腺伴随病毒载体介导Kringle5-GFP基因在眼组织中的表达

Expression of adeno-associated-virus induced angiostatin Kringles5-GFP gene in SD rats' ocular tissues

目的:探寻腺伴随病毒载体介导Kringle5基因注入玻璃体腔后,在眼球组织中的分布规律,为基因治疗视网膜病变提供实验依据。方法:将已经构建包装好的rAAV-Kringle5-GFP,滴度为2.5×1012vg/mL10μL注入SD大鼠玻璃体腔后,分别在1,5,10,20,40,60,90d处死SD大鼠,在荧光显微镜下观察GFP在玻璃体、视网膜、脉络膜、虹膜等眼组织分布及表达情况。根据表达强度从无表达到强表达分别记为-,+,++,+++,++++。结果:在注射后第1d,玻璃体中有轻度表达,随着时间的推移表达缓慢增强,分布较广泛;在玻璃体注射后第5d视网膜有表达,分布范围从视盘到周边部视网膜组织均出现且随着时间的推移表达增强;在虹膜、脉络膜组织表达也有较强表达;甚至在角膜内皮及晶状体组织也有表达。结论:选用腺伴随病毒载体介导目的基因能够在视网膜组织和色素膜组织有较强表达,腺伴随病毒载体介导目的基因是治疗ROP视网膜新生血管较为理想的载体。

AIM： To investigate the distribution of adeno-associated-virus induced angiostatin Kringles5-GFP gene in ratsocular tissues after injected in vitreous cavity and to provide experiment basis to retinopathy treatment. METHODS： After injected rAAV-Kringle5-GFP with the titer 2.5×1012 vg/mL 10μL in vitreous cavity, the SD rats were sacrificed at day 1, 5, 10, 20, 40, 60 and 90. The distribution and expression of GFP in ocular tissues such as vitreous, retina, chloridum, iris and so on were observed under green microscopy. The expression degrees were recorded as -, ＋, ＋＋, ＋＋＋ and ＋＋＋＋. RESULTS： Kringle5 expressed in vitreous at the day 1 after injection and increased with the time. After 5 days, it expressed in retina, ranged from optic disc to peripheral retinal tissues. In iris and choroid, Kringle5 showed a strong expression. It could also be found in corneal endothelium and crystalline lens. CONCLUSION： The adeno-associated-virus induced angiostatin Kringles5-GFP gene can strongly express not only in retina but also in other ocular tissues. Thus, adeno-associated virus is the best vector for gene in curing retina disease.