缺氧诱导因子一1α小干扰RNA对糖尿病大鼠视网膜血管内皮生长因子mRNA抑制作用的动态观察

Inhibition effect of interfering RNA targeting hypoxia inducible factor-lα on vascular endothelial growth factor mRNA expression in the retina of diabetic rats

目的观察缺氧诱导因子一1α（HIF一1α）特异性小干扰RNA（siRNA）对糖尿病大鼠视网膜组织中血管内皮生长因子（VEGF）mRNA表达的抑制作用。方法随机对照研究。以pSilencer2．1-U6neo为质粒载体，构建HIF-1asiRNA重组质粒。雄性Sprague—Dawley大鼠54只，随机分为正常对照组和实验组，分别为15、39只大鼠。实验组大鼠尾静脉注射链尿佐菌素（STZ）诱导建立糖尿病动物模型，再分为糖尿病模型组、空载体组和基因治疗组，分别为15、12、12只大鼠。脂质体Lipofectamine^TM 2000介导，空载体组和基因治疗组分别转染pSilencer空载体质粒和HIF-h-lasiRNA重组质粒，糖尿病模型组和正常组对照组不做转染。采用实时逆转录（RT）-聚合酶链式反应（PCR）检测各组大鼠VEGFmRNA的表达。分别于干扰后24、48、72h，1周时计算VEGFmRNA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。实时RT-PCR检测结果显示，正常对照组仅见弱的VEGFmRNA表达，糖尿病模型组及空载体组表达明显上调，基因治疗组表达下调；各时间点空载体组与糖尿病模型组比较，差异无统计学意义（t=0．669，0．142，0．151，0．025；P=0．514，0．889，0．882，0．980），基因治疗组较糖尿病模型组及空载体组表达下调，差异有统计学意义（t=8．768、13．695、11．285、8．253，t=9．437、13．554、11．436、8．228；P值均=0．000）；VEGFmRNA抑制率24、48、72h和1周时分别为32．76％、43．60％、47．70％、50．86％。结论HIF-1α siRNA重组质粒能有效抑制糖尿病大鼠视网膜中VEGFmRNA的表达。

Objective To observe the inhibition effect of the hypoxia inducible factor-lα （HIF-lα） specific siRNA on the expression of vascular endothelial growth factor （VEGF） mRNA in retinal tissues in diabetic rat. Methods This is a randomized controlled study. HIF-la specific siRNA recombinant plasmid was built in pSilencer2. 1-U6neo vector. Fifty-four healthy Sprague Dawley （SD） rats were divided into control group （15 rats） and experimental group （39 rats）. The experimental rats were induced with streptozotocin injection for diabetic retinopathy model, and then randomly divided into diabetic retinopathy （DR） group （15 rats）, vector group （12 rats） and gene therapy group （12 rats）. LipofectamineTM2000 mixed with pSilencer2. 1-U6neo plasmid or HiF-la siRNA plasmid were injected into the vitreous in the vector group and gene therapy group respectively. Nothing was transfected into DR and control group. The expression of VEGF mRNA in retinas was measured by real-time RT-PCR. The inhibition efficiency of VEGFmRNA was calculated at 24, 48, 72 hours and 1 week after injection respectively. Significant differences between groups were evaluated by one-way analysis and LSD-t analysis. Results HIF-la siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis. Real-time RT-PCR revealedthat the expression of VEGFmRNA was faint in the control group, increased obviously in the DR and vector group, decreased in the gene therapy group. There was no statistically significant between DR and vector group （t=0. 669,0.142,0.151,0. 025;P=0; 514,0. 889,0. 882,0. 980）. The expression of VEGFmRNAin the gene therapy group were obviously decreased compared with DR and vector group （t= 8. 768, 13. 695, 11.285, 8. 253; P=0.000）. The inhibition efficiency of VEGFmRNA was 32.76%, 43.60%, 47.70%, 50.86% at 24, 48, 72 hours and 1 week after injection. Conclusions The expression of VEGFmRNA can be efficiently inhibited by HIF-Iα siRNA recombinant plasmid.