Characterization and evaluation of the efficacy of cationic complex mediated plasmid DNA delivery in human embryonic palatal mesenchyme cells.
Characterization and evaluation of the efficacy of cationic complex mediated plasmid DNA delivery in human embryonic palatal mesenchyme cells.
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DOI：
10.1002/term.1873
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发表时间：
2016-11
影响因子：
3.3
通讯作者：
Elangovan, Satheesh
中科院分区：
文献类型：
--
作者：
D'Mello, Sheetal;Salem, Aliasger K.;Hong, Liu;Elangovan, Satheesh
关键词：
本研究的目的是开发和测试一种非病毒基因传递系统，该系统可用于将感兴趣的基因传递到前成骨细胞系中。用载体-质粒 DNA (pDNA) 复合物转染人胚胎腭间充质 (HEPM 1486) 细胞。我们探索了磷酸钙和聚乙烯亚胺（PEI）作为非病毒载体，并比较了它们各自的体外转染功效。使用既定方案将编码荧光素酶蛋白 (LUC) 的质粒 DNA 与 PEI（具有不同的 N/P 比率）和磷酸钙（具有不同的 Ca/P 比率）复合。然后使用 Malvern Zetasizer Nano-ZS 对制备的复合物的尺寸和表面电荷进行表征。在 HEPM 细胞中评估所制备复合物的转染效率和细胞毒性。发现在整个 N/P 比率范围内的 PEI-pDNA 复合物的尺寸 < 160 nm，而磷酸钙-pDNA 复合物相对较大。发现 N/P 比为 10 时制备的 PEI-pDNA 复合物在处理 4 小时时具有最大转染效率，且细胞毒性最小。磷酸钙-pDNA 复合物(Ca/P 200) 获得的最高转染效率比PEI-pDNA 复合物(N/P 10) 获得的最高转染效率低近12 倍。此后，使用编码增强型绿色荧光蛋白 (EGFP-N1) 或治疗相关的血小板衍生生长因子 B (PDGF-B) 的 pDNA 进一步检查用 N/P 比为 10 制备的复合物处理的 HEPM 细胞中的转基因表达。总之，PEI 是一种比磷酸钙更有效的载体，可以将感兴趣的基因传递到前成骨细胞。
The purpose of this study was to develop and test a non-viral gene delivery system that can be employed to deliver genes of interest into a pre-osteoblastic cell line. Human embryonic palatal mesenchymal (HEPM 1486) cells were transfected with vector-plasmid DNA (pDNA) complexes. We explored calcium phosphate and polyethylenimine (PEI) as non-viral vectors and compared their respective in vitro transfection efficacies. Plasmid DNA encoding luciferase protein (LUC) was complexed with PEI (with differing N/P ratios) and calcium phosphate (with differing Ca/P ratios) using established protocols. The complexes prepared were then characterized for size and surface charge using a Malvern Zetasizer Nano-ZS. The transfection efficiency and cytotoxicity of the prepared complexes were evaluated in HEPM cells. The PEI-pDNA complexes over the whole range of N/P ratios were found to be < 160 nm in size while the calcium phosphate-pDNA complexes were relatively bigger. The PEI-pDNA complexes prepared at a N/P ratio of 10 were found to have maximum transfection efficiency at 4 h of treatment with minimal cytotoxicity. The highest transfection efficiency obtained with calcium phosphate-pDNA complexes (Ca/P 200) was nearly 12-fold lower than that obtained with PEI-pDNA complexes (N/P 10). Following this, transgene expression in the HEPM cells treated with complexes prepared at a N/P ratio of 10 was further examined using pDNA coding for enhanced green fluorescent protein (EGFP-N1) or therapeutically relevant platelet-derived growth factor B (PDGF-B). In conclusion, PEI was a more effective vector for delivering genes of interest to pre-osteoblasts than calcium phosphate.
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影响因子：
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