RNA干扰对低分化鼻咽癌细胞血管内皮生长因子表达的抑制作用

Down-regulation of the vascular endothelial growth factor(VEGF) by a small interfering RNA targeting VEGF in the nasopharyngeal carcinoma cell line(CNE-2Z)

目的研究血管内皮生长因子(vascular endothe-lial growth factor,VEGF)的特异性小片段干扰RNA(siRNA)对低分化鼻咽癌细胞(nasopharyngeal carcinoma cell line,CNE-2Z)VEGF表达的抑制作用,为视网膜新生血管的治疗提供新的途径。方法体外培养人CNE-2Z,并分成正常氧状态培养组(20%)和低氧(5%)培养组。采用脂质体(LF2000)将VEGFsiRNA转染两组细胞,并采用免疫细胞化学和ELISA法检测鼻咽癌细胞在正常氧状态和低氧状态下VEGF蛋白质表达的差异。结果正常氧状态下培养的CNE-2Z细胞有VEGF165及VEGF121蛋白质的表达,主要见于细胞浆中(棕色颗粒),低氧状态下VEGF的表达增多。ELISA结果显示,正常氧状态下未转染组、转染空载体组、转染VEGFsiRNA组CNE-2Z细胞培养上清液中VEGF165的含量分别为:419.72pg/ml、410.61 pg/ml、320.76 pg/ml;低氧状态下VEGF165的含量分别为:805.61 pg/ml、798.81 pg/ml、496.56 pg/ml,转染VEGFsiRNA组VEGF的表达下调。结论VEGFsiRNA有效地、特异地抑制CNE-2Z细胞VEGF蛋白质的表达,有望成为防治视网膜血管新生性病变的一种有效方法。

Objective To investigate the inhibitory effect of a small interfeting RNA targeting the vascular endothelial growth factor （VEGF） in the nasopharyngeal carcinoma cell line （CNF-2Z） in vitro, and to explore a new approach to gene-therapy for retinal revascularization. Methods Nasopharyngeal carcinema cells were cultured in vitro and divided into a normoxia group （20%） and a hypoxia group （5%）. After VEGF siRNA was transfected into CNE-2Z by Lipofectamine 2000, VEGF expression was tested with immunohistochemical staining and ELISA. Results VEGF165 and VEGF121 proteins were expressed in the. cytoplasm of CNE-2Z cells under normoxia （brown particle） and the expression increased under hypoxia. Under normoxia, the expressions of secreting VEGF165 shown by ELISA in the non-transfection group, the pSilencer2. 1-U6 hygro transfection group and the VEGF siRNA transfection group were 419.72 pg/ml,410.61 pg/ml and 320.76 pg/ml, respeclively. Under hypoxia, the expressions of secreting VEGF165 protein in above three groups were 805.61 pg/ml,798.81 pg/ml and 496.56 pg/ml, respectively. VEGF siRNA down-regulated the expression of VEGF165. Conclusion VEGF specific siRNA can effectively down-regulate the expression of VEGF protein in CNE-2Z cells in vitro, thus it may be a potential therapeutic approach in retinal vascularization.