高糖及其联合缺氧对HIF-1α表达的影响和shRNA抑制人RPE细胞HIF-1α表达的研究
|
摘要
目的观察早期糖尿病大鼠视网膜及人视网膜色素上皮(retinal pigment epithelial，RPE)细胞在高糖及高糖联合缺氧的培养条件下缺氧诱导因子1α(hypoxia-inducible factor 1α，HIF-1α)和血管内皮生长因子(vascular endothelial growth factor，VEGF)的表达，探讨高糖及高糖联合缺氧对HIF-1α及VEGF表达的影响；并利用小发卡环RNA(small hairpin loop RNA，shRNA)使缺氧培养条件下的人RPE细胞HIF-1α基因沉默，观察对VEGF及色素上皮衍生因子(pigmentepithelium-derivedfactor，PEDF)表达的影响，探讨使缺氧条件下RPE细胞HIF-1α基因沉默能否有效地抑制RPE细胞VEGF的表达和促进PEDF的表达。
方法1)使用链尿佐菌素(streptozotocin，STZ)腹腔注射制作大鼠早期糖尿病视网膜病变(diabetic retinopathy，DR)模型。于模型建立后1月处死大鼠，取眼球做石蜡切片及视网膜铺片。用免疫组化法检测HIF-1α及VEGF在视网膜的表达。用胰酶消化视网膜并行PAS视网膜血管染色，观察管视网膜血管形态变化。2)通过使用化学缺氧诱导剂CoCl_2模拟RPE细胞缺氧环境，研究RPE细胞分别在5.56mM葡萄糖+0μM CoCl_2(对照组)、5.56mM葡萄糖+150μM CoCl_2(缺氧组)、25mM葡萄糖+0μM CoCl_2(高糖组)以及25mM葡萄糖+150μM CoCl_2(联合组)的培养条件下，通过RT-PCR检测HIF-1α及VEGF mRNA的表达，并使用Western印迹分析检测HIF-1α及VEGF蛋白水平。3)通过使用化学缺氧诱导剂CoCl_2模拟RPE细胞缺氧环境，体外转录法合成针对HIF-1αmRNA序列的两个靶点的shRNA，对缺氧(150μMCoCl_2模拟)的培养条件下人RPE细胞的HIF-1α进行干扰，通过RT-PCR检测HIF-1α、VEGF及PEDF mRNA的表达，并使用Western印迹分析检测HIF-1α、VEGF及PEDF蛋白水平。
结果1)正常大鼠视网膜HIF-1α及VEGF染色为阴性表达，但在早期糖尿病大鼠视网膜HIF-1α及VEGF染色均为阳性表达，而视网膜血管形态变化不明显。2)与对照组相比，高糖组RPE细胞HIF-1αmRNA表达无显剧性差异，但高糖组可检测出微量的HIF-1α蛋白，对照组未能检测出HIF-1α蛋白；而VEGFmRNA表达和蛋白合成均增加。与缺氧组相比，高糖组联合缺氧组RPE细胞HIF-1αmRNA表达无显剧性差异，但HIF-1α蛋白水平有极显剧性差异；同时VEGF mRNA表达和蛋白合成也均明显增加。3)针对HIF-1αmRNA的两种shRNA均能有效地抑制RPE细胞在缺氧条件下HIF-1α的表达，同时也有效地抑制VEGF mRNA和蛋白表达，并促进PEDF蛋白表达，但对PEDF mRNA无影响。
结论1)早期糖尿病大鼠视网膜的HIF-1可能由高浓度血糖诱导，从而上调VEGF的表达，在糖尿病视网膜新生血管的发生过程中可能起着重要的作用。2)高糖对体外培养的人RPE细胞HIF-1α的影响主要是促进蛋白的合成，且对HIF-1α蛋白有一定的稳定的作用，在联合缺氧条件下高糖促进合成的HIF-1α蛋白得到更好的稳定，同时也显剧促进VEGF的表达增加。3)针对HIF-1αmRNA的shRNA能有效地使缺氧条件下RPE细胞HIF-1α基因沉默，进而有效地抑制缺氧对VEGF的上调作用，同时也促进PEDF表达，从而使得VEGF／PEDF的比值更低，更有利于抑制视网膜新生血管形成。结果也揭示HIF-1与缺氧条件下PEDF翻译后的下调机制有关，是促进视网膜新生血管形成最为关键的细胞因子之一。
Objective To determine whether the retina of diabetic rats at early stage express hypoxia-inducible factor lα (HIF-1α) and vascular endothelial growth factor (VEGF), to investigate the effects of high-concentration glucose and high-concentration high with hypoxia on the expression of HIF-lα and VEGF in human retinal pigment epithelial (RPE) cell, thus to illustrate the roles of high-concentration glucose and hypoxia played in retinal neovascularization. To explore the effect of small hairpin loop RNA (shRNA) keeping HIF-1α silencing on the expression of VEGF and pigment epithelium derived factor (PEDF), thus to illustrate whether shRNA inhibitting the expression of HIF-1α could reduce retinal neovascularization by downregulation of VEGF and upregulation of PEDF in human RPE cells.
Methods 1) Thirty male Wistar rates were randomly divided into the diabetic group (n=20) and normal control group (n=10). Diabetic rats were induced by injection of streptozotocin (STZ) intraperitondally. One month after the model being builded, the eyeballs were removed for making the retinal vascular network, PAS stained, HE stained and immunohistochemically stained. 2) 150μmol CoCl_2 is used as hypoxic environment of Human RPE cells, RPE cells are cultured under 5.56 mM glucose with 0 μMCoCl_2 (control group)、 5.56mM glucose with 150 μM CoCl_2 (hypoxic group) 、 25 mM glucose with 0 μM CoCl_2 (high glucose group) and 25 mM glucose with 150 μM CoCl_2(combination group). RT-PCR was used to examine the expression of HIF-1α and VEGF mRNAs, Western blot analysis was used to measure the levels of HIF-1α and VEGF proteins. 3) Utilizing 150 μM CoCl_2 to model the hypoxia environment of RPE cells. Two target sites of HIF-1α mRNA were chosen by certain design principle. In vitro, two kind of shRNA were designed and synthesised by the two target sites, and transfected into human RPE cells in vitro. Then these cells were cultivated under hypoxia condition (150 μM CoCl_2). The mRNA expressions of HIF-1α、 VEGF and PEDF were tested by RT-PCR. And the protein levels of HIF-1α、 VEGF and PEDF were tested by western blot.
Results 1) The reaction for HIF-1α and VEGF was negative in normal retina. However, HIF-1α and VEGF express in the retina of diabetic rats at early stage. 2) As compared with under 5.56 mM glucose condition, the expression of the HIF-1α mRNA of RPE cells under 25 mM glucose condition is not different, the HIF-1α protein is able to be detected out, but the quantity of the HIF-1α protein is small, however the HIF-1α protein under 5.56 mM glucose condition is not able to be detected out; the mRNA expression and the protein synthesis of VEGF are up-regulated. As compared with hypoxic group, the expression of the HIF-1α mRNA of RPE cells under 25 mM glucose with hypoxic condition is not different, but the protein synthesis of HIF-1α is more obviously up-regulated; meanwhile, the mRNA expression and the protein synthesis of VEGF are more obviously up-regulated too. 3) After the two kind of HIF-1α-specific shRNA were respectively transfected into RPE cells, the expression of HIF-1α mRNA and the levels of HIF-1α protein both significantly decreased in RPE cells under hypoxia condition. Moreover, the expression of VEGF mRNA and the levels of protein significantly decreased too. However, the levels of PEDF protein was significantly increased, but the expression of PEDF mRNA is no significant change.
Conclusion 1) The results indicate that diabetes (or high-concentration boold glucose) increases the expression of HIF-1α in retina may play a part in retinal neovascularization. 2) high-concentration glucose mainly influence the protein synthesis of HIF-1α of RPE cell, and under high concentration glucose, HIF-1α protein is able to be accumulated; however, with under hypoxia condition, the HIF-1α protein induced by high concentration glucose is more stable, and meanwhile, the expression of VEGF is obvious increases. These suggest high concentration glucose may play a large part in retinal neovascularization, especially at ischemia stage of diabetic retinopathy. 3) Under hypoxia condition, HIF-1α-specific shRNA can effectively keep the HIF-1α gene silencing , downregulate VEGF and upregulate PEDF. These results reveal HIF-1 is concerned with posttranslational mechanism for downregulating PEDF under hypoxia condition and provide an explanation for hypoxia-provoked increases in VEGF/PEDF ratios. These results suggest HIF-1 is the most key cytokine to retinal neovascularization too.
方法1)使用链尿佐菌素(streptozotocin，STZ)腹腔注射制作大鼠早期糖尿病视网膜病变(diabetic retinopathy，DR)模型。于模型建立后1月处死大鼠，取眼球做石蜡切片及视网膜铺片。用免疫组化法检测HIF-1α及VEGF在视网膜的表达。用胰酶消化视网膜并行PAS视网膜血管染色，观察管视网膜血管形态变化。2)通过使用化学缺氧诱导剂CoCl_2模拟RPE细胞缺氧环境，研究RPE细胞分别在5.56mM葡萄糖+0μM CoCl_2(对照组)、5.56mM葡萄糖+150μM CoCl_2(缺氧组)、25mM葡萄糖+0μM CoCl_2(高糖组)以及25mM葡萄糖+150μM CoCl_2(联合组)的培养条件下，通过RT-PCR检测HIF-1α及VEGF mRNA的表达，并使用Western印迹分析检测HIF-1α及VEGF蛋白水平。3)通过使用化学缺氧诱导剂CoCl_2模拟RPE细胞缺氧环境，体外转录法合成针对HIF-1αmRNA序列的两个靶点的shRNA，对缺氧(150μMCoCl_2模拟)的培养条件下人RPE细胞的HIF-1α进行干扰，通过RT-PCR检测HIF-1α、VEGF及PEDF mRNA的表达，并使用Western印迹分析检测HIF-1α、VEGF及PEDF蛋白水平。
结果1)正常大鼠视网膜HIF-1α及VEGF染色为阴性表达，但在早期糖尿病大鼠视网膜HIF-1α及VEGF染色均为阳性表达，而视网膜血管形态变化不明显。2)与对照组相比，高糖组RPE细胞HIF-1αmRNA表达无显剧性差异，但高糖组可检测出微量的HIF-1α蛋白，对照组未能检测出HIF-1α蛋白；而VEGFmRNA表达和蛋白合成均增加。与缺氧组相比，高糖组联合缺氧组RPE细胞HIF-1αmRNA表达无显剧性差异，但HIF-1α蛋白水平有极显剧性差异；同时VEGF mRNA表达和蛋白合成也均明显增加。3)针对HIF-1αmRNA的两种shRNA均能有效地抑制RPE细胞在缺氧条件下HIF-1α的表达，同时也有效地抑制VEGF mRNA和蛋白表达，并促进PEDF蛋白表达，但对PEDF mRNA无影响。
结论1)早期糖尿病大鼠视网膜的HIF-1可能由高浓度血糖诱导，从而上调VEGF的表达，在糖尿病视网膜新生血管的发生过程中可能起着重要的作用。2)高糖对体外培养的人RPE细胞HIF-1α的影响主要是促进蛋白的合成，且对HIF-1α蛋白有一定的稳定的作用，在联合缺氧条件下高糖促进合成的HIF-1α蛋白得到更好的稳定，同时也显剧促进VEGF的表达增加。3)针对HIF-1αmRNA的shRNA能有效地使缺氧条件下RPE细胞HIF-1α基因沉默，进而有效地抑制缺氧对VEGF的上调作用，同时也促进PEDF表达，从而使得VEGF／PEDF的比值更低，更有利于抑制视网膜新生血管形成。结果也揭示HIF-1与缺氧条件下PEDF翻译后的下调机制有关，是促进视网膜新生血管形成最为关键的细胞因子之一。
Objective To determine whether the retina of diabetic rats at early stage express hypoxia-inducible factor lα (HIF-1α) and vascular endothelial growth factor (VEGF), to investigate the effects of high-concentration glucose and high-concentration high with hypoxia on the expression of HIF-lα and VEGF in human retinal pigment epithelial (RPE) cell, thus to illustrate the roles of high-concentration glucose and hypoxia played in retinal neovascularization. To explore the effect of small hairpin loop RNA (shRNA) keeping HIF-1α silencing on the expression of VEGF and pigment epithelium derived factor (PEDF), thus to illustrate whether shRNA inhibitting the expression of HIF-1α could reduce retinal neovascularization by downregulation of VEGF and upregulation of PEDF in human RPE cells.
Methods 1) Thirty male Wistar rates were randomly divided into the diabetic group (n=20) and normal control group (n=10). Diabetic rats were induced by injection of streptozotocin (STZ) intraperitondally. One month after the model being builded, the eyeballs were removed for making the retinal vascular network, PAS stained, HE stained and immunohistochemically stained. 2) 150μmol CoCl_2 is used as hypoxic environment of Human RPE cells, RPE cells are cultured under 5.56 mM glucose with 0 μMCoCl_2 (control group)、 5.56mM glucose with 150 μM CoCl_2 (hypoxic group) 、 25 mM glucose with 0 μM CoCl_2 (high glucose group) and 25 mM glucose with 150 μM CoCl_2(combination group). RT-PCR was used to examine the expression of HIF-1α and VEGF mRNAs, Western blot analysis was used to measure the levels of HIF-1α and VEGF proteins. 3) Utilizing 150 μM CoCl_2 to model the hypoxia environment of RPE cells. Two target sites of HIF-1α mRNA were chosen by certain design principle. In vitro, two kind of shRNA were designed and synthesised by the two target sites, and transfected into human RPE cells in vitro. Then these cells were cultivated under hypoxia condition (150 μM CoCl_2). The mRNA expressions of HIF-1α、 VEGF and PEDF were tested by RT-PCR. And the protein levels of HIF-1α、 VEGF and PEDF were tested by western blot.
Results 1) The reaction for HIF-1α and VEGF was negative in normal retina. However, HIF-1α and VEGF express in the retina of diabetic rats at early stage. 2) As compared with under 5.56 mM glucose condition, the expression of the HIF-1α mRNA of RPE cells under 25 mM glucose condition is not different, the HIF-1α protein is able to be detected out, but the quantity of the HIF-1α protein is small, however the HIF-1α protein under 5.56 mM glucose condition is not able to be detected out; the mRNA expression and the protein synthesis of VEGF are up-regulated. As compared with hypoxic group, the expression of the HIF-1α mRNA of RPE cells under 25 mM glucose with hypoxic condition is not different, but the protein synthesis of HIF-1α is more obviously up-regulated; meanwhile, the mRNA expression and the protein synthesis of VEGF are more obviously up-regulated too. 3) After the two kind of HIF-1α-specific shRNA were respectively transfected into RPE cells, the expression of HIF-1α mRNA and the levels of HIF-1α protein both significantly decreased in RPE cells under hypoxia condition. Moreover, the expression of VEGF mRNA and the levels of protein significantly decreased too. However, the levels of PEDF protein was significantly increased, but the expression of PEDF mRNA is no significant change.
Conclusion 1) The results indicate that diabetes (or high-concentration boold glucose) increases the expression of HIF-1α in retina may play a part in retinal neovascularization. 2) high-concentration glucose mainly influence the protein synthesis of HIF-1α of RPE cell, and under high concentration glucose, HIF-1α protein is able to be accumulated; however, with under hypoxia condition, the HIF-1α protein induced by high concentration glucose is more stable, and meanwhile, the expression of VEGF is obvious increases. These suggest high concentration glucose may play a large part in retinal neovascularization, especially at ischemia stage of diabetic retinopathy. 3) Under hypoxia condition, HIF-1α-specific shRNA can effectively keep the HIF-1α gene silencing , downregulate VEGF and upregulate PEDF. These results reveal HIF-1 is concerned with posttranslational mechanism for downregulating PEDF under hypoxia condition and provide an explanation for hypoxia-provoked increases in VEGF/PEDF ratios. These results suggest HIF-1 is the most key cytokine to retinal neovascularization too.
引文
1．曹晖，胡宏慧，许迅，等．视网膜新生血管动物模型的制备．眼科新进展，2003，23(5)：335-337．
2．张鹏，王雨生，张星，等．缺氧对人视网膜色素上皮细胞增生和VEGF表达的影响．眼科新进展，2004，24(1)：6-8．
3. Pham I, Uchida T, Planes C, et al. Hypoxia upregulates VEGF expression in alveolar epithelial cells in vitro and invivo. Am J Physiol Lung Cell Mol Physiol, 2002, 283(5): 1133-1142.
4. Semenza GL, Roth PH, Fang HM. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem, 1994, 26(38): 23757-23763.
5. Huang LE, Aramy I, Livingston DM, et al. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensit stabilization of its alpha subunit. J Biol Chem, 1996, 271(50): 32253-32259.
6. Zhong H, Aganni F, Baccala AA,et al.Increased expression hypoxia inducible factor-alpha in rat and human prostate cancer. Cancer Res, 1998, 58(23): 5280-5284.
7. Fukuda R, Hirota K, Fan F, et al. Insulin-like growth factor 1 iduces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem, 2002, 277(41): 38205-38211.
8. Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Regulation of vascular endothelial growth factor expression by advanced glycation end products. J Biol Chem, 2001, 276(47): 43836-43841.
9. Bhisitkul RB, Ruan DT, Sherwood J, et al. HIF-1 -mediated induction of VEGF in RPE cells: upregulation by constitutively active HIF-1 and inhibition by RNA interference, Invest Ophthalmol Vis Sci, 2004, 45: E-Abstract 452.
10. An WG, Kanekal M, Simon MC, et al. Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha. Nature, 1998, 392(6674):405-408.
11. Halterman MW, Miller CC, Federoff HJ, et al. Hypoxia-inducible factor-1alpha mediates hypoxia-induced delayed neuronal death that involves p53. J Neurosci, 1999, 19(16):6818-6824.
12. Stellmach V, Crawford SE, Zhou W, et al. Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor. PNAS, 2001, 98(5): 2593-2597.
13. Cao W, Tombran-tink J, Elias R, et al. In vivo protection of photoreceptors from light damage by pigment epithelium-derived factor. Invest Ophthalmol Vis Sci. 2001,42(7): 1646-1652.
14. Cao W, Tombran-tink J, Chen W, et al. Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death. J Neurosci Res. 1999, 57(6):789-800.
1 Adamis AP, Shima DT, Tolention MJ, et al. Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate. Ophthalmology, 1996, 114(1): 66-71.
2 Treins C, Giorgetti-Peraldi S, Murdaca J. Regulation of vascular endothelial growth factor expression by advanced glycation end products. J Biol Chem, 2001, 276: 43836-43841.
3 Bhisitkul RB, Ruan DT, Sherwood J, et al. HIF-1-mediated induction of VEGF in RPE cells: upregulation by constitutively active HIF-1 and inhibition by RNA interference Invest Ophthalmol Vis Sci, 2004, 45: E-Abstract 452.
4 Fukuda R, Hirota K, FanF, et al. Insulin-like growth factor linduces hypoxia-inducible factor 1-mediated vascular endothelia growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem, 2002, 277(41): 38205-38211.
5 Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Insulin stimulates hypoxia-inducible factor 1 through a phosphatidylinositol 3-kinase/target of rapamycin-dependent signaling pathway. J Biol Chem. 2002, 277(31): 27975-27981.
6 Zundel W, Schindler C, Haas-Kogan D, et al. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev, 2000, 14(4): 391-396.
7 Fukuda R, Kelly B, Semenza GL. Vascular Endothelial Growth Factor Gene Expression in Colon Cancer Cells Exposed to Prostaglandin E_2 Is Mediated by Hypoxia-inducible Factor 1. Cancer Research, 2003, 63: 2330-2334.
8 John J, Stephen C. A non-hypoxia ROS-sensitive pathway mediated TNF-dependent regulation of HIF-1α. FEBB letters, 2001, 505: 269-274.
9 Sandau KB, Fandrey J, and Brune B. Accumulation of HIF-1α under the influence of nitric oxide. Blood, 2001, 97(4): 1009-1015.
1. Salnikow K, Su WC, Blagosklonny MV, et al. Carcinogenic Metals Induce Hypoxia-inducible Factor-stimulated Transcription by Reactive Oxygen Species-independent Mechanism. Cancer Research, 2000, 60: 3375-3378.
2．王雨生，严密．可见光对原代培养人视网膜色素上皮细胞的光化学损伤．中华眼底病杂志，1996，12：174-176．
3. Treins C, Giorgetti-Peraldi S, Murdaca J. Regulation of vascular endothelial growth factor expression by advanced glycation end products. J Biol Chem, 2001, 276: 43836-43841.
4. Bhisitkul RB, Ruan DT, Sherwood J, et al. HIF-1-mediated induction of VEGF in RPE cells: upregulation by constitutively active HIF-1 and inhibition by RNA interference, Invest Ophthalmol Vis Sci, 2004, 45: E-Abstract 452.
5. Fukuda R, Hirota K, Fan F, et al. Insulin-like growth factor linduces hypoxia-inducible factor 1-mediated vascular endothelia growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem, 2002, 277(41): 38205-38211.
6. Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Insulin stimulates hypoxia-inducible factor 1 through a phosphatidylinositol 3-kinase/target of rapamycin-dependent signaling pathway. J Biol Chem. 2002, 277(31): 27975-27981.
7. Zundel W, Schindler C, Haas-Kogan D, et al. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev, 2000, 14(4): 391-396.
8. Fukuda R, Kelly B, Semenza GL. Vascular Endothelial Growth Factor Gene Expression in Colon Cancer Cells Exposed to Prostaglandin E_2 Is Mediated by Hypoxia-inducible Factor 1. Cancer Research, 2003, 63: 2330-2334.
9. John J, Stephen C. A non-hypoxia ROS-sensitive pathway mediated TNF-dependent regulation of HIF-1α. FEBB letters, 2001, 505: 269-274.
10. Sandau KB, Fandrey J, Brtine B. Accumulation of HIF-1α under the influence of nitric oxide. Blood, 2001, 97(4): 1009-1015.
11．姚毅，关明，赵秀琴，等．缺氧和高浓度葡萄糖对体外培养人视网膜色素上皮衍生因子表达的影响．中华医学杂志，2003，83(22)：1989-1992．
1 Gao G, Li Y, Zhang D, et al. Unbalanced expression of VEGF and PEDF inischemia-induced retinal neovascularization. FEBS Lett, 2001, 489: 270-276.
2 Dawson DW, Volpert OV, Gillis P, et al. Pigment Epithelium-Derived Factor: A Potent Inhibitor of Angiogenesis. Science, 1999, 285 (5425): 245-248.
3 Notari L, Miller A, Martinez A, et al. Pigment epithelium-derived factor is a substrate for matrix metalloproteinase type 2 and type 9: implications for downregulation in hypoxia. Invest Ophthalmol Vis Sci, 2005, 46(8): 2736-2747.
4 Brummelkamp TR, Bernards R, Agami R. A System for Stable Expression of Short Interfering RNAs in Mammalian Cells. Science, 2002, 296: 550-553
5 王雨生，严密．可见光对原代培养人视网膜色素上皮细胞的光化学损伤．中华眼底病杂志，1996，12：174-176．
6 Balaji K, Shannon B-D, Brian K, et al. Regulation of Colon Carcinoma Cell Invasion by Hypoxia-Inducible Factor 1. Cancer Res, 2003, 63: 1138-1143
7 Salnikow K, Su WC, Blagosklonny MV, et al. Carcinogenic Metals Induce Hypoxia-inducible Factor-stimulated Transcription by Reactive Oxygen Species-independent Mechanism. Cancer Res, 2000, 60:3375-3378
8 Fire A, Xu S, Montgomery MK, et al .Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans. Nature, 1998, 391: 806-811
9 Hannon GJ. RNA interference. Nature, 2002,418(6894):244-251.
10 Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnology, 2002, 19(5):500-505.
11 Stellmach V, Crawford SE, Zhou W, et al. Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor. PNAS, 2001, 98(5): 2593-2597.
12 Cao W, Tombran-tink J, Elias R, et al. In vivo protection of photoreceptors from light damage by pigment epithelium-derived factor. Invest Ophthalmol Vis Sci. 2001, 42(7): 1646-1652.
13 Cao W, Tombran-tink J, Chen W, et al. Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death. J Neurosci Res. 1999, 57(6):789-800.
14 Halterman MW, Miller CC, Federoff HJ, et al. Hypoxia-inducible factor-1 alpha mediates hypoxia-induced delayed neuronal death that involves p53. J Neurosci, 1999, 19(16):6818-6824.
1. Semenza GL, Roth PH, Fang HM. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem, 1994, 26(38): 23757-23763.
2. WangGL, Semenza GL. Purification and characterization of hypoxia-inducible factor 1. J Biol Chem, 1995, 270(3): 1230-1237.
3. Wang GL, Jiang B, Rue E, et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O_2 tension. Proc Natl Acad Sci USA, 1995, 92(12): 5510-5514.
4. Jiang BH, Rue E, Wang GL, et al. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J Biol Chem, 1996, 271(30): 17771-17778.
5. Goldberg MA, Dunning SP, Bunn HF Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science, 1988, 242 (4884): 1412-1415.
6. Wang GL, Semenza GL. Desferrioxamine induce erythropoiet in gene expression and hypoxia-inducible factorl DNA-binding activity: implications for models of hypoxia signal transduction.Blood, 1993, 82(12): 3610-3615
7. Bunn HF, Poyton RO. Oxygen sensing and molecular adaptation to hypoxia. Physiol Rev, 1996, 76(3): 839-885.
8. Srinivas V, Zhu X, Susana S, et al. Hypoxia-inducible factor 1alpha (HIF-1αlpha) is a non-heme iron protein. Implications for oxygen sensing. J Biol Chem, 1998, 272(29): 18019-18022.
9. Salceda S, Caro J. Hypoxia-inducible factor 1alpha (HIF-1αlpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem, 1997, 272(36): 22642-22647.
10. Wiener C, Booth G, Semenza G, In vivo expression of mRNAs encoding hypoxia-inducible factorl. Biochem Biophys Res Com, 1996, 225(2): 485-488
11. Wenger RH, Kvietikova I, Roifs A, et al. Hypoxia-inducible fact1α is regulated at the post-mRNA level. Kidney Int, 1997, 51(2): 560-563.
12. Huang LE, Aramy I, Livingston DM, et al. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensit stabilization of its alpha subunit. J Biol Chem, 1996, 271(50): 32253-32259.
13. Lando D, Peet DJ, Whelan DA, et al. Asparagine hydroxylation of the HIF transactivation domain: A hypoxic switch. Science, 2002,295 (5556): 858-861.
14. Pages G, Milanini J, Richard DE, et al. Signaling angiogenesis Via p42/p44 MAP kinase cascade. Ann N Y Acad Sci, 2000, 902: 187-200.
15. Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Regulation of vascular endothelial growth factor expression by advanced glycation end products. J Biol Chem, 2001, 276(47): 43836-43841.
16. Hur E, Chang KY, Lee E, et al. Mitogen-activated protein kinase kinase inhibitor PD98059 blocks the trans-activation but not the stabilization or DNA binding ability of hypoxia-inducible factor-1alpha. Mol Pharmacol, 2001, 59(5): 1216-1224.
17. Volmat V, Camps M, Arkinstall S, et al. The nucleus,a site for signal termination by sequestration and inactivation of p42/p4 MAP kinases.J Cell Sci, 2001, 114(Pt19): 3433-3443
18. Ravi R., Mookerjee B, Bhujwalla ZM, et al. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor-1α. Genes Dev, 2000, 14(1): 34-44.
19. Hammond EM, Denko NC, Dorie MJ. Hypoxia links ATR and p53 through replication arrest. Mol Cell Biol, 2002, 22(6): 1834-1843.
20. Hansson LO, Friedler A, Freund S, et al. Two sequence motifs from HIF-1α bind to the DNA-binding site of p53. Proc Natl Acad Sci, 2002, 99(16):10305-10309.
21. Chen D, Li M, Luo J, et al. Direct interaction between HIF-1 alpha and Mdm2 modulate p53 function. J Biol Chem, 2003, 278(16):13595-13598.
22. Ravi R, Mookerjee B, Bhujwalla ZM, et al. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-induciblefactor la. Genes Dev, 2000, 14(1): 34-44.
23. Bae MK, Ann MY, Ieong JW, et al. Jab1 interacts directly with HIF-1 alpha and regulates its stability. J Biol Chem, 2002, 277(1):9-12.
24. Zhong H, Aganni F, Baccala AA,et al.Increased expression hypoxia inducible factor-alpha in rat and human prostate cancer. Cancer Res, 1998, 58(23): 5280-5284.
25. Fukuda R, Hirota K, Fan F, et al. Insulin-like growth factor 1 iduces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem, 2002, 277(41): 38205-38211.
26. Zundel W, Schindler C, Haas-Kogan D, et al. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev, 2000, 14(4): 391-396.
27. Jiang B H, Jiang G, Zheng J Z, et al. Phosphatidylinositol 3-kinase signaling control slevels of hypoxia-inducible factor 1. Cell Growth Differ, 2001, 12(7): 363-369.
28. Semenza GL, Jiang BH, Leung SW, et al. Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor, 1, J Biol Chem, 1996, 271(51): 32529-32537.
29. Forsythe JA, Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol cell Biol, 1996, 16(9): 4604-4613.
30．曹晖，胡宏慧，许迅，等．视网膜新生血管动物模型的制备．眼科新进展，2003，23(5)：335-337．
31．张鹏，王雨生，张星，等．缺氧对人视网膜色素上皮细胞增生和VEGF表达的影响．眼科新进展，2004，24(1)：6-8．
32. Pham I, Uchida T, Planes C, et al. Hypoxia upregulates VEGF expression in alveolar epithelial cells in vitro and invivo. Am J Physiol Lung Cell Mol Physiol, 2002, 283(5): 1133-1142.
33. Adamis AP, Shima DT, Tolentino, et al. Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate. Arch Ophthalmol. 1996, 114(1): 66-71.
34. Dawson DW, Volpert OV, Gillis P, et al. Pigment Epithelium-Derived Factor: A Potent Inhibitor of Angiogenesis. Science, 1999, 285(5425): 245-248.
35. Boyd SR, Tan D, Bunce C, Gittos A, Neale MH, Hungerford JL, et al. Vascular endothelial growth factor is elevated in ocular fluids of eyes harbouring uveal melanoma: identification of apotential therapeutic window. Br J Ophthalmol, 2002, 86(4): 448-452.
36. Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Regulation of vascular endothelial growth factor expression by advanced glycation end products. J Biol Chem, 2001, 276(47): 43836-43841.
37. Bhisitkul RB, Ruan DT, Sherwood J, et al. HIF-1-mediated induction of VEGF in RPE cells: upregulation by constitutively active HIF-1 and inhibition by RNA interference, Invest Ophthalmol Vis Sci, 2004,45: E-Abstract 452.
38. Semenza GL, Agani F, Booth G, et al. Structural and functional analysis of hypoxia-inducible factor 1. Structural and functional analysis of hypoxia-inducible factor 1. Kidney Int, 1997, 51(2): 553-555.
39. JinK L, Mao XO, Nagayama T, Goldsmith PC. Induction of vascular endothelial growth factor receptors and phosphatidylinositol 3' -kinase/Akt signaling by global cerebral ischemia in the rat. Neuro science, 2000, 100(4): 713-717.
40. Vincent KA, Shyu KG, Luo Y, et al. Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1 alpha/VP 16 hybrid transcription factor. Circulation, 2000,102(18): 2255-2261.
41. Damert A, Ikeda E, Risau W. Activator-protein-1 binding potentiates the hypoxia-induciblefactor-1-mediated hypoxia-induced transcriptional activation of vascular-endothelial growth factor expression in C6 glioma cells. Biochem J, 1997, 327(pt2): 419-423.
42. Kimura H, Weisz A, Kurashima Y, et al. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide. Blood, 2000,95(1): 189-197.
43. Von Marschall Z, Cramer T, Hocker M, et al. Dual mechanism of vascular endothelial growth factor upregulation by hypoxia in human hepatocellular carcinoma. Gut, 2001, 48(1): 87-96
44. Michel G, Minet E, Mottet D, et al. Site-directed mutagenesis studies of the hypoxia-inducible factor-1alpha DNA-binding domain. Biochim Biophys Acta, 2002, 1578(123): 73-83.
45. Kim HH, Lee SE, Chung WJ, et al. Stabilization of hypoxia-inducible factor-1 alpha is involved in the hypoxic stimuli-induced expression of vascular endothelial growth factor in osteoblastic cells.Cytokine, 2002, 17(1): 14-27.
46. Sun X, Kanwar JR, Leung E, et al. Regression of solid tumors by engineered overexpression of von Hippel-Lindau tumor suppressor protein and antisense hypoxia-inducible foctor-lalpha.GeneTher, 2003,10(25): 2081-2089.
47. Gogat K, Le Get L, Van Den Berghe L, et al. VEGF and KDR gene expression during human embryonic and fetal eye development. Invest Ophthalmol Vis Sci, 2004, 45(1): 7-14.
48. Ito N, Claesson-Welsh L. Dual effects ofh eparin on VEGF binding to VEGF receptor-1 and transduction of biological responses. Angiogenesis, 1999, 3(2): 159-166.
49. Takekoshi K, Isobe K, Yashiro T, et al. Expression of vascular endothelial Growth factor(VEGF) and its cognate receptors in human pheochromocytomas. Life Sci, 2004,74(7):863-871.
50. Gerber HP, Condorelli F, Park J, et al. Differential transcriptional regulation of the two vascular endothelial growth factor receptor genes. Flt-1, but not Flk-1/KDR, is upregulated by hypoxia. J Biol Chem, 1997, 272(38): 23659-23667.
51. An WG, Kanekal M, Simon MC, et al. Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha. Nature, 1998, 392(6674): 405-408.
52. Halterman MW, Miller CC, Federoff HJ, et al. Hypoxia-inducible factor-1alpha mediates hypoxia-induced delayed neuronal death that involves p53. J Neurosci, 1999,19(16):6818-6824.
53. Stellmach V, Crawford SE, Zhou W, et al. Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor. PNAS, 2001, 98(5): 2593 - 2597.
54. Cayouette M, Smith SB, Becerra SP, et al. Pigment epithelium-derived factor delays the death of photoreceptors in mouse models of inherited retinal degenerations. Neurobiol Dis. 1999, 6(6): 523-532.
55. Cao W, Tombran-tink J, Elias R, et al. In vivo protection of photoreceptors from light damage by pigment epithelium-derived factor. Invest Ophthalmol Vis Sci. 2001,42(7): 1646-1652.
56. Cao W, Tombran-tink J, Chen W, et al. Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death. J Neurosci Res, 1999, 57(6): 789-800.
57. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans. Nature, 1998, 391(6669): 806-811.
58. Hannon GJ. RNA interference. Nature, 2002, 418(6894): 244-251.
59. Lee NS, Dohjima T, Bauer G, et al. Expression of small-interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnology, 2002, 19(5): 500-505.
2．张鹏，王雨生，张星，等．缺氧对人视网膜色素上皮细胞增生和VEGF表达的影响．眼科新进展，2004，24(1)：6-8．
3. Pham I, Uchida T, Planes C, et al. Hypoxia upregulates VEGF expression in alveolar epithelial cells in vitro and invivo. Am J Physiol Lung Cell Mol Physiol, 2002, 283(5): 1133-1142.
4. Semenza GL, Roth PH, Fang HM. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem, 1994, 26(38): 23757-23763.
5. Huang LE, Aramy I, Livingston DM, et al. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensit stabilization of its alpha subunit. J Biol Chem, 1996, 271(50): 32253-32259.
6. Zhong H, Aganni F, Baccala AA,et al.Increased expression hypoxia inducible factor-alpha in rat and human prostate cancer. Cancer Res, 1998, 58(23): 5280-5284.
7. Fukuda R, Hirota K, Fan F, et al. Insulin-like growth factor 1 iduces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem, 2002, 277(41): 38205-38211.
8. Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Regulation of vascular endothelial growth factor expression by advanced glycation end products. J Biol Chem, 2001, 276(47): 43836-43841.
9. Bhisitkul RB, Ruan DT, Sherwood J, et al. HIF-1 -mediated induction of VEGF in RPE cells: upregulation by constitutively active HIF-1 and inhibition by RNA interference, Invest Ophthalmol Vis Sci, 2004, 45: E-Abstract 452.
10. An WG, Kanekal M, Simon MC, et al. Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha. Nature, 1998, 392(6674):405-408.
11. Halterman MW, Miller CC, Federoff HJ, et al. Hypoxia-inducible factor-1alpha mediates hypoxia-induced delayed neuronal death that involves p53. J Neurosci, 1999, 19(16):6818-6824.
12. Stellmach V, Crawford SE, Zhou W, et al. Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor. PNAS, 2001, 98(5): 2593-2597.
13. Cao W, Tombran-tink J, Elias R, et al. In vivo protection of photoreceptors from light damage by pigment epithelium-derived factor. Invest Ophthalmol Vis Sci. 2001,42(7): 1646-1652.
14. Cao W, Tombran-tink J, Chen W, et al. Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death. J Neurosci Res. 1999, 57(6):789-800.
1 Adamis AP, Shima DT, Tolention MJ, et al. Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate. Ophthalmology, 1996, 114(1): 66-71.
2 Treins C, Giorgetti-Peraldi S, Murdaca J. Regulation of vascular endothelial growth factor expression by advanced glycation end products. J Biol Chem, 2001, 276: 43836-43841.
3 Bhisitkul RB, Ruan DT, Sherwood J, et al. HIF-1-mediated induction of VEGF in RPE cells: upregulation by constitutively active HIF-1 and inhibition by RNA interference Invest Ophthalmol Vis Sci, 2004, 45: E-Abstract 452.
4 Fukuda R, Hirota K, FanF, et al. Insulin-like growth factor linduces hypoxia-inducible factor 1-mediated vascular endothelia growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem, 2002, 277(41): 38205-38211.
5 Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Insulin stimulates hypoxia-inducible factor 1 through a phosphatidylinositol 3-kinase/target of rapamycin-dependent signaling pathway. J Biol Chem. 2002, 277(31): 27975-27981.
6 Zundel W, Schindler C, Haas-Kogan D, et al. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev, 2000, 14(4): 391-396.
7 Fukuda R, Kelly B, Semenza GL. Vascular Endothelial Growth Factor Gene Expression in Colon Cancer Cells Exposed to Prostaglandin E_2 Is Mediated by Hypoxia-inducible Factor 1. Cancer Research, 2003, 63: 2330-2334.
8 John J, Stephen C. A non-hypoxia ROS-sensitive pathway mediated TNF-dependent regulation of HIF-1α. FEBB letters, 2001, 505: 269-274.
9 Sandau KB, Fandrey J, and Brune B. Accumulation of HIF-1α under the influence of nitric oxide. Blood, 2001, 97(4): 1009-1015.
1. Salnikow K, Su WC, Blagosklonny MV, et al. Carcinogenic Metals Induce Hypoxia-inducible Factor-stimulated Transcription by Reactive Oxygen Species-independent Mechanism. Cancer Research, 2000, 60: 3375-3378.
2．王雨生，严密．可见光对原代培养人视网膜色素上皮细胞的光化学损伤．中华眼底病杂志，1996，12：174-176．
3. Treins C, Giorgetti-Peraldi S, Murdaca J. Regulation of vascular endothelial growth factor expression by advanced glycation end products. J Biol Chem, 2001, 276: 43836-43841.
4. Bhisitkul RB, Ruan DT, Sherwood J, et al. HIF-1-mediated induction of VEGF in RPE cells: upregulation by constitutively active HIF-1 and inhibition by RNA interference, Invest Ophthalmol Vis Sci, 2004, 45: E-Abstract 452.
5. Fukuda R, Hirota K, Fan F, et al. Insulin-like growth factor linduces hypoxia-inducible factor 1-mediated vascular endothelia growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem, 2002, 277(41): 38205-38211.
6. Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Insulin stimulates hypoxia-inducible factor 1 through a phosphatidylinositol 3-kinase/target of rapamycin-dependent signaling pathway. J Biol Chem. 2002, 277(31): 27975-27981.
7. Zundel W, Schindler C, Haas-Kogan D, et al. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev, 2000, 14(4): 391-396.
8. Fukuda R, Kelly B, Semenza GL. Vascular Endothelial Growth Factor Gene Expression in Colon Cancer Cells Exposed to Prostaglandin E_2 Is Mediated by Hypoxia-inducible Factor 1. Cancer Research, 2003, 63: 2330-2334.
9. John J, Stephen C. A non-hypoxia ROS-sensitive pathway mediated TNF-dependent regulation of HIF-1α. FEBB letters, 2001, 505: 269-274.
10. Sandau KB, Fandrey J, Brtine B. Accumulation of HIF-1α under the influence of nitric oxide. Blood, 2001, 97(4): 1009-1015.
11．姚毅，关明，赵秀琴，等．缺氧和高浓度葡萄糖对体外培养人视网膜色素上皮衍生因子表达的影响．中华医学杂志，2003，83(22)：1989-1992．
1 Gao G, Li Y, Zhang D, et al. Unbalanced expression of VEGF and PEDF inischemia-induced retinal neovascularization. FEBS Lett, 2001, 489: 270-276.
2 Dawson DW, Volpert OV, Gillis P, et al. Pigment Epithelium-Derived Factor: A Potent Inhibitor of Angiogenesis. Science, 1999, 285 (5425): 245-248.
3 Notari L, Miller A, Martinez A, et al. Pigment epithelium-derived factor is a substrate for matrix metalloproteinase type 2 and type 9: implications for downregulation in hypoxia. Invest Ophthalmol Vis Sci, 2005, 46(8): 2736-2747.
4 Brummelkamp TR, Bernards R, Agami R. A System for Stable Expression of Short Interfering RNAs in Mammalian Cells. Science, 2002, 296: 550-553
5 王雨生，严密．可见光对原代培养人视网膜色素上皮细胞的光化学损伤．中华眼底病杂志，1996，12：174-176．
6 Balaji K, Shannon B-D, Brian K, et al. Regulation of Colon Carcinoma Cell Invasion by Hypoxia-Inducible Factor 1. Cancer Res, 2003, 63: 1138-1143
7 Salnikow K, Su WC, Blagosklonny MV, et al. Carcinogenic Metals Induce Hypoxia-inducible Factor-stimulated Transcription by Reactive Oxygen Species-independent Mechanism. Cancer Res, 2000, 60:3375-3378
8 Fire A, Xu S, Montgomery MK, et al .Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans. Nature, 1998, 391: 806-811
9 Hannon GJ. RNA interference. Nature, 2002,418(6894):244-251.
10 Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnology, 2002, 19(5):500-505.
11 Stellmach V, Crawford SE, Zhou W, et al. Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor. PNAS, 2001, 98(5): 2593-2597.
12 Cao W, Tombran-tink J, Elias R, et al. In vivo protection of photoreceptors from light damage by pigment epithelium-derived factor. Invest Ophthalmol Vis Sci. 2001, 42(7): 1646-1652.
13 Cao W, Tombran-tink J, Chen W, et al. Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death. J Neurosci Res. 1999, 57(6):789-800.
14 Halterman MW, Miller CC, Federoff HJ, et al. Hypoxia-inducible factor-1 alpha mediates hypoxia-induced delayed neuronal death that involves p53. J Neurosci, 1999, 19(16):6818-6824.
1. Semenza GL, Roth PH, Fang HM. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem, 1994, 26(38): 23757-23763.
2. WangGL, Semenza GL. Purification and characterization of hypoxia-inducible factor 1. J Biol Chem, 1995, 270(3): 1230-1237.
3. Wang GL, Jiang B, Rue E, et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O_2 tension. Proc Natl Acad Sci USA, 1995, 92(12): 5510-5514.
4. Jiang BH, Rue E, Wang GL, et al. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J Biol Chem, 1996, 271(30): 17771-17778.
5. Goldberg MA, Dunning SP, Bunn HF Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science, 1988, 242 (4884): 1412-1415.
6. Wang GL, Semenza GL. Desferrioxamine induce erythropoiet in gene expression and hypoxia-inducible factorl DNA-binding activity: implications for models of hypoxia signal transduction.Blood, 1993, 82(12): 3610-3615
7. Bunn HF, Poyton RO. Oxygen sensing and molecular adaptation to hypoxia. Physiol Rev, 1996, 76(3): 839-885.
8. Srinivas V, Zhu X, Susana S, et al. Hypoxia-inducible factor 1alpha (HIF-1αlpha) is a non-heme iron protein. Implications for oxygen sensing. J Biol Chem, 1998, 272(29): 18019-18022.
9. Salceda S, Caro J. Hypoxia-inducible factor 1alpha (HIF-1αlpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem, 1997, 272(36): 22642-22647.
10. Wiener C, Booth G, Semenza G, In vivo expression of mRNAs encoding hypoxia-inducible factorl. Biochem Biophys Res Com, 1996, 225(2): 485-488
11. Wenger RH, Kvietikova I, Roifs A, et al. Hypoxia-inducible fact1α is regulated at the post-mRNA level. Kidney Int, 1997, 51(2): 560-563.
12. Huang LE, Aramy I, Livingston DM, et al. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensit stabilization of its alpha subunit. J Biol Chem, 1996, 271(50): 32253-32259.
13. Lando D, Peet DJ, Whelan DA, et al. Asparagine hydroxylation of the HIF transactivation domain: A hypoxic switch. Science, 2002,295 (5556): 858-861.
14. Pages G, Milanini J, Richard DE, et al. Signaling angiogenesis Via p42/p44 MAP kinase cascade. Ann N Y Acad Sci, 2000, 902: 187-200.
15. Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Regulation of vascular endothelial growth factor expression by advanced glycation end products. J Biol Chem, 2001, 276(47): 43836-43841.
16. Hur E, Chang KY, Lee E, et al. Mitogen-activated protein kinase kinase inhibitor PD98059 blocks the trans-activation but not the stabilization or DNA binding ability of hypoxia-inducible factor-1alpha. Mol Pharmacol, 2001, 59(5): 1216-1224.
17. Volmat V, Camps M, Arkinstall S, et al. The nucleus,a site for signal termination by sequestration and inactivation of p42/p4 MAP kinases.J Cell Sci, 2001, 114(Pt19): 3433-3443
18. Ravi R., Mookerjee B, Bhujwalla ZM, et al. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor-1α. Genes Dev, 2000, 14(1): 34-44.
19. Hammond EM, Denko NC, Dorie MJ. Hypoxia links ATR and p53 through replication arrest. Mol Cell Biol, 2002, 22(6): 1834-1843.
20. Hansson LO, Friedler A, Freund S, et al. Two sequence motifs from HIF-1α bind to the DNA-binding site of p53. Proc Natl Acad Sci, 2002, 99(16):10305-10309.
21. Chen D, Li M, Luo J, et al. Direct interaction between HIF-1 alpha and Mdm2 modulate p53 function. J Biol Chem, 2003, 278(16):13595-13598.
22. Ravi R, Mookerjee B, Bhujwalla ZM, et al. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-induciblefactor la. Genes Dev, 2000, 14(1): 34-44.
23. Bae MK, Ann MY, Ieong JW, et al. Jab1 interacts directly with HIF-1 alpha and regulates its stability. J Biol Chem, 2002, 277(1):9-12.
24. Zhong H, Aganni F, Baccala AA,et al.Increased expression hypoxia inducible factor-alpha in rat and human prostate cancer. Cancer Res, 1998, 58(23): 5280-5284.
25. Fukuda R, Hirota K, Fan F, et al. Insulin-like growth factor 1 iduces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem, 2002, 277(41): 38205-38211.
26. Zundel W, Schindler C, Haas-Kogan D, et al. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev, 2000, 14(4): 391-396.
27. Jiang B H, Jiang G, Zheng J Z, et al. Phosphatidylinositol 3-kinase signaling control slevels of hypoxia-inducible factor 1. Cell Growth Differ, 2001, 12(7): 363-369.
28. Semenza GL, Jiang BH, Leung SW, et al. Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor, 1, J Biol Chem, 1996, 271(51): 32529-32537.
29. Forsythe JA, Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol cell Biol, 1996, 16(9): 4604-4613.
30．曹晖，胡宏慧，许迅，等．视网膜新生血管动物模型的制备．眼科新进展，2003，23(5)：335-337．
31．张鹏，王雨生，张星，等．缺氧对人视网膜色素上皮细胞增生和VEGF表达的影响．眼科新进展，2004，24(1)：6-8．
32. Pham I, Uchida T, Planes C, et al. Hypoxia upregulates VEGF expression in alveolar epithelial cells in vitro and invivo. Am J Physiol Lung Cell Mol Physiol, 2002, 283(5): 1133-1142.
33. Adamis AP, Shima DT, Tolentino, et al. Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate. Arch Ophthalmol. 1996, 114(1): 66-71.
34. Dawson DW, Volpert OV, Gillis P, et al. Pigment Epithelium-Derived Factor: A Potent Inhibitor of Angiogenesis. Science, 1999, 285(5425): 245-248.
35. Boyd SR, Tan D, Bunce C, Gittos A, Neale MH, Hungerford JL, et al. Vascular endothelial growth factor is elevated in ocular fluids of eyes harbouring uveal melanoma: identification of apotential therapeutic window. Br J Ophthalmol, 2002, 86(4): 448-452.
36. Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Regulation of vascular endothelial growth factor expression by advanced glycation end products. J Biol Chem, 2001, 276(47): 43836-43841.
37. Bhisitkul RB, Ruan DT, Sherwood J, et al. HIF-1-mediated induction of VEGF in RPE cells: upregulation by constitutively active HIF-1 and inhibition by RNA interference, Invest Ophthalmol Vis Sci, 2004,45: E-Abstract 452.
38. Semenza GL, Agani F, Booth G, et al. Structural and functional analysis of hypoxia-inducible factor 1. Structural and functional analysis of hypoxia-inducible factor 1. Kidney Int, 1997, 51(2): 553-555.
39. JinK L, Mao XO, Nagayama T, Goldsmith PC. Induction of vascular endothelial growth factor receptors and phosphatidylinositol 3' -kinase/Akt signaling by global cerebral ischemia in the rat. Neuro science, 2000, 100(4): 713-717.
40. Vincent KA, Shyu KG, Luo Y, et al. Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1 alpha/VP 16 hybrid transcription factor. Circulation, 2000,102(18): 2255-2261.
41. Damert A, Ikeda E, Risau W. Activator-protein-1 binding potentiates the hypoxia-induciblefactor-1-mediated hypoxia-induced transcriptional activation of vascular-endothelial growth factor expression in C6 glioma cells. Biochem J, 1997, 327(pt2): 419-423.
42. Kimura H, Weisz A, Kurashima Y, et al. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide. Blood, 2000,95(1): 189-197.
43. Von Marschall Z, Cramer T, Hocker M, et al. Dual mechanism of vascular endothelial growth factor upregulation by hypoxia in human hepatocellular carcinoma. Gut, 2001, 48(1): 87-96
44. Michel G, Minet E, Mottet D, et al. Site-directed mutagenesis studies of the hypoxia-inducible factor-1alpha DNA-binding domain. Biochim Biophys Acta, 2002, 1578(123): 73-83.
45. Kim HH, Lee SE, Chung WJ, et al. Stabilization of hypoxia-inducible factor-1 alpha is involved in the hypoxic stimuli-induced expression of vascular endothelial growth factor in osteoblastic cells.Cytokine, 2002, 17(1): 14-27.
46. Sun X, Kanwar JR, Leung E, et al. Regression of solid tumors by engineered overexpression of von Hippel-Lindau tumor suppressor protein and antisense hypoxia-inducible foctor-lalpha.GeneTher, 2003,10(25): 2081-2089.
47. Gogat K, Le Get L, Van Den Berghe L, et al. VEGF and KDR gene expression during human embryonic and fetal eye development. Invest Ophthalmol Vis Sci, 2004, 45(1): 7-14.
48. Ito N, Claesson-Welsh L. Dual effects ofh eparin on VEGF binding to VEGF receptor-1 and transduction of biological responses. Angiogenesis, 1999, 3(2): 159-166.
49. Takekoshi K, Isobe K, Yashiro T, et al. Expression of vascular endothelial Growth factor(VEGF) and its cognate receptors in human pheochromocytomas. Life Sci, 2004,74(7):863-871.
50. Gerber HP, Condorelli F, Park J, et al. Differential transcriptional regulation of the two vascular endothelial growth factor receptor genes. Flt-1, but not Flk-1/KDR, is upregulated by hypoxia. J Biol Chem, 1997, 272(38): 23659-23667.
51. An WG, Kanekal M, Simon MC, et al. Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha. Nature, 1998, 392(6674): 405-408.
52. Halterman MW, Miller CC, Federoff HJ, et al. Hypoxia-inducible factor-1alpha mediates hypoxia-induced delayed neuronal death that involves p53. J Neurosci, 1999,19(16):6818-6824.
53. Stellmach V, Crawford SE, Zhou W, et al. Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor. PNAS, 2001, 98(5): 2593 - 2597.
54. Cayouette M, Smith SB, Becerra SP, et al. Pigment epithelium-derived factor delays the death of photoreceptors in mouse models of inherited retinal degenerations. Neurobiol Dis. 1999, 6(6): 523-532.
55. Cao W, Tombran-tink J, Elias R, et al. In vivo protection of photoreceptors from light damage by pigment epithelium-derived factor. Invest Ophthalmol Vis Sci. 2001,42(7): 1646-1652.
56. Cao W, Tombran-tink J, Chen W, et al. Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death. J Neurosci Res, 1999, 57(6): 789-800.
57. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans. Nature, 1998, 391(6669): 806-811.
58. Hannon GJ. RNA interference. Nature, 2002, 418(6894): 244-251.
59. Lee NS, Dohjima T, Bauer G, et al. Expression of small-interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnology, 2002, 19(5): 500-505.