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为了解决感光细胞转基因的长期表达，应尽量选择对有丝分裂后细胞转染有效的载体，如rAAV载体、慢病毒载体及AV载体。为了减少AV载体对宿主的免疫反应和延长目的基因的表达，新一代的AV载体剪去了病毒DNA的E1和E2区，有学者用免疫调节分子局部联合注射，抑制局部的免疫反应，如CTLA4-Ig(免疫球蛋白Cr-2a与T细胞表面受体CTA4即CD152融合形成)。研究表明，视网膜下腔联合注射rAV编码的分泌性免疫调节分子CTLA4 -Ig，可延长β苷乳糖报告基因在视网膜细胞的表达［11］。为了减少炎症反应，改善视网膜的功能，有学者联合玻璃体切割及SF6填充后，玻璃体腔内注射rAV，当rAV携带的lacZ报告基因与视网膜充分接触30分钟后，用PBS液冲洗未转染的病毒，可减少炎症反应对视网膜功能的损害［12］。
5 神经前体细胞的应用前景
在眼部，有学者将患者体内的细胞如成纤维细胞取出后经基因工程加工修饰后再植入视网膜，使转基因表达的产物作为一些缺失蛋白的来源，同时，表达的基因产物还可在细胞内通过进一步修饰以利于靶细胞如感光细胞摄取利用。这种方法对于体内转基因不能长期表达的感光细胞来说是很有前景的方法。
近年来，永生化神经前体细胞转基因移植在中枢神经系统变性疾病的治疗方面显示了很好的应用前景。永生化神经前体细胞或神经干细胞的特点是：①体外培养时不分化，能自我更新。②能够单个分离并克隆；③可在体外进行基因加工或修饰，移植到体内能使报告基因长期稳定地表达；④在体内具有多功能干细胞的特点［25］。研究表明：①移植到脑内特定部位的微环境中，能定向分化为该区特异性的神经原同时能整合到该区的神经组织中［26］；②前体干细胞在局部微环境中的增生能力很强，在病理情况下这些前体细胞在分化时可被修饰，并且不会形成肿瘤［27］；③用神经前体细胞体内转基因表达，可通过药物进行调节干预［27］。
通过近几年来的努力，已能在发育或成年的中枢神经系统中分离并扩增具有多潜能的神经原干细胞或前体细胞，目前，已建立了多种永生化神经前体细胞的细胞株：如HiB5，RN33B，RN46A，ST14A，C17-2细胞株等［25，27］，这些技术为应用干细胞样特性的细胞株作为中枢神经系统移植，基因转染提供了新的细胞来源。同时，作为基因转染的前体细胞移植到中枢神经系统后，能分泌营养因子或产生代谢酶，从而使变性的神经组织再生，这些为神经变性疾病提供了很有前途的治疗方法。眼作为神经系统的一个特殊成员，神经前体细胞的应用也应有一定前景，即使在眼内不能定向分化作为变性细胞的组织来源，也有可能从其转基因稳定表达的特性，为视网膜组织转基因治疗带来一线曙光。
本课题由国家自然科学基金资助，基金号：39770790
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