缺氧诱导因子-1α反义寡核苷酸对缺氧时视网膜血管内皮细胞增殖活性影响的研究

Effect of HIF-1α antisense oligo-deoxynucleotides on proliferation of bovine retinal endothelial cells

目的研究缺氧诱导因子-1α(HIF-1α)反义寡核苷酸(ASODN)对缺氧时体外培养的牛眼视网膜血管内皮细胞(BREC)增殖活性的影响,探讨HIF-1αASODN治疗视网膜新生血管性疾病的可行性。方法根据Genbank提供的HIF-1αcDNA序列设计HIF-1αASODN,全硫代修饰,并用脂质体包被,体外转染BREC细胞,荧光显微镜下计算转染效率。采用免疫组织化学法检测HIF-1α的表达,采用免疫组织化学法和生物荧光法分别检测细胞增殖细胞核抗原(PCNA)的表达、细胞内ATP含量以分析细胞增殖能力。结果荧光显微镜检测表明,HIF-1αASODN可成功转染BREC。免疫荧光检测显示,未转染组缺氧开始时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,两组间差异有统计学意义(P<0.01)。PCNA免疫组织化学检测和细胞内ATP含量测定结果显示,在缺氧后各检测点ASODN转染组较对照组细胞增殖活性受到抑制,抑制作用在缺氧后8h最为明显(P<0.01)。结论 HIF-1αASODN转染能有效抑制BREC细胞HIF-1α的表达,从而降低细胞增殖活性,可能对视网膜新生血管性疾病有治疗作用。

Objective To investigate the effect of hypoxia-inducible factors-1α( HIF-1α) antisense oligo-deoxynucleotides ( ASODN) on the proliferation of bovine retinal endothelial cell( BREC) and the therapy effects of HIF-1α ASODN on retinal neovasculation. Methods HIF-1α ASODN were designed and constructed and were transfected into BREC cell line. The rate of transfection was detected by fluorescence microscopy. The BREC cells were cultured in normoxic and CoCl2-induced hypoxic condition respectively. At the same time positive control( ASODN group) and control group were set for comparison. Expressions of HIF-1α were measured with immunofluorescence staining. Immunohistochemical staining of PCNA and bioluminescence-based ATP assay were used to detect proliferation of BREC cells. Results HIF-1α ASODN was successfully transfected into BREC cells. After the transfection,immunofluorescence staining revealed that the expressions of HIF-1α of antisense group was significantly lower than those of the control group. The results from the immunohistochemical staining of PCNA and bioluminescence-based ATP assay indicated that the cell proliferation of the antisense group was inhibited. The inhibition peaked at 8 hours after the hypoxic condition,P < 0. 01. Conclusions The transfection of HIF-1α ASODN could effectively down-regulate the expression of HIF-1α and reduce cell proliferation of BREC cells. The therapeutic prospect of HIF-1α ASODN on retinal neovasculation is worthy of further investigation.