Oligopeptide-mediated gene transfer into mouse corneal endothelial cells: expression, design optimization, uptake mechanism and nuclear localization.
Oligopeptide-mediated gene transfer into mouse corneal endothelial cells: expression, design optimization, uptake mechanism and nuclear localization.
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DOI：
10.1093/nar/gkp651
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发表时间：
2009-10
影响因子：
14.9
通讯作者：
George AJ
中科院分区：
文献类型：
--
作者：
Seow WY;Yang YY;George AJ
基因转移到角膜内皮细胞有可能防止角膜移植排斥反应。在这项研究中，我们转染小鼠角膜内皮细胞（MCEC）与一类新的富含精氨酸的寡肽。所述肽具有三嵌段设计，并且比商业聚乙烯亚胺标准品更有效地介导MCEC中的报告基因表达。证明每个嵌段的功能性对肽的性能有关键影响。共聚焦成像和流式细胞术的结果表明，能量依赖性内吞是主要的摄取形式，涉及多个途径。此外，摄取强烈依赖于与细胞表面硫酸乙酰肝素的相互作用。荧光共振能量转移研究表明，肽/DNA作为缔合复合物进入细胞，并且一些将在8.5小时前解离。在26 h时，也可观察到细胞核内未浓缩DNA的大规模积累。最后，作为生物相关性的证明，我们用编码功能性吲哚胺2，3-双加氧酶（IDO）的质粒转染MCEC。然后，我们证明了表达的IDO可以催化l-色氨酸的降解，这反过来抑制了增殖测定中的CD 4 + T细胞的生长。
Gene transfer to the corneal endothelium has potential in preventing corneal transplant rejection. In this study, we transfected mouse corneal endothelial cells (MCEC) with a class of novel arginine-rich oligopeptides. The peptides featured a tri-block design and mediated reporter gene expression in MCEC more efficiently than the commercial polyethylenimine standard. The functionality of each block was demonstrated to critically influence the performance of the peptide. Results from confocal imaging and flow cytometry then showed that energy-dependent endocytosis was the dominant form of uptake and multiple pathways were involved. Additionally, uptake was strongly dependent on interactions with cell-surface heparan sulphate. Fluorescence resonance energy transfer studies revealed that the peptide/DNA entered cells as an associated complex and some will have dissociated by 8.5 h. Large-scale accumulation of uncondensed DNA within the nucleus can also be observed by 26 h. Finally, as a proof of biological relevance, we transfected MCEC with plasmids encoding for the functional indoleamine 2,3-dioxygenase (IDO) enzyme. We then demonstrated that the expressed IDO could catalyse the degradation of l-tryptophan, which in turn suppressed the growth of CD4+ T-cells in a proliferation assay.
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影响因子：
6.2
作者：
Breitenkamp, Rebecca B.;Emrick, Todd
通讯作者：
Emrick, Todd
影响因子：
6.2
作者：
Gabrielson, Nathan P.;Pack, Daniel W.
通讯作者：
Pack, Daniel W.
影响因子：
4.8
作者：
Eguchi, A;Akuta, T;Nakanishi, M
通讯作者：
Nakanishi, M
影响因子：
3.9
作者：
Jun, AS;Larkin, DFP
通讯作者：
Larkin, DFP
DOI：
10.1164/ajrccm.162.supplement_3.15tac11
发表时间：
2000-10-01
影响因子：
24.7
作者：
George, AJT;Arancibia-Cárcamo, CV;Larkin, DFP
通讯作者：
Larkin, DFP