| 杨艳1,2 吕国军1 郭昕1 张德蒙1,2 于炜婷1 张英1 刘袖洞3△ 马小军1△.PMA 及乏氧诱导VEGF 上调的细胞模型构建及用于RNAi 研究[J].,2012,12(2):265-269 |
| PMA 及乏氧诱导VEGF 上调的细胞模型构建及用于RNAi 研究 |
| Construction of VEGF Up-regulated Cells Induced by PMA and Hypoxiafor RNAi |
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| DOI： |
| 中文关键词: 小鼠黑色素瘤细胞 血管内皮细胞生长因子 PMA 诱导 乏氧诱导 RNAi 模型 |
| 英文关键词: Mouse melanoma cell VEGF PMA-stimulated Hypoxia-induced RNAi model |
| 基金项目:国家自然科学基金(20876018，20736006)，教育部留学回国人员科研启动基金，
中国科学院知识创新工程重要方向项目(KJCX2.YW.M02，KJCX2-YW-210-02) |
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| 中文摘要: |
| 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%。 |
| 英文摘要: |
| 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 chitosan/
siRNA nanoparticles showed preliminary result of 40% with the established B16-F10 cell model. |
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