目的：探讨Caspase-1对小鼠氧诱导视网膜病变(oxygen-induced retinopathy，OIR)中小胶质细胞参与视网膜新生血管生成的作用及其机制。

方法：随机将12只7日龄(P7)C57BL/6J小鼠分为正常组、OIR组和OIR+VX-765组，正常组在正常氧环境中饲养，其余两组构建OIR模型； P12～P16，OIR+VX-765组和OIR组分别每天腹腔注射Caspase-1抑制剂VX-765(4mg/kg)和等量0.4%聚乙二醇(VX-765溶剂)； 于P17制作视网膜铺片行Lectin染色，比较三组间视网膜无血管区和新生血管区面积的大小； 采用免疫荧光染色法观察视网膜组织中Caspase-1的表达和活化小胶质细胞的分布。培养小胶质细胞BV-2细胞分为对照组、缺氧组及抑制剂组，抑制剂组和缺氧组分别经VX-765和0.4%聚乙二醇预处理3h后，缺氧条件下培养24h； 对照组常规培养相同时间。通过Western blot检测Caspase-1、p20(Caspase-1活化形式)、IL-1β和VEGF的蛋白表达变化； 用各组BV-2细胞培养上清液作为条件培养基，刺激培养血管内皮细胞RF/6A，进行管腔形成和细胞迁移实验，并比较各组间的差异。

结果：P17正常组小鼠视网膜血管化完全，未见明显无血管区及视网膜新生血管； OIR组视网膜无血管区和新生血管面积百分比分别为16.58%±1.14%、4.00%±0.41%； OIR+VX-765组两者明显减少，分别为12.23%±1.02%和2.16%±0.52%(P<0.01)。免疫荧光染色结果显示，Caspase-1在正常小鼠视网膜组织中表达较弱，在OIR小鼠中主要在神经节细胞层和内丛状层有明显的阳性表达，并与活化的小胶质细胞有明确的共定位。Western blot检测结果显示，缺氧处理的培养小胶质细胞BV-2中Caspase-1、p20、IL-1β和VEGF蛋白表达明显提高，而Caspase-1抑制剂则可明显下调p20、IL-1β和VEGF的蛋白表达水平(P<0.05)。管腔形成和细胞迁移实验结果显示，RF/6A细胞经缺氧组BV-2培养上清液处理后，管腔形成长度和细胞迁移数目分别为271±12和347±34个，而加入抑制剂后，二者明显减少，分别为171±22和212±27个(P<0.05)。

结论：在小鼠OIR中，Caspase-1能够调节小胶质细胞促进视网膜新生血管的生成，其作用机制可能与Caspase-1活化小胶质细胞中其下游炎性效应分子IL-1β，并释放VEGF相关。

METHODS: Twelve 7-day-old(P7)C57BL/6J mice were randomly divided into normal group, OIR group and OIR+VX-765 group. OIR models were established in OIR group and OIR+VX-765 group. Caspase-1 inhibitor VX-765(4mg/kg, dissolved in 0.4% polyethylene glycol)or 0.4% polyethylene glycol, were intraperitoneally injected from P12 to P16 into the mice of OIR+VX-765 and OIR groups, respectively. Whole retinal flatmounts of P17 mice were prepared, and Lectin staining was performed to calculate the ratio of avascular and neovascular area to retina area. The frozen sections of the posterior ocular segment were prepared, and the distribution of Caspase-1 and activated microglial cells were detected by immunofluorescence technique. Cultured BV-2 cells were divided into control group, hypoxia group and inhibitor group. The cells of inhibitor and hypoxia groups were pre-treated with VX-765 or 0.4% polyethylene glycol for 3h, and then hypoxic incubated for 24h. The expression levels of Caspase-1, p20(active form of Caspase-1), IL-1β and VEGF were detected by Western blotting. The angiogenesis and migration capacity of cultured RF/6A cells were assessed by endothelial cell tube formation assay and migration assay, after they were incubated with supernatant of those different BV-2 groups.

RESULTS:The distribution and morphology of retinal blood vessels were normal in P17 mice of the normal group, and avascular and new blood vessel cluster were found in the mice of OIR group and OIR+VX-765 group. The ratio of avascular area was 12.23%±1.02% and that of the new blood vessel area was 2.16%±0.52% in the OIR+VX-765 group, which decreased in comparison with 16.58%±1.14% and 4.00%±0.41% of the OIR group(P<0.01). Caspase-1 was rarely detected by immunofluorescence staining in the normal retina of the mice, whereas it was mainly co-located with activated microglial cells in the ganglion cell layer and the inner plexiform layer in the mice of OIR group. The expression of Caspase-1, p20, IL-1β and VEGF increased in BV-2 cells of the hypoxia group, which were down-regulated by VX-765(P<0.05), except Caspase-1. The tube length was 271±12, and the number of migrated cells was 347±34 in RF/6A cells cultured with supernatant of BV-2 cells in the hypoxia group, which significantly decreased to 171±22 and 212±27 with inhibitor of Caspase-1(P<0.05).

CONCLUSION:Caspase-1 promotes retinal neovascularization in the mice with OIR, probably by activating the downstream inflammatory factor IL-1β in microglial cells and accelerating the release of VEGF.