摘要目的 探讨缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF- 1α)在早产儿视网膜病发病中的作用,为其基因治疗寻找新的靶点.方法 将化学合成的小分子干扰RNA( smallinterference RNA,siRNA)用脂质体介导法转染大鼠视网膜血管内皮细胞,转染的细胞在含化学缺氧剂——二氯化钴的培养基中培养.培养8h后,应用荧光定量逆转录-聚合酶链反应和Western印迹技术检测转染后细胞HIF-1α mRNA和蛋白的表达量.培养24 h后应用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐比色法检测细胞的增殖活性.采用两独立样本t检验比较各转染组与阴性对照组之间的差异.结果 成功将siRNA转染至大鼠视网膜血管内皮细胞,荧光定量逆转录-聚合酶链反应技术检测显示,针对靶基因HIF-1α设计的4条平行干扰序列siRNA均能不同程度抑制HIF-1α的转录表达,siRNA1、siRNA2和siRNA4组HIF-1α mRNA的相对表达水平分别为0.1620±0.0147、0.2034±0.0251和0.3049±0.0165,分别降至空白对照组(1.0000±0.0344)的16.20％、20.34％和30.49％,与阴性对照组(0.8334±0.0242)之间差异有统计学意义(t分别为16.786、8.953和4.087,P均＜0.05).Western印迹技术显示,siRNA1和siRNA2转染的大鼠视网膜血管内皮细胞HIF-1α蛋白表达水平分别为0.4956±0.0421和0.6544±1.0032,明显低于空白对照组(3.5105±0.4084)和阴性对照组(3.4019±1.0677),差异均有统计学意义(t分别为6.861、2.893、4.567和5.072,P均＜0.05).siRNA1和siRNA2组细胞增殖抑制率分别为(49.5±2.9)％和(67.4±1.2)％,明显高于阴性对照组[(15.7±1.5)％],差异有统计学意义(t分别为2.786和6.904,P＜0.05).结论 化学合成的HIF-1α siRNA能有效抑制大鼠视网膜血管内皮细胞在缺氧条件下HIF-1α mRNA及蛋白的表达,从而降低细胞增殖活性.

abstractsObjective To investigate the effects of hypoxia-inducible factor-1α (HIF-1α)expression on pathogenesis of retinopathy of prematurity (ROP) and to find new target for gene therapy.Methods After liposome-mediated small interference RNA (siRNA) transfection into rat retinal endothelial cells,the cells were cultured in medium with CoCl2-induced hypoxic condition.Expression of HIF-1α mRNA was determined by fluorenscence quantitative reverse transcription-polymerase chain reaction(RT-PCR),HIF-1α protein expression was detected by Western Blot after cocultured for 8 hours.Cell proliferation was measured with 3-(4,5)-dimethylthiazol (-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay after cocultured for 24 hours.Difference between groups was compared with independent samples t test.Results Rat retinal vascular endothelial cells were successfully transfected with siRNA.Fluorescence quantitative RT-PCR results showed that at 48 hours of transfection,the expression of HIF-1α mRNA in the interference group of siRNA1,siRNA2 and siRNA4 were 0.1620 ± 0.0147,0.2034 ± 0.0251 and 0.3049 ± 0.0165,which were 16.20％,20.34 ％ and 30.49％ of blank control group (1.0000±0.0344),and were lower than that of negative control group (0.8334±0.0242) (t=16.786,8.953 and 4.087,P＜0.05 respectively).Western Blot results showed that HIF-1α protein expression was significantly inhibited by siRNA1(0.4956 ± 0.0421 ) and siRNA2 (0.6544 ± 1.0032) comparing with blank control group (3.5105 ±0.4084) and negative control group (3.4019 ± 1.0677) (t =6.861,2.893,4.567 and 5.072,P＜0.05 respectively).As for cellular proliferation activity,(49.5±2.9) ％ and (67.4±1.2) ％ of cells growth inhibition were observed after transfection with siRNA1 and siRNA2,which were higher than those of negative control group [(15.7±1.5) ％ ] (t=2.786 and 6.904,P＜0.05).Conclusions The synthetic HIF-1α siRNA could effectively inhibit the expression of HIF-1α gene and reduce cell proliferation in rat retinal endothelial cells under hypoxic condition.RNA interference technology targeting HIF-1α might become a new strategy for gene therapy of ROP.