2-（4-吗啉基）-8-苯基-4氢-1-苯并吡喃-4酮改变替莫唑胺耐药细胞系耐药特性的体外研究  

Study on mechanism of the resistant characteristic of temozolomide-resistant glioma cell line changed by LY294002 in vitro

作　　者：潘强[1] 朱琳[2] 刁兴涛[1] 宋纯玉[1] 刘秀云 刘振瑞 岳晓[4] 

机构地区：[1]泰山医学院附属莱芜医院神经外科,山东莱芜271199 [2]泰山医学院附属莱芜医院ICU,山东莱芜271199 [3]山东省昌乐县人民医院神经内科,262499 [4]天津市环湖医院神经外科天津市神经外科研究所,300060

出　　处：《中华实验外科杂志》2016年第9期2096-2099,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（81201973）

摘　　要：目的 观察应用2-（4-吗啉基）-8-苯基-4氢-1-苯并吡喃-4酮（LY294002）对替莫唑胺耐药胶质瘤细胞株U251/TR细胞增殖、侵袭及耐药性影响,并探讨其机制.方法 应用LY294002作用于细胞株U251/TR细胞,通过噻唑蓝（MTT）比色法检测细胞存活率,Transwell法检测细胞侵袭,流式细胞仪检测细胞周期,膜联蛋白V（Annexin V）法及原位末端标记（TUNEL）法检测细胞凋亡.Western blot法检测磷酸化丝苏氨酸蛋白激酶（p-Akt）、基质金属蛋白酶-2（MMP-2）、核因子-κB（NF-κB）、B淋巴细胞瘤/白血病-2（bcl-2）、半胱氨酰天冬氨酸特异性蛋白酶-3（Caspase-3）、细胞周期素D1（Cyclin D1）等磷脂酰肌醇3-激酶/丝苏氨酸蛋白激酶（PI3 K/Akt）信号通路相关蛋白及O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）蛋白的表达.结果 LY294002组与空白对照组和二甲基亚砜（DMSO）组相比自培养第2天起细胞生存率明显下降（P＜0.05）.LY294002组Transwell穿过细胞数[（43.60±1.54）个]较空白对照组[（81.90±1.64）个]、DMSO组[（82.10±1.07）个]明显减少（P＜0.05）.LY294002组细胞周期被明显阻滞在G0/G1期（67.44％）,S期（30.56％）明显减少（P＜0.05）,细胞凋亡率明显增加（36.05±1.99）％ （P ＜0.05）.Western blot结果显示LY294002组能有效抑制p-Akt、MMP-2、MGMT、NF-κB、bcl-2及Cyclin D1蛋白的表达,而上调Caspase-3表达（P＜0.05）.结论 靶向PI3K/Akt信号通路的LY294002显著抑制了U251/TR细胞的增殖、侵袭,并促进细胞的凋亡,其机制可能与LY294002下调了PI3K/Akt信号通路中相关蛋白及MGMT蛋白的表达有关。Objective To observe the inhibition effects and mechanism of phosphatidylinositol 3 kinase （PI3K） inhibitor LY294002 on the PI3K/serine threonine kinases （Akt） signal pathway,cell prohfeation,cell invasion and resistant characteristic in human temozolomide （TMZ）-resistant glioma cell line U251/TR cells.Methods U251/TR cells were treated with LY294002.The cell proliferation was detected by methyl thiazol tetrazolium （MTT） methods,the cell invasion was measured by Transwell assay,the cell cycle was observed by flow cytometry,and the apoptosis was detected by Annexin V and TdT-mediated dUTP-biotin nick end labeling （TUNEL）.The expression of proteins including p-protein kinase B （p-Akt）,matrix metalloproteinase-2 （MMP-2）,nuclear factor kappa B （NF-κB）,B cell lymphoma/lewkmia-2 （bcl-2）,Caspase-3,Cyclin D1 and 06-Methylguanine-DNA methyltransferase （MGMT） were dected by Western blotting.Results Compared with U251/TR cells in the dimethyl sulphoxide （DMSO） group and control group,proliferative activity was suppressed in the cells of LY294002 group starting on the second day,with an obvious decrease in viability （P 〈 0.05）.The results of the Transwell assay showed that the number of cells travelling through the matrigel were obvious reduction of LY294002 group [（43.60 ± 1.54） cells],which was statistically significant （P 〈 0.05）.The flow cytometry results displayed that there was G0/G1 arrest in the LY294002 group （67.44%）.The percentage of apoptotic cells was increased in LY294002 group （36.05 ± 1.99） %,（P 〈 0.05）.LY294002 can significantly downregulate the proteins expression of p-Akt,MMP-2,MGMT,NF-κB,bcl-2 and Cylin D1,While Caspase-3 was significantly higher （P 〈 0.05）.Conclusion LY294002 can inhibit proliferation and invasion of U251/TR cells by arresting cycle and promoting the apoptosis rate.The mechanism may be related to down-regulation of the PI3K/Akt downstream proteins and MGMT expression.

关 键 词：磷脂酰肌醇3-激酶抑制剂 脑肿瘤 神经胶质瘤 抗药性 肿瘤 磷脂酰肌醇3-激酶/丝苏氨酸蛋白激酶信号通路 

分 类 号：R735.2[医药卫生—肿瘤]