The role of cell cycle on polyplex-mediated gene transfer into a retinal pigment epithelial cell line
The role of cell cycle on polyplex-mediated gene transfer into a retinal pigment epithelial cell line
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DOI：
10.1002/jgm.693
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发表时间：
2005-04-01
影响因子：
3.5
通讯作者：
Urtti, A
中科院分区：
文献类型：
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作者：
Männistö, M;Rönkkö, S;Urtti, A
背景视网膜色素上皮(RPE)维持光感受器和视力的功能，是基因传递的重要靶点。由于某些疾病中RPE细胞的增殖失控，我们研究了细胞周期在非病毒基因导入RPE细胞系(D407)中的作用。方法将阳离子DNA复合体导入D407细胞。将细胞同步进入细胞周期的不同时相，用多聚L赖氨酸(PLL)或聚乙烯亚胺(PEI)载体转染3h，43h后观察不同报告基因(β-半乳糖苷酶、荧光素酶)或启动子(cnw、SV40、tk、pde-β)对基因表达的影响。用流式细胞仪检测细胞对单叠氮乙啶/DNA复合体对PLL或PEI的摄取。用共聚焦显微镜对荧光DNA及其配合物进行了定位。结果PLL在同步化细胞中的转染率低于PEI，且对细胞周期的依赖程度较低。PEI在G1期最低有效率，S期最高有效率。所有启动子和报告基因都表现出对细胞周期的依赖性。复合体的摄取以S期最高(80-90%)，G1期最低(5-30%)。共聚焦显微镜显示，细胞核内不同组之间的游离DNA略有不同，在那里，它主要是与载体结合的。细胞周期对荧光素酶表达的影响在稳定的细胞系中明显。结论复合体在RPE细胞系中的表达受细胞摄取和转录的影响，且这两个过程都是细胞周期依赖性的。这一结果对视网膜基因治疗具有重要意义。版权所有(C)2004 John Wiley&Sons，Ltd.
Background Retinal pigment epithelium (RPE) maintains the function of photoreceptors and eyesight and is an important target for gene delivery. Since in some diseases RPE cells proliferate uncontrollably, we investigated the role of cell cycle in non-viral gene delivery into a RPE cell line (D407).Methods D407 (human) cells were transfected with cationic DNA complexes. Cells were synchronized into different phases of the cell cycle and transfected using poly-L-lysine (PLL) or polyethyleneimine (PEI) carriers for 3 h. The effects of different reporters (beta-galactosidase, luciferase) or promoters (CNW, SV40, tk, PDE-beta) on gene expression were evaluated 43 h later. Cellular uptake of ethidium monoazide/DNA complexes with PLL or PEI was determined by flow cytometry. Fluorescent DNA and the complexes were localized with a confocal microscope. The role of cell cycle in transcription was evaluated by stable luciferase-expressing cells.Results PLL showed lower transfection levels than PEI in synchronized cells and only slight dependence on cell cycle. PEI showed minimal efficiency at G1 phase and maximum level at S phase. All promoters and reporter genes showed dependence on cell cycle. Cellular uptake of polyplexes was highest at S phase (80-90%) and lowest at G1 phase (5-30%). Confocal microscopy showed minor differences of free DNA between groups in the nucleus, where it was largely carrier-bound. Cell cycle effects on luciferase expression were clear in stable cell line.Conclusions Transfection by polyplexes in the RPE cell line is influenced by cellular uptake and transcription, and both processes are cell-cycle-dependent. The results have implications in retinal gene therapy. Copyright (c) 2004 John Wiley & Sons, Ltd.