Ad-PTEN增强LY294002对人胶质瘤细胞系U251的生长抑制作用

Ad -PTEN sensitizes inhibition effect of U251 cell lines to LY294002

目的在体外实验中，利用携带野生型PTEN基因的重组腺病毒（Ad—PTEN）和P13K的小分子抑制剂LY294002联合抑制P13K／AKT信号通路，观察两者联合对人胶质瘤细胞系U251生长有无正向协同作用及探讨产生这种作用的机制。方法15251细胞按处理方式不同分为4组：DMSO对照组、空载病毒对照组、LY294002组和联合组（LY294002＋Ad—PTEN组）。在U251细胞系中导入LY294002并转染Ad—PTEN病毒后，提取总蛋白，用Westernblotting检测PTEN表达状态及P13K、AKT表达情况；用MTT法检测Ad—PTEN和LY294002对U251生长抑制率、流式细胞仪检测细胞周期、AnnexinV法检测细胞凋亡，观察导人LY294002和转染Ad—PTEN后U251增殖能力的改变；用划痕实验、Transwell实验观察LY294002及Ad—PTEN对U251迁移和侵袭能力的影响；Westernblotting检测P13K／AKT信号通路下游相关蛋白表达水平变化。结果Westernblotting显示：与DMSO组和空载病毒组相比，LY294002组P13K、AKT蛋白表达水平明显降低，联合组（LY294002＋Ad—PTEN）的WEN蛋白明显上调，而P13K、AKT蛋白下降水平比LY294002组更为显著。联合组与LY294002组、空载病毒组、DMSO组相比细胞周期阻滞在G0／G1期；联合组凋亡率比其他三组明显增加；自培养第2天起，联合组细胞增殖速率呈下降趋势。联合组侵袭和迁移细胞的相对数少于LY294002组、空载病毒组和DMSO组。Westernblotting结果证明位于P13K／AKT下游的MMP-2、MMP-9、PCNA、Bcl-2、CyclinD1、NF—KB、FAK蛋白表达水平显著下调。结论小分子抑制剂LY294002能够有效抑制U251中P13K、AKT蛋白的表达，联合转染Ad—PTEN后不仅能提高PTEN蛋白的表达水平，对P13K、AKT的抑制更为显著。在体外实验中，联合Ad—PTEN和LY294002能够有效地通过阻滞细胞周期、减少细胞凋亡来抑制U251的增殖能力，并能够抑制细胞的侵袭和迁移能力，两者联合使用具有正向协同作用。联合Ad—PTEN和LY294002对U251的生长与侵袭的抑制作用与P13K／AKT下游的MMP-2、MMP-9、PCNA、Bcl-2、CyclinD1、NF—KB、FAK蛋白表达水平下调有关。联合腺病毒转染技术和小分子抑制剂调控P13K／AKT信号通路是治疗胶质瘤的新途径。

Objective To investigate the role of PI3K/AKT pathway on growth and invasion effect in human glioma cells U251 by Ad - PTEN and phosphatidylino - sitol 3 - kinase inhibitor LY294002. Methods Western blotting analysis was used to detect the PTEN ,PI3K and AKT protein expression ＇after LY294002 and Ad -PTEN were transfected into glioma cell lines U251. Flow cytometry, Annexin V/FITC methods, MTT methods were used to detect the proliferative activitity of the U251 cells. The invasion of U251 cells in vitro was examined by Scrath assay and Transwell cell invasion test. The difference between the combined therapy group and the single LY294002 therapy group was also examined. The downstream protein expression in PI3K/AKT signal pathway were also detected by Western blotting. Results The protein expression of PTEN in combined group were significantly higher,while PI3K,AKT（ p -ser473 ）was significantly lower than that in the other three groups （P 〈 0.05 ）. After treating, the cells in combined group were inhibited at G0/G1 phase, and the cell number decreased at S phase. The apoptosis rate in combined group was higher than that in DMSO, Ad - nonsense and LY294002 group. As compared with DMSO, Ad - nonsense and LY294002 group, the cell multiplication rate in combined group presented decreasing trend since the second day in culture（ P 〈 0.05 ）. The invasion and migration cells in combined group was lower than that in other three groups. The protein expression of PCNA ,CyclinD1 ,Bcl - 2 ,NF - KB, MMP - 2, MMP - 9, and FAK was significantly lower than that in the other three groups （ P 〈 0.05 ）. Conclusion The combined therapy of Ad - PTEN and LY294002 plays a synergistic role in inhibiting the proliferation and invasion of U251 cells.