Männistö Marjo, Rönkkö Seppo, Mättö Mikko, Honkakoski Paavo, Hyttinen Mika, Pelkonen Jukka, Urtti Arto
Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland.
J Gene Med. 2005 Apr;7(4):466-76. doi: 10.1002/jgm.693.
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).
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 (CMV, 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.
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
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.
视网膜色素上皮（RPE）维持光感受器的功能和视力，是基因递送的重要靶点。由于在某些疾病中RPE细胞会失控增殖，我们研究了细胞周期在非病毒基因递送至RPE细胞系（D407）中的作用。
用阳离子DNA复合物转染D407（人）细胞。将细胞同步到细胞周期的不同阶段，并用聚-L-赖氨酸（PLL）或聚乙烯亚胺（PEI）载体转染3小时。43小时后评估不同报告基因（β-半乳糖苷酶、荧光素酶）或启动子（CMV、SV40、tk、PDE-β）对基因表达的影响。通过流式细胞术测定用PLL或PEI的单叠氮溴化乙锭/DNA复合物的细胞摄取。用共聚焦显微镜对荧光DNA及其复合物进行定位。通过稳定表达荧光素酶的细胞评估细胞周期在转录中的作用。
在同步化细胞中，PLL的转染水平低于PEI，且对细胞周期的依赖性较小。PEI在G1期效率最低，在S期水平最高。所有启动子和报告基因均显示出对细胞周期的依赖性。多聚体的细胞摄取在S期最高（80 - 90%），在G1期最低（5 - 30%）。共聚焦显微镜显示细胞核中游离DNA在各组之间差异较小，在细胞核中其大多与载体结合。在稳定细胞系中细胞周期对荧光素酶表达的影响很明显。
RPE细胞系中多聚体的转染受细胞摄取和转录的影响，且这两个过程均依赖于细胞周期。这些结果对视网膜基因治疗具有重要意义。
