Decorin-PEI nanoconstruct attenuates equine corneal fibroblast differentiation
Decorin-PEI nanoconstruct attenuates equine corneal fibroblast differentiation
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DOI：
10.1111/vop.12060
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发表时间：
2014-05-01
影响因子：
1.6
通讯作者：
Mohan, Rajiv R.
中科院分区：
文献类型：
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作者：
Donnelly, Kevin S.;Giuliano, Elizabeth A.;Mohan, Rajiv R.
目的探讨以绿色荧光蛋白(GFP)为标记基因的聚乙烯亚胺(PEI)纳米粒子作为载体将基因导入马角膜成纤维细胞(ECF)的可能性，(Ii)PEI纳米粒子介导的核心蛋白聚糖(DCN)基因治疗能否抑制体外培养的马角膜纤维化。方法将22 kDa线性PEI与编码GFP或DCN的质粒混合，在氮磷比为15的条件下制备聚乙烯亚胺-DNA纳米粒。ECF是由供体角膜产生的，如前所述。首先，使用PEI纳米颗粒将GFP引入ECFS以确认基因传递，然后引入DCN来评估其抗纤维化效果。实时荧光定量聚合酶链式反应和酶联免疫吸附试验证实GFP基因的导入。通过定量聚合酶链式反应、免疫染色和免疫印迹检测平滑肌肌动蛋白(SMA)的mRNA和蛋白水平来量化DCN治疗后纤维化的变化。通过细胞形态、细胞存活率和TUNEL法检测细胞毒性。结果经聚乙烯亚胺-绿色荧光蛋白处理后，细胞DNA含量为2.2×104个GFP质粒拷贝数/g，裂解物GFP含量为2.1pg/100L。PEI-DCN显著减弱了转化生长因子诱导的成纤维细胞向肌成纤维细胞的转分化(SMA mRNA下降2倍；P=0.05)，并显著抑制了SMA的表达(49+/-14.2%；P
Objective To explore (i) the potential of polyethylenimine (PEI) nanoparticles as a vector for delivering genes into equine corneal fibroblasts (ECFs) using green fluorescent protein (GFP) marker gene, (ii) whether PEI nanoparticle-mediated decorin (DCN) gene therapy could be used to inhibit fibrosis in the equine cornea using an in vitro model. Procedure Polyethylenimine-DNA nanoparticles were prepared at nitrogen-to-phosphate (N-P) ratio of 15 by mixing 22kDa linear PEI and a plasmid encoding either GFP or DCN. ECFs were generated from donor corneas as previously described. Initially, GFP was introduced into ECFs using PEI nanoparticles to confirm gene delivery, then DCN was introduced to evaluate for antifibrotic effects. GFP gene delivery was confirmed with real-time qPCR and ELISA. Changes in fibrosis after DCN therapy were quantified by measuring -smooth muscle actin (SMA) mRNA and protein levels with qPCR, immunostaining, and immunoblotting. Cytotoxicity was determined by evaluating cell morphology, cellular viability, and TUNEL assay. Results Polyethylenimine-green fluorescent protein-treated cultures showed 2.2x104 GFP plasmid copies/g of cellular DNA and 2.1pg of GFP/100L of lysate. PEI-DCN delivery significantly attenuated TGF-induced transdifferentiation of fibroblasts to myofibroblasts (2-fold decrease of SMA mRNA; P=0.05) and significant inhibition of SMA (49 +/- 14.2%; P