DDR2对缺氧诱导的人视网膜色素上皮细胞HIF-1α和VEGF表达的影响

发布时间：2018-04-14 22:35

  本文选题：盘状结构域受体2 + 缺氧　； 参考：《第四军医大学》2012年硕士论文

【摘要】：【研究背景】盘状结构域受体2（DDR2）是一类受体型蛋白酪氨酸激酶（RTKS），DDR2通过与细胞外基质（ECM）相互作用参与了多种细胞的迁移，粘附，，分化，转移，并在肿瘤、自身免疫性疾病的发生、发展中发挥重要作用。DDR2被胶原激活后，可诱导受体发生自磷酸化，随后介导众多信号传导途径，调控细胞的生物学行为。脉络膜新生血管是眼内新生血管的重要表现形式之一，与眼部多种疾病有关，是造成致盲的主要原因之一，目前尚无有效的治疗方法，有关其发生机制的研究，寻找针对CNV病因，抑制新生血管生成的有效疗法，将为未来治疗CNV提供可能。目前关于DDR2是否参与了脉络膜新生血管发生发展尚不明确。本实验探讨DDR2在CNV发生中的作用。 【目的】观察缺氧状态下人RPE细胞中DDR2表达以及激活或是抑制状态的DDR2对人RPE细胞中缺氧诱导因子-1α（HIF-1α）活化和血管内皮生长因子（VEGF）表达的影响，探讨DDR2在CNV发生中的作用。 【方法】运用200μmol/L CoCl２处理RPE细胞建立化学缺氧模型，于缺氧后0，2，6，12，24h用免疫荧光，逆转录聚合酶链反应（RT-PCR），实时荧光定量分析法（Q-PCR）和免疫蛋白印迹法(Western blot)检测RPE细胞中DDR2的表达。缺氧条件下以Ⅰ型胶原（10μg／ml，35℃孵箱内孵育）刺激，分别于作用后0，2，6，12，24hRT-PCR，Q-PCR和Westernblot检测RPE细胞中HIF-1α，RT-PCR和酶联免疫吸附试验（ELISA）法观察RPE细胞培养上清中VEGF表达，缺氧条件下给RPE细胞转染siRNADDR2后分别于作用后0，2，6，12，24hRT-PCR，Q-PCR和Westernblot检测RPE细胞中HIF-1α的表达，RT-PCR和酶联免疫吸附试验（ELISA）法观察RPE细胞培养上清中VEGF表达。 【结果】正常RPE细胞细胞核，胞质，胞膜上有微弱的DDR2荧光表达，随着缺氧时间延长DDR2表达减弱；缺氧状态下，RPE细胞HIF-1α，VEGF表达随缺氧时间的延长明显增加；缺氧胶原刺激下，HIF-1α，VEGF表达随缺氧时间的延长未见明显增加。缺氧胶原刺激后，可以抑制缺氧诱导的RPE细胞HIF-1α和VEGF表达上调。缺氧条件下给RPE细胞转染siRNADDR2后，HIF-1α，VEGF表达较单纯缺氧组明显增加。 【结论】说明DDR2在缺氧状态下可以抑制RPE细胞中HIF-1α和VEGF表达上调。
[Abstract]:[background] Disc domain receptor 2 (2) is a class of tyrosine kinase tyrosine kinase (RTKS) DDR2 involved in the migration, adhesion, differentiation, metastasis, and occurrence of tumors and autoimmune diseases by interacting with ECM.After being activated by collagen, DDR2 can induce the receptor to self-phosphorylation, and then mediate many signal transduction pathways to regulate the biological behavior of cells.Choroidal neovascularization is one of the important manifestations of intraocular neovascularization, which is related to many ocular diseases and is one of the main causes of blindness.Finding effective therapy for the etiology of CNV and inhibiting neovascularization will provide the possibility for the treatment of CNV in the future.It is unclear whether DDR2 is involved in choroidal neovascularization.The purpose of this study was to investigate the role of DDR2 in the pathogenesis of CNV.[objective] to investigate the effects of DDR2 expression in human RPE cells under hypoxia and the activation or inhibition of DDR2 on the activation of hypoxia inducible factor-1 伪 (HIF-1 伪) and the expression of vascular endothelial growth factor (VEGF) in human RPE cells, and to explore the role of DDR2 in the pathogenesis of CNV.[methods] the model of chemical hypoxia was established in RPE cells treated with 200 渭 mol/L CoCl2. The expression of DDR2 in RPE cells was detected by immunofluorescence, reverse transcriptase polymerase chain reaction (RT-PCR), real time quantitative analysis (Q-PCR) and Western blot analysis at 1224 h after hypoxia.The expression of VEGF in the supernatant of RPE cells was detected by RT-PCR and Westernblot in RPE cell culture supernatant by RT-PCR and enzyme linked immunosorbent assay (Elisa), respectively, stimulated by 10 渭 g / ml of collagen 鈪

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