【摘要】：第一部分缺氧導致視網膜新生血管性疾病體外模型的建立及評估 目的：建立并評估一種新型的視網膜新生血管性疾病體外模型。 方法：原代培養(yǎng)的視網膜微血管內皮細胞取材于C57/BL6J小鼠視網膜,異硫氰酸熒光素標記的CD31進行細胞鑒定。3-6代小鼠視網膜微血管內皮細胞培養(yǎng)于厭氧袋中構建缺氧模型。缺氧時間設為0.5、1、2、3、12、24、48及72小時。血氣分析各時間點細胞培養(yǎng)液的pO2, pCO2及pH,對該模型進行評估。 結果：異硫氰酸熒光素標記的CD31鑒定視網膜微血管內皮細胞陽性率為90%。當視網膜微血管內皮細胞在厭氧袋中培養(yǎng)2小時,血氣分析結果顯示p02為5.60Kpa, pCO2為6.04Kpa, pH為7.07；當視網膜微血管內皮細胞在厭氧袋中培養(yǎng)大于2小時,血氣分析結果顯示p02為4.5Kpa, pCO2和pH無明顯變化。 結論：厭氧袋培養(yǎng)視網膜微血管內皮細胞可以模擬缺氧導致視網膜新生血管性疾病的體外模型,該方法簡單有效。 第二部分VEGF在視網膜微血管內皮細胞中自分泌作用的研究 目的：探討在缺氧導致的視網膜新生血管性疾病中,視網膜微血管內皮細胞是否通過自分泌血管內皮生長因子信號實現增殖。 方法：實驗分為缺氧組和常氧組。視網膜微血管內皮細胞培養(yǎng)于厭氧袋中為缺氧組,缺氧時間為12、24、48、72小時。設立相應時間點常氧環(huán)境下培養(yǎng)的細胞作為常氧組。CCK-8法檢測細胞增殖。流式細胞術檢測細胞調亡率。各組細胞不同時間點VEGF-A, VEGFR-2, eNOs的mRNA及蛋白水平通過實時定量PCR及Western blot檢測。 結果：CCK-8結果顯示缺氧組各時間點的視網膜微血管內皮細胞增殖能力高于常氧組(p0.05)。在缺氧環(huán)境下,48h細胞的增殖能力達高峰。流式細胞術結果顯示缺氧組各時間點視網膜微血管內皮細胞的調亡率低于常氧組。實時定量RT-PCR結果顯示缺氧組的視網膜微血管內皮細胞的VEGF-A, VEGF-R2和eNOs的mRNA水平明顯高于常氧組(p0.01),缺氧組中VEGF-A, VEGF-R2和eNOs的]mRNA水平隨時間上升,48h達高峰,隨后下降。Western blot結果顯示VEGF-A, VEGF-R2和NOs蛋白水平的變化和mRNA的變化相一致。 結論：在缺氧環(huán)境下,視網膜微血管內皮細胞可通過自分泌途徑,上調血管內皮生長因子家族成員,完成自身增殖,生成視網膜新生血管。 第三部分15-脂氧合酶-1在缺氧導致的視網膜新生血管性疾病中的作用及機制研究 目的：探討15-脂氧合酶-1在缺氧導致的視網膜新生血管性疾病中的作用機制。 方法：實驗分為四組：常氧組,缺氧組,缺氧轉染15-LOX-1組,缺氧轉染GFP對照組。缺氧環(huán)境通過厭氧袋構建。觀察時間設為12、24、48、72h。轉染效率通過熒光顯微鏡觀察。CCK-8法檢測細胞增殖。各組細胞不同時間點15-LOX-1, VEGF-A, VEGFR-2, eNOs, PPAR-r mRNA及蛋白水平通過實時定量RT-PCR及Western blot檢測。 結果：CCK-8結果顯示缺氧組各時間點的視網膜微血管內皮細胞增殖能力高于常氧組。在缺氧條件下,15-脂氧合酶-1組細胞增殖率明顯被抑制。實時定量RT-PCR結果顯示缺氧組VEGF-A, VEGF-R2,eNOs的mRNA水平明顯高于常氧組(p0.01)；但缺氧組15-LOX-1, PPAR-r的mRNA水平卻明顯低于常氧組(p0.01)。缺氧轉染15-脂氧合酶-1組VEGF-A, VEGF-R2, eNOs的mRNA水平明顯低于缺氧組(p0.01)；但其15-LOX-1, PPAR-r的mRNA水平卻明顯高于缺氧組(p0.01)。缺氧轉染GFP對照組與缺氧組之間VEGF-A, VEGF-R2, eNOs,15-LOX-1和PPAR-r的mRNA水平無統計學差異(p0.05)。Western blot結果顯示蛋白水平的變化和(?)nRNA的變化相一致。 結論：15-脂氧合酶-1和PPAR-r在視網膜血管形成過程中起負性調節(jié)作用。視網膜微血管內皮細胞過表達15-脂氧合酶-1通過抑制缺氧導致的VEGF家族的上調,從而抑制視網膜新生血管的形成。同時PPAR-r作為15-脂氧合酶-1的配體,結合VEGFR2是15-脂氧合酶-1抑制缺氧導致的視網膜新生血管的另一機制。
[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.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R774.1

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相關期刊論文 前1條

1 許建華;劉哲麗;李若溪;孔偉;張薇;;曲安奈德對缺氧條件下恒河猴視網膜血管內皮細胞增殖的抑制作用(英文)[J];國際眼科雜志;2006年02期

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