腺病毒介导的TSP-1f基因转移抑制视网膜新生血管的实验研究

Inhibition of retinal angiogenesis by adenovirus vector carrying TSP-1_f gene in mice

目的构建表达凝血酶敏感蛋白-1(thrombin sensitive protein-1,TSP-1)抗新生血管活性片段(TSP-1f)腺病毒载体,并观察其对氧诱导的小鼠视网膜新生血管形成的抑制作用。方法应用RT-PCR方法从正常人外周血单个核细胞扩增TSP-1fcDNA序列;采用AdEasy系统构建TSP-1f重组腺病毒ADV-TSP-1f。将鼠龄为7d的40只C57BL/6J新生鼠置于体积分数75%的氧环境中连续生活5d,然后回到正常环境中建立氧诱导的鼠血管增生性视网膜病变动物模型;每只鼠随机选取一眼为实验组,而对侧眼为对照组,在鼠生后12d时实验组玻璃体腔注射ADV-TSP-1f,对照组注射ADV-LacZ。1周后麻醉处死小鼠,Western印迹方法检测TSP-1f在实验组视网膜中的表达;视网膜铺片ADP酶法观察视网膜血管变化,组织学切片观察比较突破视网膜内界膜的血管内皮细胞数量。结果成功构建表达TSP-1f重组腺病毒载体并转染鼠视网膜,Western blotting检测到实验组视网膜目的基因表达。视网膜铺片实验组视网膜未见明显的无灌注区形成,新生血管几乎消失。组织切片实验组突破内界膜的内皮细胞核数目(10.18±1.74)明显减少,与对照组(48.89±2.98)相比差异有显著统计学意义(P<0.01)。结论腺病毒介导的TSP-1f对氧诱导的鼠视网膜新生血管有显著的抑制作用,为视网膜新生血管性疾病的基因治疗奠定基础。

Objective To construct an adenovirus vector carrying antiangiogenic fragment of thrombospidin-1 （TSP-1f） gene and investigate the effect of recombinant adenovirus vector thrombospidin-1 （ADV-TSP-1f） on retinal neovascularization in mice. Methods TSP-1f cDNA was amplified by RT-PCR from normal human peripheral blood mononuclear cells and was used as transgene to construct a recombinant ADV-TSP-1f by AdEasy system, Forty-seven-day-old C57BL/6J mice were exposed to volume fraction 75% oxygen for five days and then to normal situation to produce the murine model of oxygen-induced retinopathy（OIR）. Two eyes of each mouse were respectively divided into experimental group and control group at random. Eyes in experimental group received intravitreal injections of ADV-TSP-1f and eyes in control group received intravitreal injections of ADV-LacZ on 12th day postnatally. TSP-1f was detected by western blotting at one week after injection. The changes of retinal vessels of mice were observed by ADPase histochemical technique. The inhibitory effect of ADV-TSP-1f on retinal neovascularization was evaluated by counting the endotheliocyte nuclei of new vessels which extended from retina to vitreous in the tissue-slice of HE staining. Results The recombinant ADV-TSP-1f was produced successfully and TSP-1f was expressed and secreted efficiently by ADV- TSP-1f infected retina. Abnormal neovasctdarization reduced and avasctdar areas were not found in eyes of experimental group in retinal flatmotmt. The number of the endotheliocyte nuclei of new vessels extending from retina to vitreous was significantly less in the eyes in the experimental group （ 10.18 ± 1.74） than in control group （48.89±2.98）（P〈0.01）.Cenclusien Adenovirus-mediated TSP-1f gene greatly inhibits retinal angiogenesis in OIR, And determines the feasibility of ocular angiogenesis gene therapy.