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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6h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1h时表达量显著上升,4h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。
Objective To investigate the effect of HIF-1α ASODN treatment on bovine retinal microvascular endothelial cells (HUVECs) and the expression of HIF-1α in hypoxic cells. Methods HIF-1α antisense oligonucleotides were transfected into isolated bovine retinal vascular endothelial cells. CoCl2 was used to hypoxia culture. The expression of HIF-1α in different hypoxia time was detected by immunohistochemistry. Results The thiolated modified HIF-1α antisense oligonucleotides labeled with FAM were transfected into BREC cells with Lipofectin. After transfection for 6h, green fluorescent spots in the cells were observed under a fluorescence microscope. The transfection rate was calculated as (93.8 ± 3.4)%. At the beginning of untransfected group, the expression of HIF-1α was extremely low, at 1 hour hypoxia, the expression of HIF-1α increased significantly, reaching the peak at 4 hours and then decreasing at 16 hours. The expression of HIF-1α in ASODN transfection group was always at extremely low levels, with significant difference between the two groups (P <0.01). Conclusion Liposome-mediated delivery of HIF-1α ASODN into retinal vascular endothelial cells with high efficiency can significantly inhibit the expression of HIF-1α protein, which may provide a theoretical basis for the treatment of retinal neovascularization by HIF-1α ASODN.