PMA 及乏氧诱导VEGF 上调的细胞模型构建及用于RNAi 研究

目的:探讨佛波酯(PMA)与乏氧诱导对小鼠黑色素瘤细胞B16-F10中血管内皮细胞生长因子(VEGF)表达量的影响,构建适合RNA干扰(RNAi)的体外细胞模型。方法:通过酶联免疫吸附试验(ELISA法)在蛋白质水平上检测细胞分泌的VEGF量,并用激光共聚焦显微镜观察小干扰RNA(siRNA)转染的细胞胞吞及细胞形态。结果:1 M PMA处理细胞2 h能明显上调B16-F10细胞中VEGF蛋白的合成及分泌,与常规培养相比,细胞可增加50%的VEGF水平。再经乏氧诱导48 h,稳定释放到培养液里的VEGF浓度大幅提高200%,范围在55-65 pg/mL/h。结论:经PMA和乏氧诱导后,B16-F10细胞稳定的VEGF分泌量与一定时间内分泌的稳定性均表明其适合作为RNAi的体外细胞模型。初步的RNAi结果表明,TKO/siRNA纳米粒与壳聚糖/siRNA纳米粒对于VEGF的沉默效率达40%。

Construction of VEGF Up-regulated Cells Induced by PMA and Hypoxia for RNAi

Objective: To investigate the expression of vascular endothelial growth factor(VEGF) in mouse melanoma cell B16-F10 induced by PMA and hypoxia so as to set up a stable in vitro model for RNA interference.Methods: Mouse VEGF immunoassay(ELISA kit) was utilized to determine the VEGF concentration secreted by B16-F10 cell.Confocal laser scanning microscopy was used for visualization of cellular uptake of siRNA loaded nanoparticles as well as cell morphology.Results: Stimulation of B16-F10 cells with 1 M PMA for 2 h,VEGF expression increased.Following hypoxia incubation for 48 h,the concentration of VEGF increased significantly 200%,ranging from 55 to 65 pg/mL/h in comparison with routine culture.Conclusions: Combining PMA stimulation and hypoxia culture,VEGF expression in B16-F10 cells was up-regulated and could keep a stable level in 48 h,which suggested that the induced B16-F10 cells were suitable for in vitro RNAi study.Furthermore,the silencing efficiency of VEGF by TKO/siRNA nanoparticles and chi-tosan/siRNA nanoparticles showed preliminary result of 40% with the established B16-F10 cell model.