siRNA使HIF-1α基因沉默抑制大鼠视网膜体外新生血管形成的实验研究

发布时间：2018-05-28 16:13

本文选题：早产儿视网膜病变 + 小干扰RNA　；参考：《广州医学院》2012年硕士论文

【摘要】：目的探讨缺氧诱导因子-1α(hypoxia-inducible factor-1α，HIF-1α)在早产儿视网膜病变（retinopathy of prematurity，ROP）发病中的作用，为ROP的基因治疗寻找新的靶点。方法将化学合成小分子干扰RNA(small interference 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（CoCl2）的培养基中培养8h；应用荧光定量聚合酶链反应和Western印迹技术检测转染后HIF-1αmRNA和蛋白的表达量，应用四甲基偶氮唑蓝比色法对细胞增殖活性进行检测分析。正态分布计量资料用均数±标准差（x±s）表示，比较2组间是否存在差异采用t检验。结果成功将siRNA转染大鼠视网膜血管内皮细胞，荧光定量聚合酶链反应技术检测显示，4条siRNA均能不同程度抑制HIF-1α的转录表达，siRNA1、siRNA2和siRNA4组HIF-1α mRNA分别降至空白对照组的16.20%、20.34%和30.49%，与阴性对照组（83.34%）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0.496、0.654，明显低于空白对照组（3.402）和阴性对照组（3.510），差异有统计学意义（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49.50%和67.41%，明显高于阴性对照组（15.70%），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基因及蛋白的表达，从而降低细胞增殖活性。
[Abstract]:Objective to investigate the role of hypoxia-inducible factor-1 伪 (HIF-1 伪) in the pathogenesis of retinopathy of preterm infant retinopathy (ROP) and to find a new target for gene therapy of ROP. Methods the chemically synthesized small interfering RNA(small interference RNAs siRNAs were transfected into rat retinal vascular endothelial cells by liposome mediated transfection. The transfected cells were cultured for 8 hours in a medium containing chemical anoxic agent Cobalt dichloride (CoCl2). The expression of HIF-1 伪 mRNA and protein was detected by fluorescence quantitative polymerase chain reaction (FQ-PCR) and Western blot. Cell proliferation activity was determined by tetramethyl azolium blue colorimetry. The measurement data of normal distribution were expressed by mean 卤standard deviation (x 卤s). T test was used to compare the differences between the two groups. Results siRNA was successfully transfected into rat retinal vascular endothelial cells. Fluorescence quantitative polymerase chain reaction (FQ-PCR) analysis showed that all four siRNA could inhibit the transcription expression of HIF-1 伪 in different degree. The HIF-1 伪 mRNA in siRNA4 group and siRNA4 group decreased to 16.20% and 30.49% of the blank control group, respectively, and there was significant difference between the four siRNA strips and the negative control group (83.34%). The expression levels of HIF-1 伪 protein in rat retinal vascular endothelial cells transfected with siRNA1 and siRNA2 were significantly lower than those in control group (P < 0.05).Western) and negative control group (P < 0.05).Western blotting, respectively), and the difference was statistically significant. The inhibitory rates of cell proliferation in the 0.05).siRNA1 and siRNA2 groups were 49.50% and 67.41%, respectively, which were significantly higher than those in the negative control group (P < 0.05). Conclusion chemically synthesized HIF-1 伪 siRNA can effectively inhibit the expression of HIF-1 伪 gene and protein in rat retinal vascular endothelial cells (RVEC) under hypoxia.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.6

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