MEK/ERK参与大鼠脉络膜新生血管基质金属蛋白酶-2和基质金属蛋白酶-9的表达调控

Contribution of MEK/ERK pathway in regulation of MMP-2 and MMP-9 expression in rat choroidal neovascularization

目的探讨基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)和MMP-9在大鼠脉络膜新生血管(choroidal neovascularization,CNV)内的表达及其可能的调控机制。方法将23只成年雄性棕色挪威大鼠随机分为2组,一组为玻璃体内注药组,视网膜光凝后即刻玻璃体内注射3μL PD98059;另一组为单纯光凝组,单纯行视网膜光凝。观察时间为光凝后3 d、7 d和14 d。各时间点处死大鼠后摘除眼球,免疫组织化学法和免疫荧光法观察MMP-2和MMP-9在大鼠CNV内不同时间点的表达特点。眼底荧光血管造影(fundus fluorescence angiography,FFA)和HE法观察玻璃体内注射PD98059对CNV生成的作用,Western blotting检测观察注药对CNV内MMP-2和MMP-9表达的影响。结果光凝后3d,单纯光凝组光凝区即有MMP-2和MMP-9的阳性表达;光凝后7 d和14 d二者表达均逐渐增强。光凝后7 d,玻璃体内注药组MMP-2和MMP-9均显著被抑制,分别被抑制约69%和80%。Western blotting检测结果示玻璃体内注射组可显著抑制ERK的磷酸化,光凝后3d及7 d的抑制率分别为54%和60%,而对ERK总量的表达无明显作用。FFA和HE显示,玻璃体内注药组光凝后14 d减少光凝局部CNV的荧光素渗漏约51%,抑制CNV厚度达57%。免疫荧光法检测结果示光凝后7 d,玻璃体内注药组抑制MMP-2和MMP-9的表达分别为73%和64%。结论 MMP-2和MMP-9参与了大鼠CNV的生成,光凝后3 d即有阳性表达,至光凝后14 d表达进一步增强,MEK/ERK通路至少部分参与了MMP-2和MMP-9在CNV内的生成调控。

Objective To investigate the expression and regulation mechanism of matrix metaUoproteinase-2 （MMP-2） and MMP-9 in rat choroidal （CNV）. Methodu Twenty-three adult male Brown Norway （BN） rats were divided into 2 groups：the PD08059 treated group and the simple photocoagulation group. Both eyes of each animal were induced by laser photocoagulation with 532 nm laser,the ani- mals in PD98059 treated group were intravitreous injected the PD98059 timely immedi- ately after photocoagulation. At 3 days, 7 days and 14 days after photocoagulation, the expression of MMP-2 and MMP-9 were examined by HE,immunohistochemistry staining and immunofluorescence staining, the effect of intravitreous injection of PD98059 on CNV formation was observed by FFA and HE staining, and the effect of intravitreous in- jection of PD98059 on MMP-2 and MMP-9 expression were detected by Western blot- ting. R^ults At 3 days after photocoagulation,the expression of MMP-2 and MMP-9 in the simple photocoagulation group were positive,and increased at 7 days and 14 days af- ter photocoagulation. At 7 days after photocoagulation, the expression of MMP-2 and MMP-9 were obviously inhibited by PD98059,the inhibitive rate were 69% and 80% ,re- spectively. Intravitreous injection of PD08059 dramatically decreased the level of p-ERK to 54% and 60% at 3 days and 7 days after photocoagulation, respectively, while had no effect on ERK expression. PD98059 was able to inhibit the growth of CNV to 57% by HE staining and decrease the leakage to 51% by FFA at 14 days after photoeoagulafion. Im- munofluorescenee staining had the similar result as immunohistochemistry which showed PD98059 attenuated the expression of MMP-2 and MMP-9 to 73% and 64%, respectively. Conelulon MMP-2 and MMP-9 are involved in the development of CNV,positively ex- press at 3 days and increase at 14 days after photocoagulation. MEK/ERK pathway at least partly regulate the expression of MMP-2 and -9 during the development of CNV.