YKL-40在激光诱导的小鼠脉络膜新生血管模型中的表达及意义

Expression and clinical significance of YKL-40 in choroidal neovascularization model in mice induced by laser

摘要:

目的：探讨在激光诱导的小鼠脉络膜新生血管（choroidal neovascularization，CNV）模型中几丁质酶-3样蛋白-1（chiti－nase-3-like-1，YKL-40）的表达及意义。方法：将40只成年雄性C57BL/6J小鼠以随机数字表法按照造模后取材时间的不同随机分为光凝后7 d组和14 d组，各20只。设定每组小鼠左眼为正常对照眼，右眼通过532 nm激光光凝视网膜诱导小鼠脉络膜新生血管模型，分别于光凝后7 d和14 d心脏灌注2×106 F1TC-dextran荧光素，行视网膜色素上皮（retinal pigment epithelium，RPE）/脉络膜复合体铺片以及苏木精-伊红染色法（hematoxylin-eosin staining，HE）鉴定CNV模型。采用实时荧光定量PCR法检测各组小鼠视网膜神经层（简称神经视网膜）和RPE/脉络膜复合体中YKL-40与血管内皮生长因子（vascular endothelial growth factor，VEGF）的mRNA水平变化。免疫荧光染色法观察YKL-40在各组小鼠眼组织中的表达及定位。结果：脉络膜铺片及HE染色均证实激光造模成功，光凝后7 d和14 d均有CNV形成。在光凝后7 d组中，神经视网膜中YKL-40和VEGF的mRNA相对表达量明显升高[（1.939±0.209），（4.017±1.312）]，与正常组[（1.002±0.076），（1.017±0.219）]相比差异均有统计学意义（t=13.320，P=0.006；t=5.457，P=0.012），而RPE/脉络膜复合体中YKL-40和VEGF表达[（0.968±0.381），（1.192±0.354）]较正常组[（1.004±0.101），（1.021±0.240）]无明显改变（t=-4.519，P=0.053；t=2.450，P=0.134）；在光凝后14d组，神经视网膜和RPE/脉络膜复合体中YKL-40和VEGF的mRNA表达水平[（3.174±1.583），（3.045±1.430），（12.669±4.512），（8.254±2.968）]与正常组[（1.013±0.173），（1.043±0.371），（1.037±0.347），（1.078±0.462）]相比均明显升高，且差异具有统计学意义（t=4.777，P=0.041；t=3.508，P=0.039；t=4.827，P=0.040；t=12.800，P=0.006）。Pearson相关性分析提示在激光造模后YKL-40的表达与VEGF具有正相关性。免疫荧光发现YKL-40主要表达于光凝后小鼠视神经节细胞层、内丛状层和内核层，且光凝后7 d和14 d YKL-40表达[（0.141±0.004），（0.105±0.018）]与正常组（0.008±0.001）相比明显升高（F=106.388，P=0.000）。 结论：YKL-40在CNV形成过程中表达上调，且与VEGF的表达具有正相关性，因此我们推断YKL-40可能在CNV形成这一病理过程中起着重要作用。

Abstract:

Objective：To explore the expression and significance of chitinase-3-like-1（YKL-40） in choroidal neovascularization （CNV） models in mice. Methods：Forty adult male C57BL/6J mice were divided into day 7 group and day 14 group post laser coagu－lation randomly according to the sampling time，with twenty mice in each group. The left eyes of mice were set as normal control and the right eyes were established CNV models by 532 nm laser photocoagulation. Retinal pigment epithelium（RPE）/choroidal flatmounts after cardiac perfusion with FITC-dextran and HE staining on the 7th and 14th day post laser were used to identify the CNV models. The mRNA levels of YKL-40 and vascular endothelial growth factor（VEGF） in neuroretina and RPE/choroidal complex were detected by real-time PCR. The localizations and expressions of YKL-40 in eyes of each group were determined by immunofluorescence staining. Results：Both RPE/choroidal flatmount and HE staining confirmed the success of model and the CNV formation on day 7 and day 14 after laser coagulation. Real-time PCR showed that in neuroretina the expressions of YKL-40 and VEGF[（1.939±0.209），（4.017±1.312）] were higher in day 7 group than control group[（1.002±0.076），（1.017±0.219）]，and the differences were significant（t=13.320，P=0.006；t=5.457，P=0.012）. However，in RPE/choroidal complex，no significant differences（t= -4.519，P=0.053；t=2.450，P=0.134） were observed between control group[（1.004±0.101），（1.021±0.240）] and day 7 group[（0.968±0.381），（1.192±0.354）]. Futhermore，the mRNA levels of YKL-40 and VEGF in day 14 group[（3.174±1.583），（3.045±1.430），（12.669±4.512），（8.254±2.968）] were higher than those in control group[（1.013±0.173），（1.043±0.371），（1.037±0.347），（1.078±0.462）] both in neuroretina and in RPE/choroidal complex tissues，and the differences were significant（t=4.777，P=0.041；t=3.508，P=0.039；t=4.827，P=0.040；t=12.800，P=0.006）. Pearson correlation analysis showed that the expression of YKL-40 was positively correlated with VEGF in CNV models. The immunofluorescence staining showed that YKL-40 were mainly expressed in the retinal ganglion cell layer，inner plexiform layer and inner nuclear layer of mice after laser. Both in day 7 group and day 14 group，the expressions of YKL-40[（0.141±0.004），（0.105±0.018）] were higher than control group（0.008±0.001），and the differences were significant（F=106.388，P=0.000）. Conclusion：The expression of YKL-40 is up-regulated during the process of CNV formation and is positively correlated with VEGF. Therefore，we infer that YKL-40 may play a critical role in the pathological process of CNV formation.