Seitz B, Baktanian E, Gordon E M, Anderson W F, LaBree L, McDonnell P J
Doheny Eye Institute, University of Southern California, School of Medicine, Los Angeles, USA.
Graefes Arch Clin Exp Ophthalmol. 1998 Aug;236(8):602-12. doi: 10.1007/s004170050129.
To determine the potential of somatic gene transfer as a novel technique for modulating corneal wound healing on a cellular level, the successful transduction of human keratocytes should be ascertained in vitro. In addition, the ability of different polycations to increase the transduction efficiency and their antiproliferative and cytotoxic effects should be assessed.
To test transduction efficiency (X-Gal staining), cultured human keratocytes were incubated for 2 h with a retroviral vector bearing the beta-galactosidase gene, with and without the addition of polybrene or protamine sulfate. To test the antiproliferative and cytotoxic effects, cultured human keratocytes were incubated with various concentrations of polybrene and protamine sulfate (0.08 to 800 micrograms/ml) for 2, 24 and 72 h, and evaluations were performed by means of an XTT-based colorimetric assay and phase-contrast microscopy.
Human keratocytes in vitro were transduced successfully with the beta-galactosidase gene (3.5 +/- 1.0%). Transduction efficiency was significantly (P < or = 0.01) improved by addition of a polycation (from 12.3 +/- 1.7% to 18.6 +/- 2.3%), but there was no significant difference between the effects of polybrene and those of protamine sulfate. Both drugs induced a highly significant dose-dependent inhibition of proliferation (P < 0.001). ID50 ranged from 11 to 22 micrograms/ml with polybrene and from 15 to 244 micrograms/ml with protamine sulfate. Only with doses of 80 and 800 micrograms/ml did protamine sulfate produce less antiproliferative effects than polybrene (P < or = 0.04). The lowest concentrations induced no morphological signs of cytotoxicity, whereas these signs were mild at 8 micrograms/ml and moderate to severe at the highest concentrations.
Both polybrene and protamine sulfate can significantly improve the in vitro efficiency of successful retroviral vector-mediated gene transfer into keratocytes. Mild cytotoxic and moderate antiproliferative effects are to be expected in cultured keratocytes with a standard transduction procedure (8 micrograms/ml for 2 h).
为了确定体细胞基因转移作为一种在细胞水平上调节角膜伤口愈合的新技术的潜力，应在体外确定人角膜细胞的成功转导。此外，应评估不同聚阳离子提高转导效率的能力及其抗增殖和细胞毒性作用。
为了测试转导效率（X - Gal染色），将培养的人角膜细胞与携带β - 半乳糖苷酶基因的逆转录病毒载体孵育2小时，添加或不添加聚凝胺或硫酸鱼精蛋白。为了测试抗增殖和细胞毒性作用，将培养的人角膜细胞与不同浓度的聚凝胺和硫酸鱼精蛋白（0.08至800微克/毫升）孵育2、24和72小时，并通过基于XTT的比色测定法和相差显微镜进行评估。
人角膜细胞在体外成功转导β - 半乳糖苷酶基因（3.5±1.0%）。添加聚阳离子可显著（P≤0.01）提高转导效率（从12.3±1.7%提高到18.6±2.3%），但聚凝胺和硫酸鱼精蛋白的作用之间无显著差异。两种药物均诱导高度显著的剂量依赖性增殖抑制（P＜0.001）。聚凝胺的半数抑制浓度（ID50）范围为11至22微克/毫升，硫酸鱼精蛋白的ID50范围为15至244微克/毫升。仅在80和800微克/毫升剂量下，硫酸鱼精蛋白的抗增殖作用比聚凝胺小（P≤0.04）。最低浓度未诱导细胞毒性的形态学迹象，而在8微克/毫升时这些迹象轻微，在最高浓度时为中度至重度。
聚凝胺和硫酸鱼精蛋白均可显著提高逆转录病毒载体介导的基因成功转入角膜细胞的体外效率。采用标准转导程序（8微克/毫升，2小时）时，培养的角膜细胞预计会出现轻度细胞毒性和中度抗增殖作用。
