线粒体参与蓝光诱导视网膜色素上皮细胞损伤机制的体外研究

发布时间：2018-01-28 06:31

本文关键词： 蓝光照射线粒体DNA 凋亡氧化应激人视网膜色素上皮细胞　出处：《广州中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:眼底病近年来的研究热点中,年龄相关性黄斑病变(Age-related macμlar degeneration,AMD)受到越来越多的关注,AMD是一种可以造成中心视力下降甚至视力完全丧失的疾病,在疾病早期常无明显症状,但早期往往就会伴有视网膜色素上皮(retinal pigment epithelium,RPE)的萎缩。国内外均有研究表明,光损伤是AMD的重要诱因,其中蓝光是可见光中能量最高的,长时间暴露于高强度的蓝光下可导致人视网膜的光损伤,现已有研究证实蓝光可以诱导RPE细胞发生氧化损伤和细胞凋亡,其中线粒体在蓝光损伤RPE细胞中的具有重要作用,但是线粒体及线粒体DNA在RPE细胞损伤中的的作用机制和动态变化仍不清楚。本实验拟通过体外分离培养人原代RPE细胞,经过蓝光诱导损伤RPE细胞,观察在同样蓝光辐照强度下,处以不同时长的蓝光照射,观察RPE细胞因此产生的损伤变化,同时深入研究线粒体DNA参与到蓝光诱导RPE细胞损伤中的作用具体变化。研究方法:取对数生长期的原代RPE细胞用于本次实验研究,使用一定辐射强度的蓝光进行照射。根据光照时间将RPE细胞分为正常对照组(光照0 h组)和蓝光光照组细胞,蓝光光照组分别照射0.5 h、1 h、2 h、4 h、6 h、12 h、24h。免疫荧光法检测RPE细胞特有蛋白RPE65表达结果。Western blot蛋白质印迹法检测不同蓝光照射时长下RPE 细胞中的凋亡相关蛋白 Caspase-3、Cleaved Caspase-3、Caspase9、Cleaved Caspase-9 Bcl-2及Bax的相对表达量。RT-PCR技术检测凋亡相关基因Caspase-3、Caspase-9、Bcl-2及Bax的表达情况。实时荧光定量PCR技术检测线粒体DNA拷贝数的相对表达量。普通PCR扩增检测线粒体DNA4977bp基因缺失的表达情况。PCR扩增产物进行基因测序观察突变情况。研究结果:1.不同时长的蓝光照射人原代RPE细胞后,Western Blot结果显示Bax从lh开始表达水平均显著高于对照组(P0.05);Cleaved Caspase-3从2h开始表达水平均显著高于对照组(P0.05);Caspase-3、Caspase-9 Cleaved Caspase-9 从 4h开始表达水平均显著高于对照组(P0.05);Bcl-2从2h开始表达水平均较对照组下调(P0.05)。2.RT-PCR显示凋亡相关基因Bcl-2在光照2h后表达量上调,光照6h后表达量低于对照组(P0.05),Caspase-3、Caspase-9和Bax分别在光照12h、4h和4h后表达量明显上调(P0.05)。3.与对照组相比,光照2h后,线粒体DNA拷贝数表达开始下降,具有显著差异(*P0.05),呈时间依赖性降低。4.光照4h后,线粒体DNA4977bp缺失表达情况与正常组比较呈上升趋势,并且随光照时间延长,缺失情况越加严重,结果具有显著差异(*P0.05)。5.基因测序提示普通PCR扩增产物与线粒体DNA发生4977bp基因缺失后基因型相符。结论:1.蓝光照射1h后,促凋亡蛋白Bax表达增加,光照2h后,CleavedCaspase-3开始上升,抑凋亡蛋白Bcl-2开始下降,4h后Caspase-3、Caspase-9、Cleaved Caspase-9开始上升,凋亡相关基因Bcl-2在光照2h后出现上调,6小时开始出现下降,Bax、Caspase-9在光照4小时后开始上调,Caspase-3在12h后出现上升,提示在细胞经过蓝光照射后,凋亡系统被激活,并且是以线粒体介导的凋亡通路为主,从而诱导RPE细胞的凋亡。2.蓝光照射2h后,线粒体DNA拷贝数逐步下降,然而在光照4h开始,线粒体DNA4977bp基因缺失明显增加,证明了蓝光照射可以影响线粒体DNA的复制并且提高其突变率。这表明蓝光光照2h开始出现线粒体DNA损伤,损伤形式包括线粒体DNA拷贝数下降以及突变率增加,随后出现RPE细胞损伤,继而出现凋亡以及死亡。
[Abstract]:Background: the research focus in recent years fundus diseases, age-related macular degeneration (Age-related MAC lar degeneration, AMD) has attracted more and more attention, AMD is a can cause the decline of visual acuity and even complete loss of vision disease early in the disease often no obvious symptoms, but often accompanied by early retinal pigment epithelium (retinal pigment epithelium, RPE) atrophy. Some studies at home and abroad show that the optical damage is an important cause of AMD, which is the highest visible light blue energy, long time exposure to high intensity blue light can cause retinal light damage, present studies have confirmed that blue light can induce oxidative damage and apoptosis of RPE cells among them, mitochondrial damage in RPE cells plays an important role in the blue, but the mitochondria and mitochondrial DNA in RPE cell injury mechanism and the dynamic change is still not Clearly. The experiment of in vitro cultured human primary RPE cells after injury of RPE cells induced by blue light, blue light irradiation intensity observed at the same time, a blue light, so the observation of damage caused by the changes of RPE cells, and studies on mitochondrial DNA involved in blue light induced RPE cell damage in concrete change. Methods: primary RPE cells in logarithmic growth phase were used in this experiment, using the radiation intensity of blue light irradiation. According to the illumination time of RPE cells were divided into normal control group (H group, 0 light) and blue light group cells, blue light group were irradiated 0.5 h. 1 h, 2 h, 4 h, 6 h, 12 h, detection of RPE cell specific protein RPE65 24h. immunofluorescence detection of.Western protein expression results of blot blotting in different time under blue light irradiation in RPE cell apoptosis related protein Caspase-3, Cleaved Casp Ase-3, Caspase9, Cleaved Caspase-9 and Bax Bcl-2 relative expression of apoptosis related gene Caspase-3.RT-PCR technology, Caspase-9, expression of Bcl-2 and Bax. The relative expression of real-time fluorescence quantitative PCR detection of mitochondrial DNA copy number amplification. Mitochondrial DNA4977bp gene deletion PCR expression of.PCR amplified products were sequenced to observe the mutation. Results: 1. blue light irradiation time of primary RPE cells, Western Blot showed that Bax began expression level were significantly higher than the control group from LH (P0.05); Cleaved Caspase-3 expression levels were significantly higher than those in the control group from 2H (P0.05); Caspase-3 Caspase-9 Cleaved Caspase-9, the expression level began were higher than the control group from 4h (P0.05); Bcl-2 expression levels than those in the control group decreased from 2H (P0.05).2.RT-PCR showed apoptosis related gene Bcl-2 in After 2H light upregulated after 6h light expression was lower than that of the control group (P0.05), Caspase-3, Caspase-9 and Bax respectively according to 12h in light, up-regulated 4H and 4H (P0.05).3. compared with the control group, after 2H light mitochondrial DNA copy number expression began to decrease significantly the difference (*P0.05), a time dependent decrease in.4. after 4H light expression of mitochondrial DNA4977bp deletion compared with the normal group showed an upward trend, and with the increase of irradiation time, the lack of more results showed significant difference (*P0.05).5. gene sequencing suggested that genotype PCR amplified products and consistent common mitochondrial DNA 4977bp after gene deletion. Conclusion: 1. blue light 1H, the pro apoptotic protein Bax expression increased after 2H light, CleavedCaspase-3 began to rise, the antiapoptotic protein Bcl-2 began to decline after 4h Caspase-3, Caspase-9, Cleaved and Caspase-9 began to rise, apoptosis related genes Because Bcl-2 was up-regulated after 2H light 6 hours began to decline, Bax, Caspase-9 began to increase in light after 4 hours, Caspase-3 increased after 12h, suggesting that the cell apoptosis after blue light irradiation, the system is activated, and the mitochondria mediated apoptosis pathway in the apoptosis of.2.. The blue light irradiation 2H can induce RPE cells, mitochondrial DNA copy number decreased gradually, but in the light of 4h, mitochondrial DNA4977bp deletion increased significantly, that blue light can affect mitochondrial DNA replication and increase the mutation rate. This indicates that blue light began to appear 2H mitochondrial DNA damage, including damage of mitochondrial DNA copy number the mutation rate decreased and increased, followed by RPE cell injury, and apoptosis and death.

【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R774.5

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