therefore there were some large clinical trials, the high glucose metabolic memory effect, good explanation of this phenomenon. The early hyperglycemia patients with diabetes damage to various organs of the body, even if the latter glucose reaches the ideal level, the damage still persists, this phenomenon is called "metabolic memory phenomenon. A number of studies have shown that oxidative stress plays a key role in the pathogenesis of diabetic patients with cardiovascular disease. Immune response to oxidative stress stimulated by high glucose, thereby causing diabetes occurrence, development has been confirmed. At present, there are mainly 4 kinds of mechanisms involved in tissue injury caused by hyperglycemia: polyol pathway. Plus, in cells of advanced glycation end products (AGEs) increased, C protein kinase (PKC) activation, excessive activation of the hexosamine pathway. High blood glucose can be through the activation of polyol pathway and hexosamine pathway to produce excessive ROS, induced by advanced glycation end products (AGEs) formation, and the with the specific receptor (RAGE) binding. In turn, the AGEs and its specific receptor (RAGE) binding, but also promote the reactive oxygen species (ROS) generation and activation of nuclear factor kappa B pathway NF-, the cells in the inflammatory state for a long time, the acceleration of vascular atherosclerosis formation. Furthermore, extracellular in the matrix AGEs, can make the blood vessel elasticity decreased, reducing the activity of nitric oxide, endothelium-dependent diastolic function decreased. In addition, chronic hyperglycemia increased cytokine, circulating growth factor, endothelin -I, angiotensin II content, these substances and the corresponding cell surface Binding, activation of protein kinase C (PKC) pathway, and the activation of PKC pathway can induce vascular injury, inhibition of nitric oxide synthase in endothelial cells (E NOS) expression, inhibition of nitric oxide in smooth muscle cells, improve the plasminogen activator (PAI-I) expression of B NF- increased the oxidation reduction of nuclear transcription factor kappa the expression of sensitivity, improve the pro oxidant activity of NADPH oxidase, which caused the injury of vascular endothelial cells. Resveratrol (Resveratrol, Res), the chemical name is 3,5,4- three -1,2- two hydroxy styrene, is a natural polyphenolic compound. Resveratrol rich content in grape skin and red wine, can be used as active oxygen scavenger (ROS), metal chelating alloy, enzyme regulator. Some studies show that red wine or resveratrol can reduce lipid peroxidation in different brain regions in diabetic rats. At the same time, increase the levels of antioxidant enzymes and recently. The study also showed that resveratrol in different tissues as well as cardiovascular disease, inflammation, cancer, diabetes and other pathological disease has a protective effect against oxidative stress. The purpose of this study was to cultured human umbilical vein endothelial cells in vitro, to investigate the role of oxidative stress in high glucose metabolic memory for vascular endothelial cells cultured in vitro, and to explore the protection effect of resveratrol on vascular endothelial cells in high glucose metabolic memory. Methods: human umbilical vein endothelial cells (HUVECs) cultured in vitro adherent were randomly divided into 2 groups: normal control group: NG, 5.5mmol/L * D- glucose for 3 days; mannitol control group: MA, 5.5mmol/L D- 24.5mmol/L D- glucose, mannitol * 3 days; high glucose group: HG, 30mmol/L for D- glucose * 3 days; hyperglycemia group: TG, D- glucose 30mmol/L * 1 days replacement for 5.5mmol/L * D- glucose for 2 days; hyperglycemia + resveratrol group: TG+100 mol/L, 10 m Ol/L, 1 mol/L, 0.1 mol/L Res * 1 days replacement for 5.5mmol/L D- glucose * 2 days in.NG group, MA group, HG group, TG group and TG+Res group were cultured 24h, 48h and 72h cell proliferation was assessed by MTT method, determination of intracellular superoxide dismutase by WST-1 method (SOD) activity, TBA method was used to detect intracellular malondialdehyde (MDA) content. Results: 1 compared with normal control group, high glucose group significantly inhibited the proliferation of human umbilical vein endothelial cell activity (P0.01). The replacement for the normal glucose concentration after restoration of endothelial cell proliferation activity part (P0.05), but still lower than the normal control group. (P0.01). Resveratrol increased proliferation of metabolic memory cell activity in a dose dependent group form (P0.05). The control group of mannitol on endothelial cell proliferation is inhibited (P0.05).2 mannitol group of intracellular MDA content is higher than the normal control group (P0.05). And the normal control group, high glucose content of MDA group of cells Increased significantly (P0.01). The content of MDA memory cells in high glucose metabolism group was lower than that in high glucose group (P0.05), but higher than the normal control group (P0.01). Resveratrol decreased the content of MDA in high glucose group memory in endothelial cells in a concentration dependent form (P0.05.3) compared with the normal control group, mannitol group decreased the intracellular level of SOD (P0.05), high glucose group, high glucose group and resveratrol intervention memory SOD group decreased significantly (P0.01). The level of SOD in high glucose group continued intracellular metabolic memory group decreased significantly (P0.01). Resveratrol showed a concentration dependent recovery of SOD metabolism memory group in endothelial cells (P0.05). Conclusion: 1 high glucose inhibited the proliferation of HUVECs in vitro, HUVECs induced oxidative stress, the removal of high glucose stimulation, cell proliferation inhibition and oxidative stress persists. Suggest that metabolic memory effects in diabetic vascular endothelial injury mechanism of.2 resveratrol dose dependently It can restore cell proliferation inhibition and oxidative stress effect mediated by high glucose metabolism, suggesting that resveratrol may play a potential protective role in endothelial cell injury mediated by metabolic memory effect.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R587.2

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