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目的探讨携带血管生成素1(Ang-1)基因的骨髓间充质干细胞(BMSCs)对氧诱导视网膜病模型小鼠视网膜新生血管的作用。方法将出生7天的昆明小鼠随机分成空气对照组和高氧模型组(其中包括注射Ang-1+BMSCs组、单纯BMSCs组及高氧PBS组),每个亚组12只。除空气对照组之外,其余组均放入氧分压65%±5%的氧箱内饲养5天后取出。将携带Ang-1的BMSCs和不携带Ang-1的BMSCs各2 ml(内含细胞总数为1×105)在鼠龄第7、12、17日分别腹腔注入高氧模型组中的Ang-1+BMSCs组和单纯BMSCs组小鼠体内,空气对照组和高氧PBS组注入相同剂量的PBS缓冲液。于鼠龄17天时取小鼠眼球做标本固定,分别用免疫组织化学法检测Ang-1蛋白、qRT-PCR法检测Ang-1蛋白mRNA、计数突破视网膜内界膜的血管内皮细胞数及明胶墨汁灌注眼底视网膜血管,比较小鼠视网膜新生血管情况。结果高氧模型各组每只眼球均可见突出内界膜的新生血管内皮细胞。其中Ang-1+BMSCs组、单纯BMSCs组与高氧PBS组相比较,突破视网膜内界膜的血管内皮细胞核明显减少,差异有统计学意义(P<0.05);与高氧PBS组比较,Ang-1+BMSCs组及单纯BMSCs组血管走形较直,Ang-1蛋白表达量增加,免疫组织化学染色被黄染的胞浆较丰富;Ang-1+BMSCs组与单纯BMSCs组相比,前者血管走行较直,周边新生血管覆盖率降低,突破内界膜新生血管细胞数目减少,Ang-1蛋白的表达量增多。结论高氧能诱导新生鼠视网膜新生血管生成;Ang-1基因与BMSCs结合与单纯应用BMSCs相比,前者能更有效地改善视网膜新生血管的增生状况。
Objective To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) carrying angiopoietin 1 (Ang-1) gene on retinal neovascularization induced by oxygen in retinopathy model mice. Methods Kunming mice (7 days old) were randomly divided into air control group and hyperoxia model group (including Ang-1 + BMSCs group, BMSCs group alone and hyperoxia PBS group), 12 in each subgroup. Except for the air control group, all the other groups were placed in oxygen box with 65% ± 5% of oxygen partial pressure for 5 days and then removed. Ang-1 and BMSCs were injected intraperitoneally with 2 ml (containing 1 × 105 cells) of BMSCs carrying Ang-1 and Ang-1 + BMSCs group and BMSCs group, the same dose of PBS buffer was injected into the air-control group and the hyperoxia PBS group. At the age of 17 days, the eyeballs of mice were used to make the specimens fixed. The Ang-1 protein was detected by immunohistochemistry, the mRNA of Ang-1 was detected by qRT-PCR, the number of vascular endothelial cells which broke the inner limiting membrane of the retina and the gelatin ink Perfused retinal blood vessels were compared retinal neovascularization in mice. Results In each group of hyperoxia model, neovascular endothelial cells with prominent inner limiting membrane were observed in each eyeball. In Ang-1 + BMSCs group and BMSCs group alone, compared with hyperoxia-PBS group, the number of vascular endothelial nuclei that broke through the inner limiting membrane of the retina obviously decreased (P <0.05) 1 + BMSCs group and simple BMSCs group were more straight, Ang-1 protein expression increased, immunohistochemical staining was more yellow stained cytoplasm; Ang-1 + BMSCs group compared with simple BMSCs group, the former Blood vessels walk more straight, peripheral neovascularization coverage decreased, breaking through the membrane of the number of neovascularization decreased Ang-1 protein expression increased. Conclusion Hyperoxia can induce retinal neovascularization in neonatal rats. Compared with BMSCs alone, Ang-1 gene and BMSCs can effectively improve the proliferation of retinal neovascularization.