目的：观察缺氧诱导因子-1α(hypoxia inducible factor-1α，HIF-1α)小干扰RNA(siRNA)对糖尿病大鼠视网膜组织中血管内皮生长因子(vascular endothelial growth factor，VEGF)蛋白表达的抑制作用，及其用于糖尿病新生血管性疾病治疗的可行性。

方法：以pSilencer2.1-U6neo为质粒载体，构建HIF-1α.siRNA 重组质粒。选取雄性Sprague-Dawley(SD)大鼠54只，随机分为正常对照组(15只)和实验组(39只)。实验组采用尾静脉注射链尿佐菌素(STZ)的方法建立糖尿病大鼠模型，造模成功后将实验组随机分为糖尿病组(15只)、基因治疗组(12只)和空载体组(12只)。正常对照组及糖尿病组大鼠均不做转染； 基因治疗组和空载体组分别转染HIF-1α.siRNA 重组质粒和pSilencer空载体质粒。行苏木精-伊红染色(hematoxylin-eosin staining，HE染色)观察视网膜组织形态，并采用免疫组织化学法检测VEGF蛋白的表达。分别于干扰后24、48、72h，1wk时计算VEGF蛋白的抑制效率。采用单因素方差分析和LSD-t检验进行统计学分析。

结果：HIF-1α.siRNA 重组质粒经酶切、测序鉴定，确定为目的序列。HE染色显示：正常对照组大鼠视网膜各层细胞排列整齐，细胞形态基本正常。糖尿病大鼠视网膜各层细胞排列紊乱，内界膜不完整，新生血管芽、新生血管簇呈垂直状突破内界膜生长。免疫组织化学染色显示：VEGF阳性表达为细胞浆出现棕黄色颗粒，主要位于神经节细胞层。正常对照组VEGF蛋白呈弱阳性表达，而DR对照组和空载体组表达明显增强，基因治疗组较DR组和空载体组表达明显减少，差异有统计学意义(P<0.05)。VEGF蛋白抑制率24、48、72h和1wk时分别为：27.4%、40.6%、47.5%、64.5%。

结论：HIF-1α.siRNA重组质粒能够抑制糖尿病大鼠视网膜中VEGF蛋白的表达，可能成为一种治疗糖尿病性新生血管疾病的新方法。

AIM: To evaluate the inhibitory effect of hypoxia inducible factor-1α(HIF-1α)small interfering RNA(siRNA)on the expression of vascular endothelial growth factor(VEGF)protein and explore the feasibility of potential therapeutic approach for diabetic neovascular disease.

METHODS: Using pSilencer2.1-U6neo for plasmid vector, HIF-1α siRNA recombinant plasmid was constructed. There was totally 54 healthy Sprague Dawley rats in which 15 rats were chosen as normal group and 39 rats were constructed for diabetic retinopathy model by streptozotocin(STZ)which was divided into three subgroups randomly including control model group(DR group, 15 rats), vector group(12 rats)and gene therapy group(HIF-1α siRNA group, 12 rats). Nothing was transfected into DR group and normal group. The vector plasmid and HIF-1α siRNA were injected into the vitreous in vector group and HiF-1α siRNA group respectively. The retinal morphology was observed by hematoxylin-eosin(HE)staining and the expression of VEGF protein was measured by immunohistochemical staining. The inhibition efficiency of VEGF was calculated at 24, 48, 72h and 1wk after injected. Significant differences between groups were evaluated by one-way analysis of variance, followed by LSD-t analysis.

RESULTS: HIF-1α siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis. HE staining showed that the retinal cells at each layers in normal control group were arranged regularly, and cell's morphology was roughly normal. The retinal cells at each layers arranged in disorder in diabetic rat And the inner limiting membrane was not complete with neovascular buds and neovascularization cluster growing out of the inner limiting membrane vertically. Immunohistochemical staining showed that the positive expression of VEGF was brown yellow granules, which was mainly located in ganglion cell layer. It also revealed the expression of VEGF protein was weakly positive in normal control group, while the DR group and empty vector group were significantly increased. Compared with DR group and the empty vector group, gene therapy group was significantly decreased, the difference was statistically significant(P<0.05). VEGF protein level was reduced by 27.4%, 40.6%, 47.5%, 64.5% at 24, 48, 72h and 1wk.

CONCLUSION: Intravitreal injection with HIF-1α siRNA can efficiently inhibite VEGF protein in retina of diabetic rats, which may be a new method for the treatment of diabetic neovascular disease.

天津市科技支撑重点项目(No.08ZCGYSF01700)