缺氧诱导因子-1α、诱导型一氧化氮合成酶及环氧合酶-2在慢性高眼压大鼠视网膜中的表达

Expression of hypoxia-inducible factor-1α,inducible nitric oxide synthase and cyclooxygenase-2 in rat retina with chronic high ocular pressure

目的探讨缺氧诱导因子-1α(HIF-1α)、诱导型一氧化氮合成酶(iNOS)、环氧合酶-2(COX-2)的表达与高眼压视网膜损伤的关系,为青光眼视网膜缺氧提供证据。方法用巩膜静脉烧烙法在50只SPF级Wistar大鼠的双眼制作慢性高眼压模型,眼压高于造模前40%以上者为造模成功。按照视网膜取材时间的不同将动物模型眼随机分为高眼压3、7、14、21、28 d组,每组10只大鼠20只眼。另选取10只匹配大鼠作为假手术对照组,仅作球结膜切开。应用逆转录-聚合酶链反应(RT-PCR)法及Western blot法检测各组视网膜组织中HIF-1α、iNOS、COX-2 mRNA及其蛋白在大鼠视网膜中的表达。结果 HIF-1α、iNOS、COX-2 mRNA及蛋白在假手术组大鼠视网膜中呈微弱表达,造模眼HIF-1α、iNOS、COX-2 mRNA及其蛋白在视网膜中的表达水平明显升高,于7 d时达到高峰,随后有所下降,但仍明显高于正常组的表达水平。造模后3、7、14、21、28 d组大鼠视网膜中HIF-1α、iNOS、COX-2 mRNA及其蛋白的表达与假手术组相比,差异均有统计学意义(P<0.05)。结论 HIF-1α、iNOS、COX-2的低氧信号途径是青光眼神经变性发生发展中重要的病理生理机制。

Background Researches showed that hypoxia-inducible factor-1α（HIF-1α） is a transcription factor with the close relationship to hypoxia.So to study the role of HIF-1 and its relevant target gene in the damage of glaucomatous retinal ganglion cells is very important.ObjectiveThe aim of this study was to investigate the expressions of downstream gene of HIF-1,inducible nitric oxide synthase（iNOS） and cyclooxygenase-2（COX-2） in normal retina and ocular hypertension retina,and investigate the relation of glaucomatous retinal degeneration and hypoxia-responsive gene regulation.MethodsChronic ocular hypertension models were created in both eyes of 50 SPF Wistar rats by cautering two episcleral veins unilaterally with electric coagulator.Only bulbar conjunctiva was cut in other 20 eyes of 10 rabbits as sham operative group.Intraocular pressure（IOP） of animal models was measured with TONO-PENⅡ tonometor.The animals were sacrificed respectively in 3,7,14,21 and 28 days after establishment of models.Expressions of HIF-1α,iNOS and COX-2 protein in retina were detected by Western blot,and the expressions of HIF-1α,iNOS and COX-2 mRNA were assessed by reverse transcriptase-polymerase chain reaction（RT-PCR）.Use of animals followed the Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.ResultsThe IOP was more 40% after cauterization of episclera in model eyes.RT-PCR revealed that HIF-1α,iNOS and COX-2 mRNA were faintly expressed in rats retinas of sham operative group.The expressive levels of HIF-1α,iNOS and COX-2 mRNA were enhanced in 3,7,14,21 and 28 days after operation with statistically significant differences in comparison with sham operative group（P0.05,P0.01）.Western blot assay indicated that the expressions of HIF-1α,iNOS and COX-2 proteins showed the same pattern to the expressions of HIF-1α,iNOS and COX-2 mRNA.The peak expressions of HIF-1α,iNOS and COX-2 genes were on 7 days after IOP elevated then declined but still kept a higher level until 28 days.ConclusionHIF-1α,iNOS and COX-2 participates in signal transduction of retinal neurons during retinal injury on chronic high ocular pressure.