摘要
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 detect the effect of adeno-associated-virus induced Kringles5 gene on retinal neovascularization in rats with retinopathy of prematurity （ROP）, and to explore the new ways of treatment for ROP. Methods pSNAV-Kringle5-gfp carrier was constructed by subclone and adenoassociated-virus was packed to form rAAV-Kringle5-gfp. ROP model was set up under circumstances of high oxygen in 21 SD rats which were divided into experimental （21 eyes） and control group （21 eyes）. Eighteen eyes from each group was used to making the histologic section of retina, and the other 3 eyes in each group was detected by polymerase chain reaction （PCR） and Western blotting. There were 5 rats in the normal control group. AAV Kringle5 gfp with the dosage of 10μl and titer of 2. 5 × 10^12 vg/ml was injected into the eyes in experimental group, while rAAVlacZ with the same dosage and titer of 2.5 × 10^11 vg/ml was injected in to the eyes in control group. The expression of target gene in ocular tissues was observed under the fluoroscope. Twelve weeks later, the rats were executed, and the staining of Ⅷ factor related antigens in retinal vascular endothelial cells was performed and number of nucleolus of vascular endothelial cells were counted. Results The plasmid of pSNAV-Kringle5 gfp was correct according to the sequence measurement; the expression of rAAV Kringle5 gfp was found in vitreous cavity and on retina; the expression of target gene was found on the level of mRNA and protein; the number of nucleolus of vascular endothelial cells on the surface of retina was （19. 954 2± 3. 825 7） in experimental group and （7. 335 2±2.731 3） in the control group, which had significant difference between the two groups （P 〈 0.01 ）. Conclusions Adeno-associated-virus induced Kringles5 gene can inhibit the occurrence of retinal neovascularization in patients with ROP.
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
2005年第5期288-291,共4页
Chinese Journal of Ocular Fundus Diseases
