米诺环素对缺氧猴脉络膜视网膜血管内皮细胞表达血管内皮生长因子受体的影响

Effect of minocycline for expression of vascular endothelial growth factor receptor-1 and vascular endothelialgrowth factor receptor-2 in hypoxia chorioretinal endothelial cells of monkeys

目的观察缺氧状态下猴脉络膜视网膜血管内皮细胞（RF／6A）血管内皮生长因子受体（VEGFR）-1和VEGFR-2的表达情况及米诺环素的干预效果。方法培养RF／6A并将细胞分为正常对照组、缺氧对照组、米诺环素低剂量组（O．5／lmol／L）、米诺环素中剂量组（5．0μmol／L）、米诺环素高剂量组（50．0μmol／L）。实时定量聚合酶链反应（PCR）和免疫组织化学染色检测VEGFR-1、VEGFR-2的mRNA表达和蛋白表达。结果实时定量PCR检测结果显示，各组细胞中VEGFR-1mRNA的表达水平在24（F=0．17）、48（F=1．53）、72h（F=2．04）各时间点未发生显著变化，差异无统计学意义（P〉0．05）。72h后，米诺环素低（t=4．69）、中（t=20．16）、高（t=17．12）剂量组VEGFR-2mRNA表达水平与缺氧对照组比较，差异均有统计学意义（P〈O．001）。免疫组织化学检测结果显示，各组均可见表达VEGFR1、VEGFR-2蛋白的阳性细胞。VEGFR-1蛋白表达水平偏低，染色浅或中度棕色，主要位于内皮细胞胞膜和细胞质中，各组细胞中VEGFR-1蛋白的表达水平在各时间点比较，差异无统计学意义（F24h=0．251，F18h=0．340，F72h=0．589；P〉0．05）；VEGFR-2蛋白表达水平较高，各组内皮细胞胞膜上均可见棕黄色染色物质，细胞质中也见少量表达，缺氧对照组染色强度明显比正常对照组强，并且与米诺环素处理各组间VEGFR-2蛋白表达比较，差异有统计学意义（F24h=19．147，F48h=14．893，F72h=11．984；P〈0．05）。结论缺氧RF／6AVEGFR-2表达上调，米诺环素对其有一定抑制作用。

Objective To observe the expression of vascular endothelial growth factor receptor-1 （VEGFR-1） and VEGFR-2 in hypoxic ehorioretinal endothelial cells of monkeys （RF/6A）, and to evaluate the effect of minocycline. Methods RF/6A was cultured and divided into four groups： control group, hypoxia group, hypoxia and low dose of minocyeline group （0.5 μmol/L）, hypoxia and medium dose of minocycline group （5 μmol/L）, and hypoxia and high dose of minocycline group （50 μmol/L）. Real-time reverse transcription-polymerase chain reaction （RT-PCR） and immunohistopathological staining were used to measure the mRNA and protein expression of VEGFR-1 and VEGFR-2, respectively. Results RT-PCR showed that the expression of VEGFR-1 mRNA did not vary significantly between groups （F24 h = 0. 17, F48 h =1. 53, F72 h = 2.04 ; P〉0.05）. Compared with hypoxia group, the expression of VEGFR-2 mRNA in all minocycline treated groups were significantly down-regulated （low minocycline, medium minocycline, high minocycline： t=4.69, 20.16, 17. 12; P〈0. 001）. The immunohistopathological study showed the cells with positive staining of VEGFR 1 can be observed in all groups, and the staining was relatively weak and mainly located in cell membrane and cytoplasm. The optical density value analysis showed that the protein expression of VEGFR-1 did not vary significantly between groups at all time points（F24 h = 0. 251, F48h= 0. 340, F72 h = 0. 589 ; P〉 0.05）. The VEGFR-2 positive staining cells were also observed in all groups, and the staining was relatively high. Brown staining particles of VEGFR-2 were observed in the cell membrane with minor staining particles in cytoplasm. The staining density of VEGFR-2 was significantly higher in hypoxia group than control group. Compared with the hypoxia group, the protein expression ofVEGFR 2 in minocycline treated groups was significantly lower （F24 h = 19. 147, F48 h = 14. 893, F72h = 11. 984; P〈0.05）. Conclusion The expression of VEGFR 2 is up-regulated in RF/6A, and minocycline somewhat shows an inhibition effect.