Pyk2在高氧诱导小鼠视网膜新生血管模型中的表达研究

Variation of expression of Pyk2 in oxygen-induced retinal mice model

目的探讨高氧诱导小鼠视网膜新生血管模型中富含脯氨酸的酪氨酸激酶2（Pyk2）基因表达水平的变化。方法实验研究。选取7日龄C57BI／6J新生小鼠144只，分为高氧组和对照组，每组72只。高氧组小鼠在密闭氧箱内以（75±2）％氧浓度饲养5d后置于室内空气环境中；对照组小鼠一直处于室内空气环境中饲养。两组小鼠于出生后12、14、17d分别处死8只，进行组织病理学切片检查和视网膜铺片。实时荧光定量PCR检测Pyl（2和血管内皮生长因子（VEGF）在视网膜组织中的表达水平。多组间均数比较采用两因素方差分析，组间均数比较采用SNK-q检验；同一时间点两组间数据比较采用t检验。结果组织病理学切片检查结果可见，高氧组小鼠出生后14d（15．36±3．69）和17d（29．63±4．69）有大量突破视网膜内界膜的内皮细胞核及血管芽，与对照组（0．97±1．00、0．83±0．79）相比差异有统计学意义（t14d=-20．629，P14c=0．000；t17d=-33．814，P17d=0．000）。视网膜铺片检查结果可见，高氧组小鼠出生后12d视网膜血管明显收缩、阻塞，视网膜大片区域无灌注，出生后14d新生血管开始形成，出生后17d新生血管形成达到高峰。实时荧光定量PCR检测结果可见，与对照组（Pyk2：12d：1．00±0．14，14d：0．71±0．20，17d：0．67±0．15；VEGF：12d：1．00±0．13，14d：0．88±0．14，17d：0．94±0．15）相比，高氧组出生后12dPyk2（0．05±0．03）和VEGF（0．10±0．06）mRNA表达量均降低（t=15．706，P=0．000；t=15．911，P=0．000），而出生后14d和17dPyk2（1．11±0．22、1．68±0．30）和VEGF（2．10±0．41、4．85±0．46）表达量增高（Pyk2：t14d=-3．376，P14d=0．007；t17d=-7．358，P17d=0．000；VEGF：t14d：-6．904，P14d：0．000；t17d=-19．667，P17d=0．000）。结论高氧诱导小鼠出生后14d和17d视网膜组织中Pyk2的表达水平增高，Pyk2可能参与高氧诱导的视网膜新生血管形成。

Objective To analyze the variation of expression of proline-rich tyrosine kinase 2 （Pyk2） in the oxygen-induced retinal neovascularization mice model. Methods Experimental study. One hundred and forty-four C57BL/6J mice were divided equally into the hyperoxia group and the control group. In the hyperoxia group,72 mice （7-day-old） were exposed to （75±2）% oxygen for 5 days and then moved to room air;in the control group ,72 mice were simply raised in room air. These mice were sacrificed on the 12th, 14th, 17th days and their eyeballs were collected for the preparation of pathological section,retina flat mounting and RNA extraction. The expression of Pyk2 and vascular endothelial growth factor （VEGF） mRNA in the retina were measured by real-time PCR. ANOVA was used in conjunction with SNK-q test to assess statistical significance at different time within groups; t-test was used to assess statistical significance between two groups at the same time point. Results Pathological sections showed that there were many endothelial cell nucleus and vascular buds on the 14th day （15.36 ±3.69） and 17th day （29.63 ± 4. 69） in hyperoxia group. There was significant difference between the control group （0. 97 ±1.00,0. 83 ±0. 79） and hyperoxia group （ ti4 d = - 20. 629, P14 d = 0. 000 ; t17 d = - 33. 814, P17 d = 0. 000 ） . Retina flat mounting showed that on the 12~ day, hyperoxia group showed vascular occlusion, vasoconstriction and large non-perfusion areas,neovascularization appeared and reached the peak on the 17th day. Real-time PCR showed that on the 12th day in hyperoxia group,Pyk2 mRNA （0. 05 -＋0. 03） and VEGF mRNA （0. 10 -＋0. 06） were lower （Pyk2：t = 15.706,P =0. 000; VEGF：t = 15. 911 ,P =0. 000）. However,on the 14th day and the 17th day,Pyk2 mRNA （ 1.11±0. 22,1.68 ± 0. 30） and VEGF mRNA （2. 10± 0. 41,4. 85 ±0. 46 ） increased significantly （ Pyk2 ： t14 d = -3. 376, P14 d = 0. 007 ; t17 d = -7. 358, P17 d = 0. 000 ; VEGF, t14 d = -6. 904, Pl4d =0. 000;t17d = - 19. 667,P7d =0. 000）. Conclusion Comparing to the control group,expression of Pyk2 in retinal tissue increases on the 14th day and 17th day in the hyperoxia group, indicating that the expression of Pyk2 is correlated with neovascularization.