LL4 siRNA对缺氧血管内皮祖细胞和脉络膜-视网膜血管内皮细胞增殖、迁移和血管管腔形成的影响
Effect of DLL4 siRNA on proliferation,migration and tube formation of endothelial precursor cells and choroid-retinal endothelial cells under hypoxic conditions
【目的】 Delta样配体4 (DLL4)是Notch信号通路血管内皮特异性配体，在调节生理及病理性血管形成中起重要作用。有研究表明 DLL4-Notch信号通路受缺氧调节，可通过抑制血管分支形成促进血管成熟抑制过度血管化。脉络膜新生血管(CNV) 是一种病理性新生血管，已知缺氧在其形成中起着重要作用。本实验旨在研究DLL4在脉络膜新生血管形成中的作用和机制。 【方法】 用200 μmol/L CoCl2造成体外细胞化学性缺氧，以探究DLL4 对缺氧条件下CNV形成过程中的主要参与细胞-血管内皮祖细胞（EPC）和脉络膜-视网膜内皮细胞 (RF/6A细胞) 生物学功能的影响。DLL4 siRNA转染细胞后, 利用RT-PCR, real-time PCR和蛋白印迹技术研究缺氧条件下RF/6A细胞中DLL4和其下游主要基因HES1，HEY1的mRNA和蛋白表达变化。并进一步研究DLL4 siRNA对缺氧下EPC和RF/6A细胞参与新生血管形成的生物学功能（包括细胞增殖，移行以及管腔形成）的影响。实验设立三组对照：分别为无转染的空白对照、转染试剂对照及Scrambled阴性对照。 【结果】缺氧导致体外RF/6A细胞中DLL4表达上调。转染DLL4siRNA-duplex 1后，DLL4 mRNA的表达水平较Scrambled阴性对照组下降91.4% ，其蛋白表达亦相应减少，而三个对照组间DLL4表达无明显差异。 此外，缺氧条件下转染siRNA-duplex 1后，RF/6A细胞中DLL4-Notch信号通路下游的HES1和 HEY1基因的mRNA表达较Scrambled阴性对照组分别下降75.1% 和86.3%。进一步研究发现, DLL4低表达可促进缺氧下RF/6A 细胞增殖，导致进入细胞周期S期。DLL4 siRNA可促进缺氧下EPC和RF/6A细胞移行及血管管腔形成(P <0.05)。 【结论】DLL4是新生血管分支形成及血管出芽的负向调节因子。缺氧条件下DLL4信号途径在血管内皮祖细胞和脉络膜血管内皮细胞的增殖、迁移和血管管腔形成中发挥着重要作用，DLL4可能成为脉络膜新生血管疾病治疗的新靶点。
【Objective】 DLL4 (Delta-like 4) is an endothelium specific Notch ligand and has been shown to function as a regulating factor during physiological and pathological angiogenesis. Choroidal neovascularization (CNV) is a pathological form of angiogenesis in which hypoxia is thought to play an important role. However, the role of DLL4 in the development of CNV is still poorly understood. 【Methods】 The study utilized chemical hypoxia induced by 200μM CoCl2 to observe the expression of DLL4 in endothelial precursor cells and choroid-retinal endothelial cells (RF/6A cells) which are the primary cells involved in CNV. After transfection of a DLL4 siRNA, mRNA and protein expression of DLL4 and key downstream genes, including HES1 and HEY1, in hypoxic RF/6A cells were investigated by RT-PCR, real-time PCR, and Western blot analysis. The effects of the DLL4 siRNA on the biological function of hypoxic endothelial precursor cells and RF/6A cells during angiogenesis, including cell proliferation, migration and, tube formation, were investigated. 【Results】 The results in vitro showed that hypoxic conditions lead to upregulation of DLL4 expression in RF/6A cells. After transfection, siRNA-duplex1 targeting DLL4 depleted the DLL4 mRNA levels by as much as 91.4％ compared with the scrambled siRNA control, and DLL4 protein expression was similarly effected. In addition, after transfection of hypoxic RF/6A cells with the DLL4 siRNA-duplex1, the mRNA levels of HES1 and HEY1, which function downstream of DLL4-Notch signaling, were lowered by 75.1％ and 86.3％, respectively, compared with the scrambled siRNA control. Furthermore, knockdown of DLL4 expression significantly promoted the proliferation of hypoxic RF/6A cells and led to their arrest in the S phase of the cell cycle. Migration and tube formation of hypoxic endothelial precursor cells and RF/6A cells were significantly induced by the DLL4 siRNA, compared with the scrambled siRNA (P <0.05). 【Conclusion】 DLL4 functions as a negative regulator of angiogenic branching and sprouting. Based on our results, DLL4 signaling appears to play an essential role in the biological behavior of endothelial precursor cells and choroid vascular endothelial cells under hypoxia. As such, DLL4 may represent a novel target for CNV therapy in the future.
何花、李斌
眼科学基础医学分子生物学
LL4siRNA脉络膜新生血管血管内皮祖细胞脉络膜-视网膜内皮细胞缺氧
LL4siRNAchoroidal neovascularizationendothelial precursor cellschoroid-retinal endothelial cellshypoxia
何花,李斌.LL4 siRNA对缺氧血管内皮祖细胞和脉络膜-视网膜血管内皮细胞增殖、迁移和血管管腔形成的影响[EB/OL].(2013-02-26)[2025-08-02].http://www.paper.edu.cn/releasepaper/content/201302-445.点此复制
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