晚期糖基化终末产物通过线粒体功能障碍和Fas-FasL途径诱导人视网膜色素上皮细胞凋亡的机制研究

发布时间：2018-05-01 01:28

本文选题：晚期糖基化终末产物 + 人视网膜色素上皮细胞　；参考：《武汉大学》2016年博士论文

【摘要】：糖尿病视网膜病变是糖尿病最常见的微血管并发症之一,己成为致盲的主要眼疾。糖尿病患者体内持续的或不受控制的高血糖会逐渐损害视网膜血管细胞、视网膜上皮细胞、神经细胞和神经胶质,导致糖尿病视网膜病变。随着糖尿病发病率逐年增长,越来越多的糖尿病患者将经受糖尿病视网膜病变的折磨。晚期糖基化终末产物(AGEs)是指在非酶促条件下,蛋白质、氨基酸、脂类或核酸等的游离氨基与葡萄糖或其他还原糖的醛基通过Maillard反应系列产生一组稳定的终末产物；许多研究表明AGEs在糖尿病视网膜病变发生和发展的过程中起着重要作用,但糖尿病视网膜病变的发病机制尚未完全阐明,我们的实验将致力于研究AGEs在诱导人视网膜色素上皮细胞凋亡时,发挥了怎样的作用,为临床防治糖尿病视网膜病变提供新的思路。第一部分AGEs诱导人ARPE-19细胞凋亡的有效性研究目的：探讨用AGE-BSA处理ARPE-19细胞,观察细胞的生存能力和凋亡情况。方法：1.将培养的ARPE-19细胞用浓度为50、100、150、200μg/mL的AGE-BSA分别处理24小时和48小时,阴性对照组中加入200 μg/mLBSA,采用MTT比色法分析AGE-BSA对细胞存活率的影响,采用流式细胞仪检测分析AGE-BSA对细胞凋亡率的影响。2.分别在ARPE-19细胞培养24小时和48小时时加入AGE-BSA处理,对照组中加入200 μg/mLBSA,采用MTT比色法分析AGE-BSA后处理对细胞存活率的影响,采用凋亡检测试剂盒分析AGE-BSA对细胞凋亡率的影响。结果：1.与阴性对照组比较,用150 μg/mL或200 μg/mL AGE-BSA处理的细胞,细胞存活比例分别减少了22%(p0.05)和33%(p0.01),差异有统计学意义,而且,不同浓度AGE-BSA处理的两组细胞之间也有明显的差异；当细胞中AGE-BSA浓度达到100μg/mL时,与阴性对照组相比,细胞的凋亡比例显著升高(p0.05),同时也发现,当AGE-BSA浓度超过100 μg/mL时,细胞的凋亡比例与其呈正相关性。2.用AGE-BSA处理的细胞,和阴性对照组相比,细胞的存活比例在24小时减少了20%,在48小时减少了40%；而细胞的凋亡比例在24小时和48小时分别上升了60%和110%。结论：用AGE-BSA处理ARPE-19细胞可以减少细胞的存活并有效地诱导细胞凋亡。第二部分AGEs通过线粒体功能障碍诱导人视网膜细胞凋亡的研究目的：研究线粒体功能障碍在AGEs诱导细胞凋亡过程中的作用。方法：1.用小鼠线粒体呼吸链复合物Ⅳ试剂盒检测经AGE-BSA后处理的人ARPE-19细胞线粒体呼吸链复合物Ⅳ的活性；用线粒体膜电位试剂盒检测人ARPE-19细胞线粒体膜电位水平。2.将ARPE-19细胞培养48小时后,用AGE-BSA进行处理,加入BSA的细胞设为对照组,采用Western blot技术检测Bcl-2、 Bcl-xl、Bax、Cytc和Caspase3的表达水平的变化。结果：1.与阴性对照组比较,用200μ g/mL AGE-BSA处理的人ARPE-19细胞的线粒体呼吸链复合体Ⅳ活性降低了30%(p0.01),线粒体膜电位水平降低了25%(p0.05)。2.和阴性对照组相比,ARPE-19细胞的Bcl-2和Bcl-xl的表达水平分别下调了67%(p0.01)和43%(p0.05),而Bax的表达水平却上调了122%(p0.01)；3.与凋亡相关的关键分子Cytc的释放和活化的Caspase 3的表达与阴性对照组也有显著差异,二者的表达均增加,分别上调了43%(p0.01)和188%(p0.01)。结论：AGEs通过降低线粒体呼吸链复合体Ⅳ活性和线粒体膜电位水平以及下调Bcl-2、Bcl-xl的表达水平,而上调Bax的表达水平,导致线粒体功能障碍；从而使Cytc释放增加和Caspase 3活化最终激活人视网膜ARPE-19细胞的凋亡。第三部分AGEs通过Fas-FasL信号诱导人视网膜细胞凋亡的研究目的：探讨Fas-FasL信号通路在AGEs诱导细胞凋亡过程中的作用。方法：1.将ARPE-19细胞用不同浓度的AGE-BSA处理,将加入不同浓度BSA的细胞设为阴性对照组,采用Western blot技术检测Fas、Fas-L、活化的caspase8和β-actin的表达水平。2.将ARPE-19细胞用不同浓度的α-Fas+AGE-BSA共处理,采用Western blot技术检测Cytc释放、活化caspase8、活化caspase3的表达水平。3.将合成的scramble SiRNA、SiRNA-Fas和SiRNA-FasL转染到ARPE-19细胞,通过Western blot技术检测Fas和Fas-L的mRNA表达水平的变化。4.分别将上述3种SiRNA转染到用200 μ g/mL AGE-BSA处理的人ARPE-19细胞,采用Western blot技术检测Fas、Fas-L、活化Caspase8和活化Caspase 3的表达情况。结果：1.当细胞中AGE-BSA的浓度超过100 μ g/mL时,相较于空白组、BSA组和50 μ g/mLAGE-BSA组,Fas和FasL的表达均有显著的统计学差异；200μg/mL AGE-BSA处理组的FasL的表达比100 μ g/mLAGE-BSA处理组升高了50%(p0.01),200 μ g/mL AGE-BSA处理组的活化caspase8的表达比其他组提升了42.8%(p0.01)。2. Cytc释放、活化caspase8和活化caspase3随α-Fas剂量的增加而增加；50ng/ml α-Fas共处理组相比于20ng/ml α-Fas共处理组,Cytc释放升高了60%(p0.01)；活化caspase8的表达在50ng/ml α-Fas共处理组比10ng/ml α-Fas共处理组增加了40%(p0.01)；而活化caspase3的表达在20ng/ml α-Fas共处理组和50ng/ml α-Fas共处理组分别上调了45%(p0.01)和45%(p0.01)相比于10ng/ml α-Fas共处理组。3. SiRNA-Fas和SiRNA-FasL有效地抑制了Fas和Fas-L的mRNA表达水平。4. SiRNA-Fas+AGE-BSA处理组Fas的表达下降了20%(p0.01), SiRNA-FasL+AGE-BSA处理组FasL的表达降低了13%(p0.01)；因此,SiRNA-Fas+AGE-BSA处理组和SiRNA-FasL+AGE-BSA处理组均有效减少了活化Caspase8和活化Caspase 3的表达。结论：AGE-BSA处理细胞,能够促进Fas、Fas-L的表达及Fas-FasL信号诱导活化Caspase8;外源性的α-Fas能加强AGE-BSA诱导Cytc释放、活化caspase8和活化caspase3的作用；SiRNA介导的Fas或FasL基因沉默能够有效地抑制AGE-BSA诱导的Caspase 8和Caspase 3的激活。
[Abstract]:Diabetic retinopathy is one of the most common microvascular complications of diabetes. It has become a major blindness disease. Continuous or uncontrolled hyperglycemia in diabetic patients will gradually damage retinal vascular cells, retinal epithelial cells, nerve cells, and neuroglia, leading to diabetic retinopathy. The rate of disease is increasing year by year. More and more diabetic patients will suffer from diabetic retinopathy. Late glycosylation end product (AGEs) refers to a group of stable terminals through the Maillard reaction series of free amino groups of protein, amino acids, lipids, or nucleic acids, such as protein, amino acids, lipids, or nucleic acids, or other reductive glycosylated aldehyde groups. Many studies have shown that AGEs plays an important role in the development and development of diabetic retinopathy, but the pathogenesis of diabetic retinopathy has not been fully elucidated. Our experiments will be devoted to the role of AGEs in inducing apoptosis of human retinal pigment epithelial cells and to prevent and cure diabetes in clinical practice. Disease retinopathy provides new ideas. Part one AGEs induced the effectiveness of apoptosis in human ARPE-19 cells. Objective: To explore the use of AGE-BSA to treat ARPE-19 cells and to observe the viability and apoptosis of the cells. Methods: 1. the cultured ARPE-19 cells were treated with AGE-BSA with a concentration of 50100150200 mu g/mL for 24 hours and 48 hours, respectively. The negative control group was added to 200 g/mLBSA, and the effect of AGE-BSA on the cell survival rate was analyzed by MTT colorimetry. The effect of AGE-BSA on the apoptosis rate was analyzed by flow cytometry..2. was added to AGE-BSA treatment at 24 hours and 48 hours in ARPE-19 cells, and the control group was added to 200 Mu g/mLBSA, and AGE-BS was analyzed by the MTT colorimetry. The effect of A postprocessing on cell survival rate was analyzed by the apoptosis detection kit. Results: 1. compared with the negative control group, the cell survival ratio decreased by 22% (P0.05) and 33% (P0.01) in the cells treated with 150 g/mL or 200 mu g/mL AGE-BSA, and the difference was statistically significant, and the different concentrations of AGE-BS were statistically significant. There was also significant difference between the two groups of cells treated with A. When the concentration of AGE-BSA in the cell reached 100 g/mL, the cell apoptosis ratio was significantly higher than that in the negative control group (P0.05). At the same time, it was found that when the concentration of AGE-BSA exceeded 100 u g/mL, the cell apoptosis ratio was positively correlated with the.2. treated cells with AGE-BSA, and negative pairs. The survival ratio of cells decreased by 20% in 24 hours and 40% in 48 hours, while the percentage of cell apoptosis increased by 60% and 110%. in 24 hours and 48 hours, respectively. The use of AGE-BSA to treat ARPE-19 cells could reduce cell survival and induce apoptosis effectively. Second part of AGEs was induced by mitochondrial dysfunction. Study on the apoptosis of human retinal cells: study the role of mitochondrial dysfunction in the process of AGEs induced apoptosis. Methods: 1. the viability of mitochondrial respiratory chain complex IV in human ARPE-19 cells treated by AGE-BSA was detected by the mitochondrial respiratory chain complex IV kit in mice; the mitochondrial membrane potential kits were used to detect the viability of human ARPE-19 cells. The mitochondrial membrane potential level of human ARPE-19 cells.2. cells were cultured for 48 hours and treated with AGE-BSA. The cells added to BSA were set as the control group. The changes in the expression of Bcl-2, Bcl-xl, Bax, Cytc and Caspase3 were detected by Western blot technique. Results: 1. compared with the negative control group, the persons treated with 200 mu blot were treated. The cell mitochondrial respiratory chain complex IV activity decreased by 30% (P0.01), the mitochondrial membrane potential level decreased by 25% (P0.05).2. and the negative control group, the expression level of Bcl-2 and Bcl-xl in ARPE-19 cells decreased by 67% (P0.01) and 43% (P0.05), but the level of Bax (P0.01) was up 122% (P0.01); 3. key molecules associated with apoptosis The expression of Cytc release and activation of Caspase 3 was also significantly different from that in the negative control group. The expression of the two was increased by 43% (P0.01) and 188% (P0.01). Conclusion: AGEs increased the expression of Bax by lowering the level of mitochondrial respiratory chain complex IV and mitochondrial membrane potential and the expression level of Bcl-2, Bcl-xl. Leveling, resulting in mitochondrial dysfunction, resulting in increased Cytc release and activation of Caspase 3 activation eventually activating the apoptosis of human retinal ARPE-19 cells. Third the purpose of part AGEs to induce apoptosis in human retinal cells through Fas-FasL signal: explore the role of Fas-FasL signaling pathway in the process of apoptosis induced by AGEs. Method: 1. ARPE-19 The cells were treated with different concentrations of AGE-BSA, and the cells with different concentrations of BSA were set into negative control groups. The Western blot technique was used to detect Fas, Fas-L, activated caspase8 and the expression level of beta -actin. ARPE-19 cells were treated with alpha -Fas+AGE-BSA in different concentrations. The expression level of Caspase3.3. transfected the synthesized scramble SiRNA, SiRNA-Fas and SiRNA-FasL into ARPE-19 cells. The Western blot technique was used to detect the changes of Fas and Fas-L mRNA expression levels. The expression of spase8 and activated Caspase 3. Results: 1. when the concentration of AGE-BSA in the cells exceeded 100 g/mL, the expressions of Fas and FasL were significantly different compared to those in the blank group, BSA and 50 mu g/mLAGE-BSA, and the FasL expression in the 200 micron AGE-BSA treatment group was 50% higher than that of the 100 micron g/. 200 mu. The expression of activated caspase8 in the g/mL AGE-BSA treatment group increased by 42.8% (P0.01).2. Cytc than in the other groups, and activated caspase8 and activated Caspase3 increased with the increase of the alpha -Fas dose; 50ng/ml alpha -Fas co processing group was 60% higher than that of 20ng/ml alpha co processing group. The co processing group increased by 40% (P0.01), while the expression of activated Caspase3 increased by 45% (P0.01) and 45% (P0.01) in the 20ng/ml alpha -Fas co processing group and the 50ng/ml alpha -Fas co processing group, respectively. The expression of Fas in the iRNA-Fas+AGE-BSA treatment group decreased by 20% (P0.01), and the expression of FasL in the SiRNA-FasL+AGE-BSA treatment group decreased by 13% (P0.01); therefore, both the SiRNA-Fas+AGE-BSA treatment group and the SiRNA-FasL+AGE-BSA treatment group effectively reduced the expression of activated Caspase8 and activated Caspase 3. Conclusion: AGE-BSA processing cells can promote the tables of Fas. Fas-FasL signals induce activation of Caspase8, and exogenous alpha -Fas can enhance AGE-BSA induced Cytc release, activation of caspase8 and activation of Caspase3; SiRNA mediated Fas or FasL gene silencing can effectively inhibit AGE-BSA induced Caspase 8 and activating 3.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R774.1;R587.2

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