高糖诱发氧化应激反应激活TNFR1受体促内皮祖细胞凋亡的实验研究

发布时间：2018-03-08 21:01

本文选题：内皮祖细胞　切入点：高糖　出处：《四川医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:糖尿病(Diabetes mellitus,DM)是因遗传和坏境因素共同作用而引起的高血糖以及碳水化合物、脂肪和蛋白质等代谢失衡的慢性疾病。它不仅引起糖代谢紊乱,而且引起血管病变。内皮祖细胞(Endothelial progenitor cells,EPCs)在维持血管健康和促进受损血管的重建过程中起着重要作用。当前,在糖尿病血管新生受损的机制研究中,EPCs功能失调相关因素已经受到广泛重视,其中,氧化应激与EPCs功能失调关系密切。已有研究表明:糖尿病患者的高血糖环境可以使EPCs凋亡增多,数量减少,而且凋亡增加可能是糖尿病血管病变中EPCs减少的主要机制。细胞凋亡是由基因控制的一种自主性、程序性的死亡过程,它可以由多种刺激因素引起并且主要通过死亡受体介导的途径介导凋亡,在死亡受体介导的凋亡途径中,肿瘤坏死因子受体(Tumor necrosis factor receptor,TNFR)是当前研究发现的最大的死亡受体家族,其凋亡信号传递过程中,主要是由TNFR1介导的。有研究报道高糖能诱导EPCs中TNFR1表达,这种效应能被抗氧化剂抑制。因此,本实验主要通过观察体外高糖是否通过氧化应激反应激活TNFR1及其下游的凋亡信号通路,进而对大鼠骨髓源性EPCs凋亡产生影响,来探讨高糖对EPCs的作用和影响。方法:用颈部脱臼法处死SD大鼠,将其置于75%酒精中,浸泡并消毒15 min。然后将消毒后的大鼠放置于超净工作台上,用动物解剖器械分离大鼠的股骨和胫骨,分离完成后,用含1%肝素的无菌PBS液冲洗骨髓腔,直到冲洗液变无色透明为止,收集好冲洗液,并用离心机进行高速离心,离心后收集试管底部的细胞,选择使用Ficoll密度梯度离心法进行大鼠骨髓源性单核细胞的分离;分离后,将其接种于6孔板中,3天后进行首次换液,以后每隔3-4天换液,连续使用倒置显微镜观察细胞的形态变化并且使用激光共聚焦显微镜观察鉴定经Di I-ac LDL和FITC-UEA-1双荧光染色的EPCs;用流式细胞仪检测经过不同含糖浓度(5.5、15、30、60 mmol/L)处理后的EPCs凋亡率,挑出最佳浓度;使用高糖(30 mmol/L)和氧化应激拮抗剂Tempol、TNFR1受体拮抗MAB430共同作用,流式细胞仪检测细胞凋亡率;使用分子探针(DCFH-DA)检测不同糖浓度处理后EPCs的活性氧(Reactive oxygen species,ROS)含量变化;使用Western blotting检测不同糖浓度和TNFR1受体拮抗MAB430处理EPCs后,其细胞表面TNFR1受体以及由其介导的细胞内信号通路相关蛋白(TRADD、TRAF2、RIP、NF-k Bp65、Caspase3)表达的情况。结果:在使用M199培养基培养3天后,EPCs大部分贴壁,呈圆形,逐渐增大伸展,培养至7天后,细胞生长迅速,镜下可见其呈集落样生长,培养至14天后,观察可发现细胞出现“鹅卵石”样形态分布。通过激光共聚焦显微镜观察细胞可见经过Di I-ac-LDL处理后阳性的EPCs为红色荧光,结合了FITC-UEA-1后的EPCs为绿色荧光,双染法阳性的细胞为正在分化的EPCs,表明所培养的细胞为EPCs。在高糖刺激EPCs的早期(24-48h),高糖组与对照组之间比较,没有明显的凋亡增加,而随着高糖刺激时间的延长,即高糖刺激的晚期(72-96h),可以发现高糖刺激组与正常对照组比较,凋亡细胞数目增加,高糖组(15mmol/L,30mmol/L,60mmol/L)与对照组进行组内比较,具有统计学意义(P0.01);在同一时间点,将30mmol/L,60mmol/L的高糖组分别与15mmol/L高糖组相比,早期没有发现明显凋亡,而在刺激的晚期,仅发现60mmol/L的高糖组与15mmol/L的高糖组比较,细胞凋亡增加,具有统计学意义(P0.01),在分别使用抗氧化剂Tempol、TNFR1受体拮抗剂MAB430处理EPCs24h、72h后,在与高糖组(30mmol/L)分别在24h,72h比较后,发现早期凋亡没有明显变化,而在晚期(72h)后,发现凋亡有明显下降,且数值具有显著统计学意义(P0.01),同时在实验的早期,高糖组与对照组比较,ROS的值没有明显的增加,而随着刺激时间的延长,刺激的晚期(72h),发现高糖组ROS增加明显,与对照组比较,具有显著统计学意义(P0.01),在刺激的早期和晚期,分别用Tempol、MAB430(TNFR1受体拮抗剂)处理后,发现与高糖组比较,早期ROS值没有明显的降低,而晚期ROS值有明显的降低,并且与高糖组比较,差异具有显著统计学意义(P0.01),高糖(30mmol/l)刺激早期24h,TNFR1蛋白及其凋亡信号通路相关蛋白(TRAF2、TRADD、RIP、Caspase3)表达没有上调,而在刺激的72h后,相关蛋白表达明显上调,同时将上述蛋白表达量在72h与24h进行比较,差异具有显著统计学意义(P0.01),氧化应激拮抗剂Tempol、TNFR1受体拮抗剂MAB430在72h能够降低上述TNF凋亡信号通路相关蛋白的表达,蛋白表达量有统计学意义(P0.01),而随着高糖(30mmol/l)的刺激,NF-κBp65蛋白相对表达量是逐渐降低的,其蛋白表达量在72h与24h进行比较,差异具有显著统计学意义(P0.01),在用Tempol、MAB430处理后,无论是早期还是晚期,与高糖组(30mmol/l)相比,其蛋白表达量是升高的,且差异具有显著统计学意义(P0.01)。结论:1.高糖通过诱发内皮祖细胞氧化应激反应促进其凋亡,凋亡主要发生在刺激的后期,且具有浓度和时间的依赖性。2.高糖通过诱发内皮祖细胞氧化应激激活其TNFR1受体及其衔接蛋白TRADD,进而可能通过激活NF-κB p65转录因子,从而使得与凋亡直接相关的Caspase3蛋白表达增强,导致内皮祖细胞发生凋亡。
[Abstract]:Objective: diabetes mellitus (Diabetes, mellitus, DM) is due to genetic and environmental factors caused by high blood sugar and carbohydrate, fat and protein metabolism in chronic disease. It is not only caused by the imbalance of glucose metabolism, but also cause vascular disease. Endothelial progenitor cells (Endothelial progenitor cells, EPCs) plays an important role in maintaining the health of blood vessels and promote injured vascular reconstruction process. At present, the study on Mechanism of diabetes impaired angiogenesis in EPCs dysfunction and related factors have received wide attention, among them, oxidative stress and EPCs dysfunction closely. Previous studies have shown that high levels of blood glucose in patients with diabetes mellitus can make EPCs apoptosis increased, and reduce the number of apoptosis may be the main mechanism of EPCs in diabetic angiopathy. Reduce apoptosis is an independent gene control, programmed death The process, it can be caused by a variety of stimuli and is mainly mediated by death receptor pathway in apoptosis, apoptosis mediated by death receptor, tumor necrosis factor receptor (Tumor necrosis factor receptor, TNFR) is the current findings of the largest death receptor family, the apoptosis signal transduction process, is mainly mediated by TNFR1. Studies have reported that high glucose can induce expression of TNFR1 in EPCs, this effect can be antioxidants. Therefore, this experiment mainly through the observation of the apoptosis signal pathway is mediated by oxidative stress in vitro high glucose activated TNFR1 and its downstream, and thus have an impact on the apoptosis of bone marrow derived EPCs rats, and to explore the influence of the effect of high glucose on EPCs. Methods: SD rats were killed by cervical dislocation, placed in 75% alcohol immersion disinfection, and 15 min. and then sterilized rats placed on the clean bench On animal anatomy instrument isolated from rat femur and tibia, separation after flushing the bone marrow cavity with sterile PBS solution containing 1% heparin, until the rinse solution becomes colorless and transparent, collect the washing liquid, and high-speed centrifugal centrifuge, the centrifugal collection after the bottom of the tube cell, choose to use the Ficoll density gradient centrifugation separation of bone marrow derived mononuclear cells in rats; after separation, it was inoculated in 6 well plates, 3 days after the first medium change after every change of liquid, 3-4 days of continuous use inverted microscope was used to observe the morphological changes of the cells and the use of laser confocal microscope identification by Di I-ac LDL and FITC-UEA-1 double fluorescence EPCs staining; flow cytometry was used to detect the effect of glucose concentration (5.5,15,30,60 mmol/L) EPCs apoptosis rate after treatment, select the best concentration; using high glucose (30 mmol/L) and oxidative stress of TNFR1 receptor antagonist Tempol. The interaction of antagonistic MAB430, cell apoptosis was detected by flow cytometry; using molecular probes (DCFH-DA) EPCs active oxygen in different glucose concentration after treatment (Reactive oxygen species ROS detection) content changes; using Western blotting to detect different concentrations of sugar and TNFR1 receptor antagonism of MAB430 after EPCs treatment, the cell surface receptors and by TNFR1 the mediated intracellular signal pathway related proteins (TRADD, TRAF2, RIP, NF-k, Bp65, Caspase3). Results: the expression of M199 in the culture medium after 3 days of culture, most of the EPCs adherent, rounded, increasing stretch, after 7 days of culture, cells grow rapidly, which is visible under the microscope colony growth, after 14 days of culture, observation can be found in the cells "cobblestone" distribution. By laser confocal microscopy after Di cells treated with I-ac-LDL positive red fluorescence of EPCs, combined with FITC-UE A-1 EPCs after green fluorescence double staining method was positive for differentiation of EPCs, showed that the cultured cells of early EPCs. in high glucose stimulated EPCs (24-48h), a comparison between the high glucose group and the control group, no significant increase of apoptosis, with high glucose stimulation time extended, which is stimulated by high glucose late (72-96h), can be found in high glucose group compared with normal control group, the number of apoptotic cells increased in high glucose group (15mmol/L, 30mmol/L, 60mmol/L) and control group for comparison group, with statistical significance (P0.01); at the same time point, 30mmol/L, 60mmol/L respectively compared with high glucose group 15mmol/L high glucose group early, there is no obvious apoptosis in stimulation of late, only found relatively high glucose group high glucose group and 15mmol/L 60mmol/L, increased apoptosis, with statistical significance (P0.01), respectively in the use of antioxidants Tempol, TNFR1 receptor antagonist MAB430 EPCs24h, 72h, and in the high glucose group (30mmol/L) respectively in 24h, 72h, found that early apoptosis did not change significantly, whereas in the late phase (72h), found that apoptosis has decreased significantly, with statistical significance (P0.01), and the value at the same time early in the experiment, compared with the high glucose group control, ROS was not significantly increased, with the prolongation of stimulation time, stimulation of the late (72h), high glucose group ROS increased significantly, compared with the control group, there was a statistically significant (P0.01), in the early and late stimulation, respectively Tempol, MAB430 (TNFR1 receptor antagonist) treatment compared with high glucose group, found that early ROS value was not obviously decreased, and the late ROS value decreased significantly, and compared with high glucose group, the difference was statistically significant (P0.01), high glucose (30mmol/l) stimulation of the early 24h, TNFR1 protein and apoptosis signal pathway related protein (TRAF2, TRADD, RIP, Caspase3) expression did not increase, and in the stimulation of 72h, protein expression was up-regulated, and compare the amount of 72h and 24h in the protein expression, the difference was statistically significant (P0.01), oxidative stress and expression of TNFR1 receptor antagonist Tempol and antagonist MAB430 can reduce the TNF the apoptosis signal pathway related proteins in 72h, protein expression was statistically significant (P0.01), with high glucose (30mmol/l) stimulation, NF- kappa expression of Bp65 protein was decreased, compared with 24h in 72h protein expression, the difference was statistically significant (P0.01), in Tempol after MAB430 treatment, whether early or late, compared with the high glucose group (30mmol/l), the protein content is increased, and the difference was statistically significant (P0.01). Conclusion: 1. high glucose oxidation by endothelial progenitor cells induced by stress induced In the late apoptosis, the apoptosis in stimulation, and has a time and concentration dependent.2. high glucose activation of the TNFR1 receptor and its adaptor protein TRADD induced by oxidative stress and endothelial progenitor cells, possibly through activation of NF- kappa B transcription factor p65, which is directly related to apoptosis and Caspase3 protein expression, leading to endothelial progenitor cell apoptosis.

【学位授予单位】：四川医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

【共引文献】