海藻糖衍生物DMBT对湿性老年黄斑变性的作用及机制研究

发布时间：2018-03-24 03:01

本文选题：湿性黄斑变性　切入点：脉络膜血管新生　出处：《山东大学》2017年硕士论文

【摘要】：老年黄斑变性是世界范围内老年群体最常见的致盲疾病。脉络膜新生血管是湿性老年黄斑变性的的主要标志,也是最主要的致盲原因。人眼底感光细胞和视网膜色素上皮细胞与上层视网膜血管隔开,由下层脉络膜血管供养,单层紧密排列的视网膜色素上皮细胞即血-视网膜屏障起到保护感光细胞和物质转换等作用。视网膜缺氧导致的视网膜色素上皮细胞分泌血管内皮生长因子(vascular endothelial growth factor,VEGF)增多,作用在邻近的脉络膜血管内皮细胞上,促使其增殖、迁移、形成新生血管是眼底新生血管类疾病的共同病理特点,新生血管会吸收光,一方面引起视物变形,另一方面不成熟的新生血管会出现出血和渗出,最终导致失明。如何抑制低氧条件下视网膜色素上皮细胞过量分泌的VEGF是治疗脉络膜血管新生的关键。我院药化所刘兆鹏教授以日本科学家Igarashi从放线菌Nonomuraea代谢产物中分离得到的活性化合物Brartemicin为先导化合物,设计合成多组海藻糖类的衍生物,并对这些化合物进行了初步活性筛选。发现6,6'-双(2,3-二甲氧基苯甲酰基)-α,α-D-海藻糖(6,6.-bis(2,3-dimethoxybenzoyl)-α,α-D-trehalose,DMBT,M=670.61 g/mol)的体外抗肿瘤血管新生的活性比Brartemicin的更强。前期研究发现,DMBT的毒性较低,能通过下调肿瘤细胞中VEGF和p-VEGF的表达,抑制内皮细胞小管形成和迁移的能力,具有较强的体内抗肿瘤血管新生的活性,并且能抑制鸡胚尿囊膜血管生成,这提示该化合物对于眼底血管新生类疾病可能也有作用。但DMBT对脉络膜血管新生是否有作用还不清楚。本课题通过体内外实验,研究化合物DMBT对湿性老年黄斑变性即脉络膜血管新生的治疗作用,并研究其分子机制,为得到高效低毒、具有我国自主知识产权的治疗湿性老年黄斑变性的药物奠定基础。利用MTT实验检测化合物DMBT在2-128 μmol/L时对ARPE-19和RF/6A细胞的抑制率,结果显示抑制率均低于20%,而且8-32 μmol/L DMBT对细胞增殖和细胞活力无明显影响。划痕实验和管形成实验显示,在缺氧微环境中,使用含有8和32 μmol/L DMBT的ARPE-19细胞条件培养基对RF/6A细胞进行培养,与无条件培养基的RF/6A培养组相比,能够明显抑制RF/6A细胞的迁移和管形成,说明DMBT可能通过作用于ARPE-19细胞影响RF/6A细胞的迁移和管形成。532 nm激光损伤C57BL/6小鼠眼底脉络膜,构建小鼠脉络膜血管损伤模型。脉络膜lectin488荧光染色结果显示,与阴性对照组相比,玻璃体内注射0.2和1 mmol/LDMBT 1μL,可以显著抑制脉络膜血管的新生且无明显毒副作用。ELISA实验检测ARPE-19细胞分泌到细胞外的血管内皮生长因子VEGF的水平,结果发现8和32 μmol/L的DMBT能抑制由于缺氧导致的ARPE细胞分泌的VEGF的量的升高且具有剂量依赖性,活性氧抑制剂N-乙酰半胱氨酸(N-acetyl-cysteine,NAC)也可以在一定程度上影响VEGF的分泌。Western blotting实验显示,DMBT给药组降低了模型鼠视网膜中VEGF蛋白的表达。表明DMBT在体内外都可以抑制视网膜色素上皮细胞对VEGF的分泌。通过活性氧试剂盒检测发现8和32μmol/L DMBT能抑制由于缺氧所导致的活性氧的升高。提示DMBT可能通过活性氧相关的通路降低VEGF的分泌。Western blotting实验结果显示,32 μmol/L DMBT能抑制ARPE-19细胞在缺氧微环境中细胞内上调的p-ERK1/2,核内Nrf2,HO-1和HIF-1α蛋白的表达,这进一步表明DMBT作用机制与抑制细胞活性氧相关。另外ELISA检测发现500 μmol/L的NAC对ARPE-19细胞分泌VEGF的作用弱于32 μmol/L的DMBT,说明DMBT对VEGF表达的影响不只与活性氧有关,接下来我们利用Western blotting检测了 DMBT对p-Akt和p-NF-KB的表达的影响,发现DMBT能抑制p-Akt和p-NF-κB的表达。结论:海藻糖衍生物DMBT在缺氧状态下,能够通过对视网膜色素上皮细胞的作用,间接抑制脉络膜血管新生,且细胞毒性较低。通过影响ERK1/2/Nrf2和Akt/NF-KB活性氧依赖通路影响下游HIF-1α的表达,抑制VEGF的分泌。从而起到抑制脉络膜血管新生的作用。由此DMBT以其高效低毒的特点有潜力成为抗脉络膜血管新生类疾病的候选化合物之一。
[Abstract]:Age related macular degeneration is the most common disease of the elderly blindness worldwide. Choroidal neovascularization is the main symbol of wet AMD, and it is also the main cause of blindness. Retinal photoreceptor cells and separated from human retinal pigment epithelial cells and the retinal vessels, supported by the lower choroidal vascular function, retinal pigment epithelial cells closely packed monolayers the blood retinal barrier to protect photoreceptor cells and material conversion. Retinal hypoxia induced retinal pigment epithelial cells secrete vascular endothelial growth factor (vascular endothelial, growth factor, VEGF) increased role in choroidal vascular endothelial cells on the adjacent, prompting the proliferation, migration, angiogenesis is common the pathological features of fundus neovascular diseases, neovascularization will absorb light, on the one hand cause metamorphopsia, on the other hand is not mature The neovascularization will appear bleeding and exudation, eventually lead to blindness. How to suppress the retinal pigment epithelial cells under hypoxic conditions excessive secretion of VEGF is critical for the treatment of choroidal neovascularization. Our hospital medicine Professor Liu Zhaopeng by Japanese scientists Igarashi from actinomycetes Nonomuraea isolated metabolites in the activity of compound Brartemicin as the lead compound design, synthesis of multiple groups of seaweed sugar derivatives of these compounds were preliminary screening. The discovery of 6,6'- double (2,3- two methoxy benzoyl) - alpha, alpha -D- trehalose (6,6.-bis (2,3-dimethoxybenzoyl) - alpha, alpha -D-trehalose, DMBT, M=670.61, g/mol) anti angiogenesis activity than in vitro Brartemicin stronger. The preliminary study found that the toxicity of DMBT is low, can down regulate expression of VEGF and p-VEGF in tumor cells, inhibition of endothelial cell migration and tubule formation can Force, with a strong anti angiogenesis activity, and can inhibit angiogenesis in chick chorioallantoic membrane, suggesting that the compound may also have an effect on the angiogenesis of fundus diseases. But DMBT on choroidal neovascularization is a role is not clear. This paper through the experiments to study the therapeutic effect of compound DMBT wet age-related macular degeneration or choroidal neovascularization, and study its molecular mechanism, in order to obtain high efficiency and low toxicity, lay the foundation treatment with our own intellectual property rights of wet age related macular degeneration therapeutics. In 2-128 mol/L of ARPE-19 and the inhibition rate of RF/6A cells by DMBT MTT assay compounds, results showed that the inhibition the rate was lower than 20%, and 8-32 mol/L DMBT had no obvious effect on cell proliferation and cell viability. Scratch assay and tube formation assay showed that under hypoxic conditions, containing 8 and 3 2 mol/L DMBT ARPE-19 cell conditioned medium on RF/6A cells were cultured in conditioned medium compared with no RF/6A culture group, could significantly inhibit RF/6A cell migration and tube formation, indicating that DMBT may be acting on the effect of ARPE-19 cell RF/6A cell migration and tube formation of.532 nm laser damage of C57BL/6 mice retinal choroid to construct mouse choroidal vascular injury model. Choroidal lectin488 staining results showed that compared with the negative control group, intravitreal injection of 0.2 and 1 mmol/LDMBT 1 L can significantly inhibit choroidal neovascularization, and no obvious side effects by.ELISA assay, ARPE-19 cells secreted into the extracellular levels of vascular endothelial growth factor VEGF the results showed that 8, and 32 mol/L DMBT can inhibit the secretion of ARPE cell induced by hypoxia due to the amount of VEGF increased in a dose-dependent manner, ROS inhibitor N Acetylcysteine (N-acetyl-cysteine, NAC) to some extent can also affect the secretion of VEGF.Western blotting experiment, DMBT decreased the expression of VEGF protein in retina of rats in the model group. Show that DMBT can inhibit the retinal pigment epithelial cells on the secretion of VEGF both in vitro and in vivo. The activated oxygen detection kit and found 8 32 mol/L DMBT inhibited due to elevated ROS caused by hypoxia. It suggests that DMBT may play an active oxygen related pathway reduces the secretion of VEGF.Western blotting results showed that 32 mol/L DMBT could inhibit ARPE-19 cells in hypoxic microenvironment within the cell nucleus of the upregulation of p-ERK1/2, Nrf2, HO-1 and HIF-1 protein expression and this further indicates that the action mechanism of DMBT and inhibition of ROS. Also ELISA detected 500 mol/L NAC on VEGF secretion of ARPE-19 cells less than 32 Mu mol /L DMBT, shows the influence of DMBT on the expression of VEGF is not only related to active oxygen, then we use Western blotting to detect the effects of DMBT on expression of p-Akt and p-NF-KB, found that DMBT could decrease the expression of p-Akt and p-NF- K B. Conclusion: Trehalose derivatives DMBT under anoxic condition, can pass on retinal pigment epithelial cells effect of indirect inhibition of choroidal neovascularization, and low cytotoxicity. The effects of ERK1/2/Nrf2 and Akt/NF-KB affect the expression of ROS dependent pathway downstream of HIF-1 alpha, inhibit the secretion of VEGF. In order to inhibit choroidal neovascularization. Thus DMBT with its characteristics of high efficiency and low toxicity have become one of the potential candidate compounds against choroid angiogenesis diseases.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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