氧诱导小鼠视网膜新生血管发生中乙酰肝素酶及串珠素的表达

Expression of heranase and perlecan in the retina of mouse with oxygen-induced retinopathy

目的研究小鼠视网膜新生血管发生过程中乙酰肝素酶(HPA)及其作用底物串珠素在视网膜中的表达。方法将65只新生幼鼠于生后7d在体积分数(75±2)%高氧环境中饲养5d后,再置于相对低氧环境中诱导产生视网膜新生血管为高氧诱导组。另外65只新生幼鼠在正常环境中饲养作为正常对照组。分别在小鼠生后第12、13、17、21、30天通过荧光素眼底血管造影(FFA)和视网膜病理切片观察视网膜新生血管的形态变化。通过逆转录聚合酶链反应(RT-PCR)分别检测不同时间点视网膜组织中HPA和串珠素在mRNA水平的变化,Western blot检测不同时间点视网膜组织中HPA在蛋白水平的表达变化。结果高氧诱导组与正常对照组的HPA mRNA表达水平差异有统计学意义(Fgroup=16.303,P=0.000),各时间点的HPAmRNA表达水平差异有统计学意义(Ftime=18.614,P=0.000),生后第12、13、17、21天相应时间点2组小鼠视网膜HPAmRNA的表达水平差异均有统计学意义(P=0.001,P=0.000,P=0.000,P=0.001)。高氧诱导组与正常对照组HPA蛋白表达水平差异有统计学意义(Fgroup=458.134,P=0.000),各时间点的HPA蛋白表达水平差异有统计学意义(Ftime=78.466,P=0.000)。高氧诱导组与正常对照组的串珠素mRNA表达水平差异有统计学意义(Fgroup=7.351,P=0.013),各时间点的串珠素mRNA表达水平差异有统计学意义(Ftime=9.098,P=0.000)。生后第13、17、21天的高氧诱导组小鼠视网膜串珠素mRNA的表达水平与正常对照组相应时间点,差异均有统计学意义(P=0.048,P=0.000,P=0.003)。伴随着视网膜新生血管的增多和消退,HPA在基因和蛋白水平及串珠素在基因水平的表达均呈先升高后降低的趋势。结论HPA及其作用底物可能是促进视网膜新生血管生长的重要因素。

Background Heparanase degrade heparan sulfate side chains of heparan sulfate proteoglycans in the extraeellular matrix. Heparanase induces angiogenesis and likely promotes the vascularization of tumor. Objective The present study is to investigate the expression of heparanase and perleean in retinas with oxygen-induced retinopathy. Methods Sixty- five clean neonatal C57BL/6J mice were raised in a hyperbaric oxygen box with a volume percentage of 75% ＋ 2% for 5 days and then returned to the normal air room. Another 65 matched mice were raised in the normal environment as controls. Evans blue was infused by the superior vena cava in all the mice on postnatal days 12,13,17,21 and 30, afterwards fluorescein angiography was performed and then the mice were sacrificed. The retinas of mice were isolated and prepared and the retinal vessels were examined under a fluorescent microscope and optical microscope. Heranase and perlecan InRNA was detected using reverse transcription PCR （RT-PCR）. Heranase and perleean proteins were detected by Western blot. The analysis of variance was used to compare the mRNA and the protein levels of heranase and perleean between the experimental and control groups. Results The expression of heparanase mRNA in the retinas of different ages of mice and the different groups showed significant differences （Fgroup = 16. 303,P = 0. 000; Ftime = 18. 614, P = 0. 000; F interaction= 11. 299,P = 0. 000） , and the expression of heparanase mRNA was significantly enhanced in mice from postnatal days 12,13,17 and 21 compared with normal control mice （P = 0. 001,0. 000, 0. 000,0. 001 ,respectively）. The expression of heparanase protein in the retinas of different ages of mice and the different groups followed the same tendency （ F group = 458. 134, P = 0. 000 ; F time = 78. 466, P = 0. 000 ; Finteraction = 7l. 398, P = 0. 000 ）. The expression of perleean mRNA in the retinas of different ages of mice and the different groups showed significant differences （F group =7. 351 ,P =0. 013;F time=9. 098,P =0. 000;Finteraction =3. 349,P =0. 000）,and increase in differences also were clearly seen in mice from postnatal days 13,17 and 21 compared with normal control mice （P = 0. 048,0. 000,0. 003, respectively）. Conclusion The expression of heparanase and perleean is associated with the development and progression of retinal neovascularization, and perlecan and heparanase together produce a synergistic effect. Heparanase and perlecan may participate in the angiogenesis of oxygen-induced retinopathy.