HIF-1α反义寡核苷酸脂质体的制备和对视网膜血管内皮细胞HIF-1α表达的影响

目的应用HIF-1αASODN处理牛眼视网膜微血管内皮细胞,观察细胞摄取ASODN的情况和对缺氧时细胞HIF-1α表达的影响.方法对分离的牛视网膜血管内皮细胞进行HIF-1α反义寡核苷酸的转染,CoCl2模拟缺氧培养,采用免疫组化检测不同缺氧时间HIF-1α的表达.结果硫代化修饰的标有FAM的HIF-1α反义寡核苷酸能被Lipofectin转染进BREC细胞.转染6 h后,在荧光显微镜下可观察到细胞内的绿色荧光斑.计算细胞转染率为(93.8±3.4)%.未转染组开始时HIF-1α有极低表达,在缺氧1 h时表达量显著上升,4 h达到高峰,16h后下降.而ASODN转染组HIF-1α的表达始终在极低水平,两组间有显著性差异(P＜0.01).结论以脂质体为载体能高效率地将HIF-1αASODN转染至视网膜血管内皮细胞内,明显抑制HIF-1α蛋白质的表达,这可能为HIF-1αASODN用于视网膜新生血管的治疗提供理论依据.

Preparation of liposomes/HIF-1α ASODN complexes and its inhibiting effect on HIF-1αexpression in bovine retinal endothelial cell

[Objective] To study inhibition of HIF-1α expression in bovine retinal endothelial cell(BREC) by HIF-1α fully phosporothioated antisense oligodeoxynucleotide (HIF-1α ASODN). [Methods] BRECs were incubated with HIF-1α ASODN and were cultured in COCl2-induced hypoxic condition. HIF-1α expression was detected with immunohistochemistry. [Results] Phosphorothioate ODNs labeled with FAM was transfected into BRECs by Lipofectin. Six hours later, many green fluorescence spots were observed in the cell with the fluromicroscope, and the transfective effect was (93.8 ± 3.4)%. In no-ASODN group, the expression of HIF-1α was very low at 0 h. But it began to increase significantly at 1 h after hypoxia, and reached the peak at 4 h, 16 h later, it declined. While in ASODN group, the expression of HIF-1α was always at very low level. The two groups had significant difference (P ＜0.01). [ Conclusions ] HIF-1α antisense oligonucleotides can suppress HIF-1α expression of bovine retinal endothelial cell effectively. It has good prospect in resisting retinal angiogenesis treatment.