Polymer transfected primary myoblasts mediated efficient gene expression and angiogenic proliferation.
Polymer transfected primary myoblasts mediated efficient gene expression and angiogenic proliferation.
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DOI：
10.1016/j.jconrel.2009.09.021
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发表时间：
2010-02-25
影响因子：
10.8
通讯作者：
Kim, Sung Wan
中科院分区：
文献类型：
--
作者：
Ou, Mei;Kim, Tae-il;Yockman, James W.;Borden, Bradley A.;Bull, David A.;Kim, Sung Wan
本研究旨在评估聚合物介导转染原代成肌细胞的体外基因表达效率和治疗效果。自体原代成肌细胞移植可能通过肌再生改善梗死心肌的功能。此外，原代成肌细胞可以携带编码血管生成因子的外源性血管生成基因，以促进治疗性血管生成。病毒载体由于诱导炎症反应、致瘤突变和基因组整合而限制了临床应用。为了克服这些问题，合成了两种新型的可生物降解的聚二硫醚-胺（poly（cystaminebisacrylamide-diaminohexane）[poly（CBA-DAH）]和poly（cystaminebisacrylantide-diaminohexane-arginine）[poly（CBA-DAH-R）]作为基因载体。在这项研究中，原代成肌细胞分离和纯化大鼠骨骼肌。基于使用荧光素酶测定和共聚焦显微镜的优化的聚合物介导的转染程序，这两种聚（二硫胺）在原代成肌细胞中诱导比支链聚（乙烯亚胺）（bPEI，25 kDa）高16倍的荧光素酶表达和高得多的绿色荧光蛋白表达。通过流式细胞术，poly（CBA-DAH）和poly（CBA-DAH-R）分别促进97%和99%的荧光标记的聚合物/pDNA复合物的细胞摄取率，这比bPEI 25 kDa（87%）高。两种聚（二硫胺）的细胞毒性比bPEI 25 kDa小得多。体外时间过程和共培养实验证实了聚合物工程化原代成肌细胞具有刺激内皮细胞增殖的能力。这些数据证实了聚二硫胺是将VEGF 165转染到原代成肌细胞中的安全可行的多聚基因载体。聚合物工程原代成肌细胞在缺血性心脏病的治疗中具有潜在的应用价值。
This study was designed to assess the in vitro gene expression efficiency and therapeutic effectiveness of polymer mediated transfection of primary myoblasts. Autologous primary myoblasts transplantation may improve the function of infarcted myocardium via myogenesis. In addition, primary myoblasts can carry exogenous angiogenic genes that encode angiogenic factors to promote therapeutic angiogenesis. Viral vectors have limited clinical application due to the induction of inflammatory reactions, tumorigenic mutations and genome integration. To overcome these problems, two new biodegradable poly(disulfide—amine)s, poly(cystaminebisacryamide-diaminohexane) [poly(CBA-DAH)] and poly(cystaminebisacryantide-diaminohexane-arginine) [poly(CBA-DAH-R)], were synthesized as polymer carriers for gene delivery. In this study, primary myoblasts were isolated and purified from rat skeletal muscles. Based on an optimized polymer mediated transfection procedure using a luciferase assay and confocal microscopy, these two poly(disulfide amine)s induced up to 16-fold higher luciferase expression and much higher green fluorescence protein expression than branched poy(ethylenimine) (bPEI, 25kDa) in primary myoblasts. By flow cytometry, poly(CBA-DAH) and poly(CBA-DAH-R) promote rates of cellular uptake of florescence-labeled polymer/pDNA complexes of 97% and 99%, respectively, which are rats higher than that of bPEI 25kDa (87%). Both poly(disulfide amine)s were much less cytotoxic than bPEI 25kDa. The in vitro time course and co-culture experiments verified that polymer engineered primary myoblasts have the ability to stimulate endothelial proliferation. These data confirmed that poly(disulfide amine)s are the safe and feasible polymeric gene carriers to transfect VEGF165 into primary myoblasts. Polymer engineered primary myoblasts have potential for therapeutic application in the treatment of ischemic heart diseases.
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