Tat PTD-endostatin: A novel anti-angiogenesis protein with ocular barrier permeability via eye-drops.
Tat PTD-endostatin: A novel anti-angiogenesis protein with ocular barrier permeability via eye-drops.
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DOI：
10.1016/j.bbagen.2015.01.019
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发表时间：
2015-06
期刊：
影响因子：
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通讯作者：
Xinke Zhang;Yan Li;Yanna Cheng;Haining Tan;Zhi-wei Li;Y. Qu;Guoying Mu;Fengshan Wang
中科院分区：
文献类型：
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作者：
Xinke Zhang;Yan Li;Yanna Cheng;Haining Tan;Zhi-wei Li;Y. Qu;Guoying Mu;Fengshan Wang
内皮细胞增殖和血管生成的特异性抑制剂dendostatin已通过眼内注射被证明对眼部新血管疾病有影响。为了增加其对眼屏障的渗透性，使其在无创给药（滴眼液）下对眼底血管生成疾病有效，采用基因工程方法将内皮抑素与Tat ptdvia融合。方法Tat PTD -内皮抑素大部分以包体体形式在大肠杆菌中表达，通过包体体变性、复折叠、层析等一系列操作制备纯化活性的Tat PTD -内皮抑素。通过细胞增殖实验和鸡胚绒毛膜尿囊膜实验研究了Tat ptd -内皮抑素的抗血管生成活性。此外，利用荧光显微镜和流式细胞术研究了其转运能力和进入细胞的具体机制。用免疫组织化学方法研究其穿透眼屏障的能力。建立小鼠脉络膜新生血管模型，研究Tat - ptd -内皮抑素的药效学。结果获得的Tat - ptd -内皮抑素具有良好的抗血管生成活性，在细胞易位方面优于Es。巨噬细胞作用可能是Tat - ptd -内皮抑素进入细胞的主要途径。ptd -内皮抑素经滴眼后可穿越眼屏障到达视网膜。此外，滴眼液对脉络膜新生血管有抑制作用。结论stat - ptd -内皮抑素具有良好的眼穿透能力和抗血管生成作用。PTD是一种很有前途的眼部给药工具，PTD内皮抑素是治疗眼底血管生成疾病的潜在药物。
BackgroundEndostatin, a specific inhibitor of endothelial cell proliferation and angiogenesis, has been proved to have effects on ocular neovascular diseases by intraocular injection. In order to increase its permeability to ocular barriers and make it effective on fundus oculi angiogenesis diseasesvianon-invasive administration (eye drops), endostatin was fused to Tat PTDviaa genetic engineering method.MethodsMost of the Tat PTD– endostatin was expressed as inclusion bodies inEscherichia coli, so pure and active Tat PTD–endostatin was prepared by a series of operations, including inclusion body denaturation, refolding and chromatography. The anti-angiogenesis activity of Tat PTD–endostatin was investigated by cell proliferation experiments and chick embryo chorioallantoic membrane assay. In addition, its translocating ability and concrete entry mechanism into cells were also investigated by fluorescence microscope and flow cytometry. The penetrating ability to ocular barriers was also studied by immunohistochemistry. A mouse choroidal neovascularization model was established to investigate the pharmacodynamics of Tat PTD–endostatin.ResultsThe obtained Tat PTD–endostatin had excellent anti-angiogenesis activity and was superior to Es in cellular translocating. Macropinocytosis may be the dominant route of entry of Tat PTD–endostatin into cells. Tat PTD–endostatin could cross ocular barriers and arrive at the retina after eye-drop administration. In addition, it displayed inhibitory effects on choroidal neovascularizationviaeye drops.ConclusionsTat PTD–endostatin possessed excellent ocular penetrating ability and anti-angiogenesis effects.General significanceTat PTD is a promising ocular delivery tool, and Tat PTD–endostatin is a potential drug for curing fundus oculi angiogenesis diseases.