本文選題：盤狀結(jié)構(gòu)域受體2 + 缺氧��； 參考：《第四軍醫(yī)大學》2012年碩士論文

【摘要】：【研究背景】盤狀結(jié)構(gòu)域受體2（DDR2）是一類受體型蛋白酪氨酸激酶（RTKS），DDR2通過與細胞外基質(zhì)（ECM）相互作用參與了多種細胞的遷移，粘附，，分化，轉(zhuǎn)移，并在腫瘤、自身免疫性疾病的發(fā)生、發(fā)展中發(fā)揮重要作用。DDR2被膠原激活后，可誘導受體發(fā)生自磷酸化，隨后介導眾多信號傳導途徑，調(diào)控細胞的生物學行為。脈絡膜新生血管是眼內(nèi)新生血管的重要表現(xiàn)形式之一，與眼部多種疾病有關(guān)，是造成致盲的主要原因之一，目前尚無有效的治療方法，有關(guān)其發(fā)生機制的研究，尋找針對CNV病因，抑制新生血管生成的有效療法，將為未來治療CNV提供可能。目前關(guān)于DDR2是否參與了脈絡膜新生血管發(fā)生發(fā)展尚不明確。本實驗探討DDR2在CNV發(fā)生中的作用。 【目的】觀察缺氧狀態(tài)下人RPE細胞中DDR2表達以及激活或是抑制狀態(tài)的DDR2對人RPE細胞中缺氧誘導因子-1α（HIF-1α）活化和血管內(nèi)皮生長因子（VEGF）表達的影響，探討DDR2在CNV發(fā)生中的作用。 【方法】運用200μmol/L CoCl２處理RPE細胞建立化學缺氧模型，于缺氧后0，2，6，12，24h用免疫熒光，逆轉(zhuǎn)錄聚合酶鏈反應（RT-PCR），實時熒光定量分析法（Q-PCR）和免疫蛋白印跡法(Western blot)檢測RPE細胞中DDR2的表達。缺氧條件下以Ⅰ型膠原（10μg／ml，35℃孵箱內(nèi)孵育）刺激，分別于作用后0，2，6，12，24hRT-PCR，Q-PCR和Westernblot檢測RPE細胞中HIF-1α，RT-PCR和酶聯(lián)免疫吸附試驗（ELISA）法觀察RPE細胞培養(yǎng)上清中VEGF表達，缺氧條件下給RPE細胞轉(zhuǎn)染siRNADDR2后分別于作用后0，2，6，12，24hRT-PCR，Q-PCR和Westernblot檢測RPE細胞中HIF-1α的表達，RT-PCR和酶聯(lián)免疫吸附試驗（ELISA）法觀察RPE細胞培養(yǎng)上清中VEGF表達。 【結(jié)果】正常RPE細胞細胞核，胞質(zhì)，胞膜上有微弱的DDR2熒光表達，隨著缺氧時間延長DDR2表達減弱；缺氧狀態(tài)下，RPE細胞HIF-1α，VEGF表達隨缺氧時間的延長明顯增加；缺氧膠原刺激下，HIF-1α，VEGF表達隨缺氧時間的延長未見明顯增加。缺氧膠原刺激后，可以抑制缺氧誘導的RPE細胞HIF-1α和VEGF表達上調(diào)。缺氧條件下給RPE細胞轉(zhuǎn)染siRNADDR2后，HIF-1α，VEGF表達較單純?nèi)毖踅M明顯增加。 【結(jié)論】說明DDR2在缺氧狀態(tài)下可以抑制RPE細胞中HIF-1α和VEGF表達上調(diào)。
[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 偽 (HIF-1 偽) 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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