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目的观察腺伴随病毒载体介导血管抑素Kringle5基因对SD大鼠早产儿视网膜病变(ROP)模型视网膜新生血管的影响,探寻治疗ROP的新方法。方法亚克隆构建pSNAV-Kringle5-gfp载体,腺伴随病毒包装形成rAAV-Kringle5-gfp。21只鼠高氧环境下建立ROP模型,随机分为实验组和对照组(各21只眼)。两组各取18只眼作视网膜组织切片;各剩3只眼作聚合酶链反应(RT-PCR)和Westernblotting检测。另设正常对照5只大鼠。实验组每只眼注入滴度为2.5×1012vg/ml的rAAV-Kringle5-gfp10μl,对照组每只眼注入滴度为2.5×1011vg/ml的rAAVLacZ10μl。荧光显微镜下观察目的基因在眼组织中的表达。12周后,麻醉下处死大鼠,行血管内皮细胞因子相关抗原染色、血管内皮细胞细胞核记数。结果pSNAV-Kringle5-gfp载体经测序正确;腺伴随病毒载体介导血管抑素Kringle5在玻璃体腔及视网膜有大量表达;目的基因在mRNA水平及蛋白水平均有表达;实验组、阴性对照组视网膜表面内皮细胞细胞核分别为(19.9542±3.8257)、(7.3352±2.7313)个,两组比较差异有统计学意义(P<0.01)。结论腺伴随病毒载体介导血管抑素Kringle5对ROP视网膜新生血管具有抑制作用。
Objective To observe the effect of adeno-associated virus vector-mediated angiostatin Kringle5 gene on retinal neovascularization in retinopathy of prematurity (ROP) in SD rats and to explore a new method for the treatment of ROP. Methods The pSNAV-Kringle5-gfp vector was constructed by subcloning and the adeno-associated virus was packaged to form rAAV-Kringle5-gfp. ROP models were established in 21 rats under hyperoxia. They were randomly divided into experimental group and control group (21 eyes each). Eighteen eyes of each group were used for retinal tissue sections. The remaining three eyes were examined by polymerase chain reaction (RT-PCR) and Western blotting. Another set of normal control 5 rats. In the experimental group, 10 μl of rAAV-Kringle5-gfp with a titer of 2.5 × 1012 vg / ml was injected into each eye and 10 μl of rAAVLacZ with a titer of 2.5 × 1011 vg / ml was injected into each eye of the control group. The expression of the target gene in the eye tissue was observed under a fluorescence microscope. After 12 weeks, the rats were sacrificed under anesthesia, stained with vascular endothelial cell factor-associated antigen, and counted in the nuclei of vascular endothelial cells. Results The gene of pSNAV-Kringle5-gfp was correctly sequenced. The adeno-associated virus vector-mediated expression of Kringle5 in the vitreous and the retina was highly expressed. The target gene was expressed at the mRNA and protein levels. The retinal surface The number of nuclei in endothelial cells was (19.9542 ± 3.8257) and (7.3352 ± 2.7313), respectively. The difference between the two groups was statistically significant (P <0.01). Conclusion Adeno-associated virus vector-mediated angiostatin Kringle5 has an inhibitory effect on ROP retinal neovascularization.