Yang Jin, Liu Tian Jin, Lu Yi
Department of Ophthalmology, Eye and ENT Hospital, Fudan University. Shanghai, China.
Curr Eye Res. 2007 Jan;32(1):33-42. doi: 10.1080/02713680601112793.
Posterior capsule opacification (PCO) is the most common complication after phacoemulsification cataract surgery. Hyperplasia of the lens epithelial cell after phacoemulsification is thought to be an important feature contributing to PCO. In this study,we investigated the feasibility of killing the human lens epithelial cells (HLECs) by lentivirus-mediated herpes simplex virus thymidine kinase (HSV-tk) gene/ganciclovir (GCV) in HLECs and studied the bystander effect. HLECs were infected with lentiviral vectors coexpressing HSV-tk and enhanced green fluorescent protein (EGFP) or expressing EGFP alone and treated with ganciclovir. Infection efficiency was assessed by fluorescence microscopy, fluorescence-activated cell sorting, and reverse transcription PCR. The cytotoxicity of the HSV-tk/GCV suicide gene therapy system was assessed by DNA ladder and electron microscopy. The time effect and bystander effect of HLEC growth inhibition were evaluated with cell proliferation assay. Lentiviral vector-mediated stable integration and efficient expression of HSV-tk in HLECs, with infection efficiency exceeding 95% GCV at concentrations of 15 approximately 25 mug/ml, significantly induced apoptosis or necrosis of infected HLECs. GCV also killed normal cells mixed with HSV-tk infected cells. The bystander effect markedly increased the cytotoxicity of the HSV-tk/GCV system. Our results suggest that bicistronic lentiviral vectors can efficiently integrate several genes into HLECs and may be a gene therapy platform. Lentivirus-mediated suicide gene therapy might be a feasible treatment strategy to prevent capsule opacification.
后囊膜混浊（PCO）是白内障超声乳化手术后最常见的并发症。超声乳化术后晶状体上皮细胞增生被认为是导致PCO的一个重要特征。在本研究中，我们调查了慢病毒介导的单纯疱疹病毒胸苷激酶（HSV-tk）基因/更昔洛韦（GCV）在人晶状体上皮细胞（HLECs）中杀死HLECs的可行性，并研究了旁观者效应。HLECs分别用共表达HSV-tk和增强型绿色荧光蛋白（EGFP）的慢病毒载体或单独表达EGFP的慢病毒载体进行感染，并用更昔洛韦处理。通过荧光显微镜、荧光激活细胞分选和逆转录PCR评估感染效率。通过DNA梯状条带和电子显微镜评估HSV-tk/GCV自杀基因治疗系统的细胞毒性。用细胞增殖试验评估HLEC生长抑制的时间效应和旁观者效应。慢病毒载体介导的HSV-tk在HLECs中稳定整合并高效表达，感染效率超过95%，15至25μg/ml浓度的GCV可显著诱导被感染的HLECs凋亡或坏死。GCV也能杀死与HSV-tk感染细胞混合的正常细胞。旁观者效应显著增加了HSV-tk/GCV系统的细胞毒性。我们的结果表明，双顺反子慢病毒载体可以有效地将多个基因整合到HLECs中，可能是一个基因治疗平台。慢病毒介导的自杀基因治疗可能是预防晶状体后囊膜混浊的一种可行治疗策略。
