αA晶体蛋白在缺氧状态下对视网膜的血管内皮细胞血管生成作用

摘要

Objective This study sought to treat a simian retinal vascular endothelial cell line RF/6 A with cobalt chloride (CoCl2), simulating a cellular hypoxic microenvironment. Then the effects of αA-crystallin (CRYAA) expression on the behavior of RF/6 A cells, in particular the endothelial cell angiogenesis under the hypoxic condition, was examined. Methods Real-time quantitative PCR was used to screen the small interfering RNA (siRNA) that could effectively inhibit CRYAA expression from three different siRNAs. The expression of CRYAA protein was examined by Western blotting analysis. The effects of knocking down CRYAA expression on RF/6 A cell's proliferative, migratory and tubulogenic capabilities induced by the hypoxia were examined by the cell counting kit-8, Transwell assay and Matrigel assay, respectively. Results CRYAA gene expression was up-regulated in the RF/6 A cells under the CoCl2-induced hypoxia (P ＜ 0.01) . However, only siRNA945 significantly inhibited the up-regulated expression of CRYAA (P ＜ 0.001) . Western blottingconfirmed a similar trend among the experimental groups of CRYAA protein as that of CRYAA transcript.The CoCl2-induced hypoxia enhanced the proliferation, migration and tube formation capabilities of RF/6 A cells, whereas siRNA945 significantly compromised these hypoxia-induced angiogenic capabilities of the retinal vascular endothelial cells (all P ＜ 0.001) . Conclusion Under the hypoxic microenvironment simulated by CoCl2, knocking down CRYAA profoundly inhibited the hypoxia-induced angiogenesis of retinal vascular endothelial cells.%目的 拟在体外用氯化钴处理猴视网膜血管内皮细胞系RF/6A,以模拟细胞缺氧微环境,检测αA-晶体蛋白 (CRYAA) 的表达是否导致缺氧环境下RF/6A细胞的行为学改变,验证CRYAA对细胞血管生成的影响.方法 采用实时荧光定量PCR从3种序列不同的小干扰RNA (siRNA) 中筛选能有效抑制CRYAA表达的siRNA,并通过蛋白印迹实验检测CRYAA蛋白水平的表达情况;分别采用细胞计数试剂盒-8、Transwell实验和Matrigel实验检测降低CRYAA表达对缺氧诱导的RF/6A细胞的增殖、迁移及管腔形成的作用.结果 缺氧环境下,RF/6A细胞中CRYAA的基因表达较正常对照组显著上调,而仅有siRNA945可抑制CRYAA的表达上调,差异有统计学意义 (P ＜0.01).蛋白印迹法验证了各组CRYAA在蛋白水平的表达趋势与核酸水平一致.单纯缺氧环境能诱导RF/6A细胞增殖、迁移及管腔成形的能力增加,而siRNA945可大幅度降低缺氧所诱导的内皮细胞血管生成能力的增强,差异有统计学意义 (均P ＜0.001).结论 在氯化钴模拟的缺氧微环境下,敲减CRYAA可显著抑制缺氧所诱导的视网膜血管内皮细胞的血管生成能力.