NYD-SP15在年龄相关性黄斑变性中的作用研究
发布时间：2019-04-13 18:57
【摘要】：背景及目的：在年龄相关性黄斑变性(AMD)的发病过程中,视网膜色素上皮(RPE)细胞渐进性功能障碍或凋亡起着至关重要的作用。NYD-SP15是胞苷脱氨酶家族成员之一,具有潜在地抑制细胞增殖的功能。最近发现该基因在RPE细胞中呈内源性低表达,但是具体功能尚不清楚。本实验目的是初步研究NYD-SP15对ARPE-19细胞的功能影响及作用机制,为探求NYD-SP15在AMD发生发展的作用提供基础。方法：构建慢病毒稳定转染的NYD-SP15过表达及干扰ARPE-19细胞系,通过CCK-8法、体外创伤划痕愈合实验、流式细胞术及caspase-3、caspase-8、caspase-9蛋白活性水平检测研究其增殖、迁移能力以及凋亡情况。通过基因芯片初步研究NYD-SP15过表达ARPE-19细胞系的全基因组变化及生物学功能。利用过氧化氢诱导NYD-SP15干扰ARPE-19细胞系的氧化应激损伤模型,通过流式细胞术检测细胞凋亡水平及细胞内ROS含量。蛋白印迹实验检测Akt通路、MAPKs通路和Keap-1/Nrf2/HO-1通路蛋白表达水平。结果：成功构建并鉴定慢病毒稳定转染的NYD-SP15过表达及干扰的ARPE-19细胞系。NYD-SP15过表达ARPE-19细胞系的细胞增殖、迁移能力较对照组明显减慢,细胞周期S期到G2/M期阻滞,并出现明显的细胞凋亡,伴caspase-3和caspase-9通路活化增加。基因芯片结果发现,NYD-SP15过表达ARPE-19细胞与对照组相比,有254个基因下调,仅57个基因上调,呈现普遍全基因组表达受抑制现象。GO分析发现NYD-SP15可能影响ARPE-19细胞的基因转录调控功能,而信号通路分析筛选出Akt以及MAPKs通路可能是NYD-SP15行使其负调控功能的主要通路。蛋白免疫印迹结果表明NYD-SP15过表达能明显地抑制ARPE-19细胞中Akt及MAPKs通路蛋白Erkl/2、p-38和JNK的表达水平。此外,正常培养的NYD-SP15干扰ARPE-19细胞增殖能力及细胞周期没有明显变化,但是在饥饿条件下细胞增殖活性增加。而经过氧化氢诱导氧化应激损伤后,与对照组相比,干扰组细胞生存率提高,且细胞内ROS水平降低,提示细胞抗氧化应激能力增强。蛋白免疫印迹结果发现NYD-SP15干扰组细胞中,核Nrf2及HO-1蛋白表达明显增加,Akt、Erk1/2及p38活化水平明显增加,而JNK活化水平降低。结论：NYD-SP15能够明显地抑制Akt或MAPKs通路蛋白表达从而影响ARPE-19细胞功能正常发挥,或诱导细胞凋亡。此外,NYD-SP15干扰后可通过激活Akt途径依赖的Nrf2/HO-1通路保护ARPE-19细胞抵抗氧化应激损伤。因此提示NYD-SP15对ARPE-19细胞具有负调控作用,抑制NYD-SP15病理性高表达能够提高ARPE-19细胞的抗氧化应激损伤能力。
[Abstract]:Background & objective: the progressive dysfunction or apoptosis of retinal pigment epithelium (RPE) cells plays an important role in the pathogenesis of age-related macular degeneration (AMD). NYD-SP15 is a member of cytidine deaminase family. It has the potential to inhibit the proliferation of cells. Recently, it has been found that the gene is endogenous low expression in RPE cells, but the specific function is not clear. The purpose of this study was to study the functional effects of NYD-SP15 on ARPE-19 cells and its mechanism, and to provide a basis for exploring the role of NYD-SP15 in the development of AMD. Methods: lentivirus stable transfection of NYD-SP15 over-expression and interference of ARPE-19 cell line were constructed. CCK-8 assay, wound healing test in vitro, flow cytometry and caspase-3,caspase-8, were performed. The activity level of caspase-9 protein was measured to study the proliferation, migration and apoptosis of the cells. The genomic changes and biological functions of NYD-SP15 overexpressing ARPE-19 cell lines were studied by microarray. The oxidative stress injury model of ARPE-19 cell line induced by hydrogen peroxide was induced by NYD-SP15. The level of apoptosis and the content of ROS in the cell line were detected by flow cytometry. The protein expression levels of Akt pathway, MAPKs pathway and Keap-1/Nrf2/HO-1 pathway were detected by Western blot assay. Results: the ARPE-19 cell line stably transfected with lentivirus was successfully constructed and identified. The cell proliferation and migration ability of NYD-SP15 overexpressing ARPE-19 cell line were significantly slower than those of the control group. Cell cycle arrest from S phase to G2 / M phase showed obvious apoptosis, accompanied by increased activation of caspase-3 and caspase-9 pathways. The results of gene chip showed that there were 254 genes down-regulated and only 57 genes up-regulated in NYD-SP15-overexpressing ARPE-19 cells compared with the control group. Go analysis showed that NYD-SP15 might affect the transcriptional regulation of ARPE-19 cells. Signal pathway analysis showed that Akt and MAPKs pathway may be the main pathways for NYD-SP15 to exercise its negative regulatory function. Western blot showed that the overexpression of NYD-SP15 could significantly inhibit the expression of Akt and MAPKs pathway proteins Erkl/2,p-38 and JNK in ARPE-19 cells. In addition, NYD-SP15 interfered with the proliferation and cell cycle of ARPE-19 cells in normal culture, but the cell proliferation activity increased under the condition of starvation. After oxidative stress injury induced by hydrogen oxide, the survival rate of the cells in the interference group was higher than that in the control group, and the level of ROS in the cells decreased, suggesting that the ability of anti-oxidative stress of the cells was enhanced. Western blot showed that the expression of nuclear Nrf2 and HO-1 protein increased significantly, while the activation level of Akt,Erk1/2 and p38 increased significantly, while the level of JNK activation decreased in NYD-SP15 interference group. Conclusion: NYD-SP15 can significantly inhibit the expression of Akt or MAPKs pathway proteins and thus affect the normal function of ARPE-19 cells or induce apoptosis. In addition, NYD-SP15 interference can protect ARPE-19 cells from oxidative stress damage by activating Akt pathway-dependent Nrf2/HO-1 pathway. It is suggested that NYD-SP15 has a negative regulatory effect on ARPE-19 cells, and inhibiting the pathological high expression of NYD-SP15 can improve the ability of anti-oxidative stress damage of ARPE-19 cells.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R774.5
本文编号：2457839
[Abstract]:Background & objective: the progressive dysfunction or apoptosis of retinal pigment epithelium (RPE) cells plays an important role in the pathogenesis of age-related macular degeneration (AMD). NYD-SP15 is a member of cytidine deaminase family. It has the potential to inhibit the proliferation of cells. Recently, it has been found that the gene is endogenous low expression in RPE cells, but the specific function is not clear. The purpose of this study was to study the functional effects of NYD-SP15 on ARPE-19 cells and its mechanism, and to provide a basis for exploring the role of NYD-SP15 in the development of AMD. Methods: lentivirus stable transfection of NYD-SP15 over-expression and interference of ARPE-19 cell line were constructed. CCK-8 assay, wound healing test in vitro, flow cytometry and caspase-3,caspase-8, were performed. The activity level of caspase-9 protein was measured to study the proliferation, migration and apoptosis of the cells. The genomic changes and biological functions of NYD-SP15 overexpressing ARPE-19 cell lines were studied by microarray. The oxidative stress injury model of ARPE-19 cell line induced by hydrogen peroxide was induced by NYD-SP15. The level of apoptosis and the content of ROS in the cell line were detected by flow cytometry. The protein expression levels of Akt pathway, MAPKs pathway and Keap-1/Nrf2/HO-1 pathway were detected by Western blot assay. Results: the ARPE-19 cell line stably transfected with lentivirus was successfully constructed and identified. The cell proliferation and migration ability of NYD-SP15 overexpressing ARPE-19 cell line were significantly slower than those of the control group. Cell cycle arrest from S phase to G2 / M phase showed obvious apoptosis, accompanied by increased activation of caspase-3 and caspase-9 pathways. The results of gene chip showed that there were 254 genes down-regulated and only 57 genes up-regulated in NYD-SP15-overexpressing ARPE-19 cells compared with the control group. Go analysis showed that NYD-SP15 might affect the transcriptional regulation of ARPE-19 cells. Signal pathway analysis showed that Akt and MAPKs pathway may be the main pathways for NYD-SP15 to exercise its negative regulatory function. Western blot showed that the overexpression of NYD-SP15 could significantly inhibit the expression of Akt and MAPKs pathway proteins Erkl/2,p-38 and JNK in ARPE-19 cells. In addition, NYD-SP15 interfered with the proliferation and cell cycle of ARPE-19 cells in normal culture, but the cell proliferation activity increased under the condition of starvation. After oxidative stress injury induced by hydrogen oxide, the survival rate of the cells in the interference group was higher than that in the control group, and the level of ROS in the cells decreased, suggesting that the ability of anti-oxidative stress of the cells was enhanced. Western blot showed that the expression of nuclear Nrf2 and HO-1 protein increased significantly, while the activation level of Akt,Erk1/2 and p38 increased significantly, while the level of JNK activation decreased in NYD-SP15 interference group. Conclusion: NYD-SP15 can significantly inhibit the expression of Akt or MAPKs pathway proteins and thus affect the normal function of ARPE-19 cells or induce apoptosis. In addition, NYD-SP15 interference can protect ARPE-19 cells from oxidative stress damage by activating Akt pathway-dependent Nrf2/HO-1 pathway. It is suggested that NYD-SP15 has a negative regulatory effect on ARPE-19 cells, and inhibiting the pathological high expression of NYD-SP15 can improve the ability of anti-oxidative stress damage of ARPE-19 cells.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R774.5
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