15-脂氧合酶-1在缺氧导致的视网膜新生血管性疾病中的作用机制研究
发布时间：2018-11-25 14:25
【摘要】：第一部分缺氧导致视网膜新生血管性疾病体外模型的建立及评估 目的：建立并评估一种新型的视网膜新生血管性疾病体外模型。 方法：原代培养的视网膜微血管内皮细胞取材于C57/BL6J小鼠视网膜,异硫氰酸荧光素标记的CD31进行细胞鉴定。3-6代小鼠视网膜微血管内皮细胞培养于厌氧袋中构建缺氧模型。缺氧时间设为0.5、1、2、3、12、24、48及72小时。血气分析各时间点细胞培养液的pO2, pCO2及pH,对该模型进行评估。 结果：异硫氰酸荧光素标记的CD31鉴定视网膜微血管内皮细胞阳性率为90%。当视网膜微血管内皮细胞在厌氧袋中培养2小时,血气分析结果显示p02为5.60Kpa, pCO2为6.04Kpa, pH为7.07；当视网膜微血管内皮细胞在厌氧袋中培养大于2小时,血气分析结果显示p02为4.5Kpa, pCO2和pH无明显变化。 结论：厌氧袋培养视网膜微血管内皮细胞可以模拟缺氧导致视网膜新生血管性疾病的体外模型,该方法简单有效。 第二部分VEGF在视网膜微血管内皮细胞中自分泌作用的研究 目的：探讨在缺氧导致的视网膜新生血管性疾病中,视网膜微血管内皮细胞是否通过自分泌血管内皮生长因子信号实现增殖。 方法：实验分为缺氧组和常氧组。视网膜微血管内皮细胞培养于厌氧袋中为缺氧组,缺氧时间为12、24、48、72小时。设立相应时间点常氧环境下培养的细胞作为常氧组。CCK-8法检测细胞增殖。流式细胞术检测细胞调亡率。各组细胞不同时间点VEGF-A, VEGFR-2, eNOs的mRNA及蛋白水平通过实时定量PCR及Western blot检测。 结果：CCK-8结果显示缺氧组各时间点的视网膜微血管内皮细胞增殖能力高于常氧组(p0.05)。在缺氧环境下,48h细胞的增殖能力达高峰。流式细胞术结果显示缺氧组各时间点视网膜微血管内皮细胞的调亡率低于常氧组。实时定量RT-PCR结果显示缺氧组的视网膜微血管内皮细胞的VEGF-A, VEGF-R2和eNOs的mRNA水平明显高于常氧组(p0.01),缺氧组中VEGF-A, VEGF-R2和eNOs的]mRNA水平随时间上升,48h达高峰,随后下降。Western blot结果显示VEGF-A, VEGF-R2和NOs蛋白水平的变化和mRNA的变化相一致。 结论：在缺氧环境下,视网膜微血管内皮细胞可通过自分泌途径,上调血管内皮生长因子家族成员,完成自身增殖,生成视网膜新生血管。 第三部分15-脂氧合酶-1在缺氧导致的视网膜新生血管性疾病中的作用及机制研究 目的：探讨15-脂氧合酶-1在缺氧导致的视网膜新生血管性疾病中的作用机制。 方法：实验分为四组：常氧组,缺氧组,缺氧转染15-LOX-1组,缺氧转染GFP对照组。缺氧环境通过厌氧袋构建。观察时间设为12、24、48、72h。转染效率通过荧光显微镜观察。CCK-8法检测细胞增殖。各组细胞不同时间点15-LOX-1, VEGF-A, VEGFR-2, eNOs, PPAR-r mRNA及蛋白水平通过实时定量RT-PCR及Western blot检测。 结果：CCK-8结果显示缺氧组各时间点的视网膜微血管内皮细胞增殖能力高于常氧组。在缺氧条件下,15-脂氧合酶-1组细胞增殖率明显被抑制。实时定量RT-PCR结果显示缺氧组VEGF-A, VEGF-R2,eNOs的mRNA水平明显高于常氧组(p0.01)；但缺氧组15-LOX-1, PPAR-r的mRNA水平却明显低于常氧组(p0.01)。缺氧转染15-脂氧合酶-1组VEGF-A, VEGF-R2, eNOs的mRNA水平明显低于缺氧组(p0.01)；但其15-LOX-1, PPAR-r的mRNA水平却明显高于缺氧组(p0.01)。缺氧转染GFP对照组与缺氧组之间VEGF-A, VEGF-R2, eNOs,15-LOX-1和PPAR-r的mRNA水平无统计学差异(p0.05)。Western blot结果显示蛋白水平的变化和(?)nRNA的变化相一致。 结论：15-脂氧合酶-1和PPAR-r在视网膜血管形成过程中起负性调节作用。视网膜微血管内皮细胞过表达15-脂氧合酶-1通过抑制缺氧导致的VEGF家族的上调,从而抑制视网膜新生血管的形成。同时PPAR-r作为15-脂氧合酶-1的配体,结合VEGFR2是15-脂氧合酶-1抑制缺氧导致的视网膜新生血管的另一机制。
[Abstract]:The establishment and evaluation of the in vitro model of retinal neovascular disease caused by hypoxia of the first part Objective: To establish and evaluate a new in vitro new type of retinal neovascular disease Methods: The primary cultured retinal microvessel endothelial cells were cultured in C57/ BL6J mouse retina and fluorescein isothiocyanate labeled CD31 for cell identification. The 3-6-generation mouse retinal microvessel endothelial cells were cultured in an anaerobic bag. The hypoxia time is set to 0. 5, 1, 2, 3, 12, 24, 48. and 72 hours. The blood gas analyzes the pO2, pCO2 and the pH of the cell culture solution at each time point, and the model Type assessment. Results: The CD31 labeled with fluorescein isothiocyanate identified the retinal microvessel endothelium The positive rate of the cells was 90%. When the microvessel endothelial cells were cultured in an anaerobic bag for 2 hours, the blood gas analysis showed that p02 was 5.60Kpa, pCO2 was 6.04Kpa, and the pH was 7.07; when the microvessel endothelial cells were cultured in an anaerobic bag for more than 2 hours, the blood gas analysis showed that p02 was 4.5Kpa, pCO2 Conclusion: The cultured retinal microvessel endothelial cells can simulate the body of neovascular disease caused by hypoxia. The external model is simple and effective. The second part of VEGF is in the retina. The purpose of the study on the self-secretion in vascular endothelial cells: to investigate whether the retinal microvessel endothelial cells pass through the retinal neovascular disease caused by hypoxia Self-secretion of vascular endothelial growth factor signal Methods: The experiment was divided into two groups: the hypoxia group and the normal oxygen group. The retinal microvessel endothelial cells were cultured in the anaerobic bag for hypoxia. The time of hypoxia was 12, 24, 48, 72 hours. in that normal oxygen environment of the corresponding time point Cells as the normal oxygen group. CCK-8 Cell proliferation was detected by flow cytometry. The expression of VEGF-A, VEGFR-2, eNOs and the level of protein were measured by flow cytometry. The results were as follows: CCK-8 showed the retina of each time point in the hypoxia group. The proliferation of microvessel endothelial cells was higher than that of normal oxygen group (p0.05). 5) In the hypoxia environment, the proliferation ability of the 48h cells reached a peak. The flow cytometry showed that the hypoxia group The expression of VEGF-A, VEGF-R2 and eNOs in the retinal microvessel endothelial cells of the hypoxia group was significantly higher than that of the normal oxygen group (p0.01), and the VEGF-A, VEGF-R2 and eNOs in the hypoxia group were significantly higher than that of the normal oxygen group (p0.01). The expression of VEGF-A, VEGF-R, VEGF-A, VEGF-R, VEGF-A and VEGF-R were detected by Western blot. Conclusion: Under the condition of hypoxia, the vascular endothelial cells of the retina can be up-regulated by the autocrine pathway. A member of the skin growth factor family, to complete its self-proliferation and to generate a retinal neovascularization. The role of oxygen-in-1-1 in the retinal neovascular disease caused by hypoxia and its mechanism in the study of its mechanism: The mechanism of the action of 15-lipoxygenase-1 in the retinal neovascular disease caused by hypoxia. 4 groups: normoxic group, hypoxia group, deficiency Oxygen Transfection of 15-LOX-1 and Hypoxia Transfection of GF P control group, anaerobic environment through anaerobic bag constructed. The observation time is set to 12, 2 4, 48, 72h. The transfection efficiency was observed by fluorescence microscope. The cell proliferation was detected by CCK-8 method. The different time points of each group were 15-LOX-1, VEGF-A, VEGFR-2, eNOs, PPAR-r. mRNA and protein levels were detected by real-time quantitative RT-PCR and Western blot. Results: The results of CCK-8 showed that the retinal microvessel endothelium was fine at all time points in the hypoxia group. The results of real-time quantitative RT-PCR showed that the level of VEGF-A, VEGF-R2 and eNOs in the hypoxic group was significantly higher than that of the normal oxygen group (p0.01). The mRNA levels of 15-LOX-1 and PPAR-r in group 15-LOX-1 and PPAR-r were lower than that of normal oxygen group (p0.01). The levels of VEGF-A, VEGF-R2 and eNOs in the 15-lipoxygenase-1 group were significantly lower than those in the hypoxia group (p0.01). The expression of VEGF-A, VEGF-R2, eNOs, 15-LOX-1 and PPAR-r between the control group and the hypoxia group was higher than that of the hypoxia group (p0.01). No statistical difference (p0.05) The results showed that the changes of the protein level and (?) nRNA were consistent with the Western blot. Conclusion: 15-lipoxygenase-1 and PPAR-r play a negative role in the formation of retinal vessels. 5-lipoxygenase-1 inhibits the formation of retinal neovascularization by inhibiting the up-regulation of the VEGF family resulting from hypoxia, while PPAR-r acts as a 15-lipoxygenase-1.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1
本文编号：2356433
[Abstract]:The establishment and evaluation of the in vitro model of retinal neovascular disease caused by hypoxia of the first part Objective: To establish and evaluate a new in vitro new type of retinal neovascular disease Methods: The primary cultured retinal microvessel endothelial cells were cultured in C57/ BL6J mouse retina and fluorescein isothiocyanate labeled CD31 for cell identification. The 3-6-generation mouse retinal microvessel endothelial cells were cultured in an anaerobic bag. The hypoxia time is set to 0. 5, 1, 2, 3, 12, 24, 48. and 72 hours. The blood gas analyzes the pO2, pCO2 and the pH of the cell culture solution at each time point, and the model Type assessment. Results: The CD31 labeled with fluorescein isothiocyanate identified the retinal microvessel endothelium The positive rate of the cells was 90%. When the microvessel endothelial cells were cultured in an anaerobic bag for 2 hours, the blood gas analysis showed that p02 was 5.60Kpa, pCO2 was 6.04Kpa, and the pH was 7.07; when the microvessel endothelial cells were cultured in an anaerobic bag for more than 2 hours, the blood gas analysis showed that p02 was 4.5Kpa, pCO2 Conclusion: The cultured retinal microvessel endothelial cells can simulate the body of neovascular disease caused by hypoxia. The external model is simple and effective. The second part of VEGF is in the retina. The purpose of the study on the self-secretion in vascular endothelial cells: to investigate whether the retinal microvessel endothelial cells pass through the retinal neovascular disease caused by hypoxia Self-secretion of vascular endothelial growth factor signal Methods: The experiment was divided into two groups: the hypoxia group and the normal oxygen group. The retinal microvessel endothelial cells were cultured in the anaerobic bag for hypoxia. The time of hypoxia was 12, 24, 48, 72 hours. in that normal oxygen environment of the corresponding time point Cells as the normal oxygen group. CCK-8 Cell proliferation was detected by flow cytometry. The expression of VEGF-A, VEGFR-2, eNOs and the level of protein were measured by flow cytometry. The results were as follows: CCK-8 showed the retina of each time point in the hypoxia group. The proliferation of microvessel endothelial cells was higher than that of normal oxygen group (p0.05). 5) In the hypoxia environment, the proliferation ability of the 48h cells reached a peak. The flow cytometry showed that the hypoxia group The expression of VEGF-A, VEGF-R2 and eNOs in the retinal microvessel endothelial cells of the hypoxia group was significantly higher than that of the normal oxygen group (p0.01), and the VEGF-A, VEGF-R2 and eNOs in the hypoxia group were significantly higher than that of the normal oxygen group (p0.01). The expression of VEGF-A, VEGF-R, VEGF-A, VEGF-R, VEGF-A and VEGF-R were detected by Western blot. Conclusion: Under the condition of hypoxia, the vascular endothelial cells of the retina can be up-regulated by the autocrine pathway. A member of the skin growth factor family, to complete its self-proliferation and to generate a retinal neovascularization. The role of oxygen-in-1-1 in the retinal neovascular disease caused by hypoxia and its mechanism in the study of its mechanism: The mechanism of the action of 15-lipoxygenase-1 in the retinal neovascular disease caused by hypoxia. 4 groups: normoxic group, hypoxia group, deficiency Oxygen Transfection of 15-LOX-1 and Hypoxia Transfection of GF P control group, anaerobic environment through anaerobic bag constructed. The observation time is set to 12, 2 4, 48, 72h. The transfection efficiency was observed by fluorescence microscope. The cell proliferation was detected by CCK-8 method. The different time points of each group were 15-LOX-1, VEGF-A, VEGFR-2, eNOs, PPAR-r. mRNA and protein levels were detected by real-time quantitative RT-PCR and Western blot. Results: The results of CCK-8 showed that the retinal microvessel endothelium was fine at all time points in the hypoxia group. The results of real-time quantitative RT-PCR showed that the level of VEGF-A, VEGF-R2 and eNOs in the hypoxic group was significantly higher than that of the normal oxygen group (p0.01). The mRNA levels of 15-LOX-1 and PPAR-r in group 15-LOX-1 and PPAR-r were lower than that of normal oxygen group (p0.01). The levels of VEGF-A, VEGF-R2 and eNOs in the 15-lipoxygenase-1 group were significantly lower than those in the hypoxia group (p0.01). The expression of VEGF-A, VEGF-R2, eNOs, 15-LOX-1 and PPAR-r between the control group and the hypoxia group was higher than that of the hypoxia group (p0.01). No statistical difference (p0.05) The results showed that the changes of the protein level and (?) nRNA were consistent with the Western blot. Conclusion: 15-lipoxygenase-1 and PPAR-r play a negative role in the formation of retinal vessels. 5-lipoxygenase-1 inhibits the formation of retinal neovascularization by inhibiting the up-regulation of the VEGF family resulting from hypoxia, while PPAR-r acts as a 15-lipoxygenase-1.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1
【参考文献】
相关期刊论文 前1条
1 许建华;刘哲丽;李若溪;孔伟;张薇;;曲安奈德对缺氧条件下恒河猴视网膜血管内皮细胞增殖的抑制作用(英文)[J];国际眼科杂志;2006年02期
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