Postn基因沉默抑制缺氧环境下咽癌Fadu细胞间质化和细胞迁移能力

发布时间：2018-03-26 18:11

本文选题：下咽肿瘤　切入点：细胞低氧　出处：《中华临床医师杂志(电子版)》2016年22期

【摘要】：目的观察缺氧环境下咽癌Fadu细胞间质化表型的改变与Postn蛋白表达的相关性。方法采用25μmol/L氯化钴(Co Cl2)化学缺氧方法缺氧24 h,模拟Fadu缺氧环境。采用免疫荧光法检测25μmol/L Co Cl2作用下,Postn蛋白的表达,观察上皮间质化(EMT)相关蛋白的表达变化。通过构建postn si RNA真核表达质粒,瞬时转染Fadu细胞,观察Postn沉默对Fadu间质化蛋白标记表达的影响,采用划痕实验观察Postn沉默对Fadu细胞迁移能力。结果免疫荧光结果显示,25μmol/L Co Cl2作用细胞24 h,Postn表达明显高于正常培养组,Vimentin、Snail和Twist蛋白表达也明显高于对照组,表明低氧环境可促进Fadu细胞增殖和提高细胞间质化程度。采用Postn si RNA真核表达载体转染Fadu细胞,免疫荧光和实时定量PCR结果显示,构建的si RNA表达载体的基因沉默效率为75.43%,转染阳性的Fadu细胞Vimentin。划痕实验证实,Postn基因干扰转染阳性组,细胞迁移率与转染Control质粒的对照组相比明显降低(P0.01)。结论 Postn蛋白高表达与Fadu细胞低氧环境下的细胞增殖和间质化有关,沉默Postn基因能够明显抑制Fadu细胞的间质化和迁移浸润能力。
[Abstract]:Objective to observe the correlation between the interstitial phenotype of pharyngeal carcinoma Fadu cells and the expression of Postn protein under anoxic environment. Methods 25 渭 mol/L Cobalt Chloride Co Cl2) chemical anoxic method was used to anoxic for 24 h and simulated Fadu anoxic environment. Immunofluorescence assay was used to detect the expression of Postn protein. Expression of Postn protein in the presence of 25 渭 mol/L Co Cl2. To observe the expression of EMT-related proteins in epithelial mesenchymal cells, the eukaryotic expression plasmid of postn si RNA was constructed and transfected into Fadu cells, and the effect of Postn silencing on the expression of Fadu interstitial protein markers was observed. The effect of Postn silencing on the migration of Fadu cells was observed by scratch test. Results Immunofluorescence results showed that the expression of Postn and Twist protein was significantly higher than that in normal culture group after treated with 25 渭 mol/L Co Cl2 for 24 h. The results showed that hypoxic environment could promote the proliferation of Fadu cells and increase the degree of interstitial cells. Postn si RNA eukaryotic expression vector was used to transfect Fadu cells. The results of immunofluorescence and real-time quantitative PCR showed that, The gene silencing efficiency of the constructed si RNA expression vector was 75.43. The positive Fadu cells were confirmed by scratch test. Conclusion the high expression of Postn protein is related to the proliferation and interstitial of Fadu cells in hypoxic environment. Silencing Postn gene can significantly inhibit the interstitial and migration ability of Fadu cells.
【作者单位】：吉林大学第一医院耳鼻咽喉头颈外科;
【基金】：吉林省卫计委科技项目(2009Z021)
【分类号】：R739.63

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