本文選題：濕性黃斑變性　切入點(diǎn)：脈絡(luò)膜血管新生　出處：《山東大學(xué)》2017年碩士論文

【摘要】：老年黃斑變性是世界范圍內(nèi)老年群體最常見的致盲疾病。脈絡(luò)膜新生血管是濕性老年黃斑變性的的主要標(biāo)志,也是最主要的致盲原因。人眼底感光細(xì)胞和視網(wǎng)膜色素上皮細(xì)胞與上層視網(wǎng)膜血管隔開,由下層脈絡(luò)膜血管供養(yǎng),單層緊密排列的視網(wǎng)膜色素上皮細(xì)胞即血-視網(wǎng)膜屏障起到保護(hù)感光細(xì)胞和物質(zhì)轉(zhuǎn)換等作用。視網(wǎng)膜缺氧導(dǎo)致的視網(wǎng)膜色素上皮細(xì)胞分泌血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF)增多,作用在鄰近的脈絡(luò)膜血管內(nèi)皮細(xì)胞上,促使其增殖、遷移、形成新生血管是眼底新生血管類疾病的共同病理特點(diǎn),新生血管會(huì)吸收光,一方面引起視物變形,另一方面不成熟的新生血管會(huì)出現(xiàn)出血和滲出,最終導(dǎo)致失明。如何抑制低氧條件下視網(wǎng)膜色素上皮細(xì)胞過量分泌的VEGF是治療脈絡(luò)膜血管新生的關(guān)鍵。我院藥化所劉兆鵬教授以日本科學(xué)家Igarashi從放線菌Nonomuraea代謝產(chǎn)物中分離得到的活性化合物Brartemicin為先導(dǎo)化合物,設(shè)計(jì)合成多組海藻糖類的衍生物,并對(duì)這些化合物進(jìn)行了初步活性篩選。發(fā)現(xiàn)6,6'-雙(2,3-二甲氧基苯甲�；�)-α,α-D-海藻糖(6,6.-bis(2,3-dimethoxybenzoyl)-α,α-D-trehalose,DMBT,M=670.61 g/mol)的體外抗腫瘤血管新生的活性比Brartemicin的更強(qiáng)。前期研究發(fā)現(xiàn),DMBT的毒性較低,能通過下調(diào)腫瘤細(xì)胞中VEGF和p-VEGF的表達(dá),抑制內(nèi)皮細(xì)胞小管形成和遷移的能力,具有較強(qiáng)的體內(nèi)抗腫瘤血管新生的活性,并且能抑制雞胚尿囊膜血管生成,這提示該化合物對(duì)于眼底血管新生類疾病可能也有作用。但DMBT對(duì)脈絡(luò)膜血管新生是否有作用還不清楚。本課題通過體內(nèi)外實(shí)驗(yàn),研究化合物DMBT對(duì)濕性老年黃斑變性即脈絡(luò)膜血管新生的治療作用,并研究其分子機(jī)制,為得到高效低毒、具有我國自主知識(shí)產(chǎn)權(quán)的治療濕性老年黃斑變性的藥物奠定基礎(chǔ)。利用MTT實(shí)驗(yàn)檢測(cè)化合物DMBT在2-128 μmol/L時(shí)對(duì)ARPE-19和RF/6A細(xì)胞的抑制率,結(jié)果顯示抑制率均低于20%,而且8-32 μmol/L DMBT對(duì)細(xì)胞增殖和細(xì)胞活力無明顯影響。劃痕實(shí)驗(yàn)和管形成實(shí)驗(yàn)顯示,在缺氧微環(huán)境中,使用含有8和32 μmol/L DMBT的ARPE-19細(xì)胞條件培養(yǎng)基對(duì)RF/6A細(xì)胞進(jìn)行培養(yǎng),與無條件培養(yǎng)基的RF/6A培養(yǎng)組相比,能夠明顯抑制RF/6A細(xì)胞的遷移和管形成,說明DMBT可能通過作用于ARPE-19細(xì)胞影響RF/6A細(xì)胞的遷移和管形成。532 nm激光損傷C57BL/6小鼠眼底脈絡(luò)膜,構(gòu)建小鼠脈絡(luò)膜血管損傷模型。脈絡(luò)膜lectin488熒光染色結(jié)果顯示,與陰性對(duì)照組相比,玻璃體內(nèi)注射0.2和1 mmol/LDMBT 1μL,可以顯著抑制脈絡(luò)膜血管的新生且無明顯毒副作用。ELISA實(shí)驗(yàn)檢測(cè)ARPE-19細(xì)胞分泌到細(xì)胞外的血管內(nèi)皮生長因子VEGF的水平,結(jié)果發(fā)現(xiàn)8和32 μmol/L的DMBT能抑制由于缺氧導(dǎo)致的ARPE細(xì)胞分泌的VEGF的量的升高且具有劑量依賴性,活性氧抑制劑N-乙酰半胱氨酸(N-acetyl-cysteine,NAC)也可以在一定程度上影響VEGF的分泌。Western blotting實(shí)驗(yàn)顯示,DMBT給藥組降低了模型鼠視網(wǎng)膜中VEGF蛋白的表達(dá)。表明DMBT在體內(nèi)外都可以抑制視網(wǎng)膜色素上皮細(xì)胞對(duì)VEGF的分泌。通過活性氧試劑盒檢測(cè)發(fā)現(xiàn)8和32μmol/L DMBT能抑制由于缺氧所導(dǎo)致的活性氧的升高。提示DMBT可能通過活性氧相關(guān)的通路降低VEGF的分泌。Western blotting實(shí)驗(yàn)結(jié)果顯示,32 μmol/L DMBT能抑制ARPE-19細(xì)胞在缺氧微環(huán)境中細(xì)胞內(nèi)上調(diào)的p-ERK1/2,核內(nèi)Nrf2,HO-1和HIF-1α蛋白的表達(dá),這進(jìn)一步表明DMBT作用機(jī)制與抑制細(xì)胞活性氧相關(guān)。另外ELISA檢測(cè)發(fā)現(xiàn)500 μmol/L的NAC對(duì)ARPE-19細(xì)胞分泌VEGF的作用弱于32 μmol/L的DMBT,說明DMBT對(duì)VEGF表達(dá)的影響不只與活性氧有關(guān),接下來我們利用Western blotting檢測(cè)了 DMBT對(duì)p-Akt和p-NF-KB的表達(dá)的影響,發(fā)現(xiàn)DMBT能抑制p-Akt和p-NF-κB的表達(dá)。結(jié)論:海藻糖衍生物DMBT在缺氧狀態(tài)下,能夠通過對(duì)視網(wǎng)膜色素上皮細(xì)胞的作用,間接抑制脈絡(luò)膜血管新生,且細(xì)胞毒性較低。通過影響ERK1/2/Nrf2和Akt/NF-KB活性氧依賴通路影響下游HIF-1α的表達(dá),抑制VEGF的分泌。從而起到抑制脈絡(luò)膜血管新生的作用。由此DMBT以其高效低毒的特點(diǎn)有潛力成為抗脈絡(luò)膜血管新生類疾病的候選化合物之一。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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