PI3K/Akt/mTOR信号通路抑制剂对体外培养血管瘤内皮细胞的影响  

Effects and mechanism of inhibitors of PI3K/Akt/mTOR signaling pathway on hemangioma vascular endothelial cell in vitro

作　　者：闰陶然 俞松[1] 李堂江[1] 徐艳朋[1] 赵兴[1] 苏凯[1] Yan Taoran;Yu Song;Li Tangjiang;Xu Yanpeng;Zhao Xing;Su Kai(Department of Pediatric Orthopedics,Affiliated Hospital,Zunyi Medical College,Zunyi 563003,China)

机构地区：[1]遵义医学院附属医院小儿矫形外科,563003

出　　处：《中华小儿外科杂志》2018年第11期835-840,共6页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金(81160330,81460476).

摘　　要：目的以PI3K/Akt/mTOR信号转导通路为靶点,观察PBK抑制剂LY294002和mTOR抑制剂RAD001对体外培养血管瘤内皮细胞增殖与凋亡的影响,并检测PI3K/Akt/mTOR信号轴中效应分子以探讨血管瘤消长的分子机制。方法采用组织块结合酶消化法培养血管瘤内皮细胞、传代培养,并用第Ⅷ凝血因子相关抗原对其鉴定。待细胞处于对数生长期,使用无血清培养孵育48h行同步化处理后,分别加入RAD001、LY294002及RAD001+LY294002作为干预组,加入完全内皮细胞培养基作为空白对照组。以上4组继续孵育24h后收集细胞,采用蛋白免疫印迹方法检测各组细胞p-Akt、p-mTOR、p-ERK及p-eIF4E蛋白的表达,流式细胞仪检测各组细胞的周期分布及凋亡率;分析p-Akt、p-mTOR、p-ERK及p-eIF4E蛋白的表达水平与血管瘤内皮细胞周期及凋亡变化的相关性。结果1.RAD001、LY294002及RAD001+LY294002干预24h后,内皮细胞凋亡率分别为(13.80±1.73)%、(15.03±1.05)%、(36.06±2.67)%,较对照组(3.90±0.2b)%有明显升高,差异具有统计学意义(￡=一9.82,一18.03,一20.82,P<O.01),且RAD001+LY294002组凋亡率显著高于对照组、LY294002组、RAD001组(t=一20.82,一12.12,一12.7,P<O:01);G0/G1期细胞比例分别为(74.29±1.59)%、(74.57±0.59)%、(82.28±3.01)%,较对照组(67.15±1.49)%有明显升高,差异具有统计学意义(t=一5.67,一8.02,一7.79,P<0.01),且RAD001+LY294002组高于对照组、LY294002组、RAD001组(t=一7.79,一4.35,一4.06,P<0.05)。2.蛋白免疫印迹检测显示,RAD001干预后,p-Akt、p-ERK、p-eIF4E蛋白表达分别为(O.75±0.04)、(0.54±0.08)、(1.50±0.34),较对照组(0.43±0.08)、(0.27±0.04)、(0.84±0.17)增加,差异具有统计学意义(t=一6.13,一5.49,一3.03,P<O.05);p-mTOR(O.40±0.04)比对照组(0.77±0.07)明显降低,差异具有统计学意义(t=7.91,P<0.01)。LY294002干预后,p-Akt、p-ERK、p-eW4E、p-耐r0R蛋白表达分别为(0.31±0.08)、(0.26±0.04)、(O.78±0.18)、(0.42±0.05),与对照组比较,蛋Objective To explore the effects of proliferation and apoptosis of hemangioma vascular endothelial cell after a pre-treatment of PI3K inhibitor LY294002and mTOR inhibitor RAD001 targeting the PI3K/Akt/mTOR signaling pathways and to elucidate the molecular mechanism of hemangioma succession through detecting the effector molecules of PI3K/Akt/mTOR pathway. Methods HemECs were detected by tissue culturing through inspecting the antigen of factor Ⅷ When cells reached the logarithmic phase,HemECs were cultured for 48h without serum and this process was intended for cellular synchronizatiorL HemECs were treated with RAD001,LY294002 and RAD001+LY294002as treatment group and cultured medium with serum regarded as control group.After 24h,through analyzing the relationships between cell apoptosis and protein level of p- Akt,p-mTOR,p-ERK and p-elF4E,the protein expressions of p-Akt,p-mTOR,p-ERK and p- eIF4E were examined by Western blot (WB).Flow cytometry (FCM)was used for examining cycling distribution and apoptotic index.Results After a 24h treatment of with RAED0I,LY294002and RAD001+LY294002,the indice of apoptosis cells from treatment group RAD001were (13.80± 1.73)%,LY294002(15.03±1.05)%,and RAD001+LY294002(36.06±2.67)%were higher than control group (3.90±0.20)%,and the differences were statistically significant (P<0.01). Furthermore,the apoptotic index of union group was higher than that of control group: LY294002 & RAD001group (P<0.01).After 1-day treatment of cells with RAD001,LY294002and RAD001+LY294002%the apoptotic indice in G0/Gi phase were RAD001(74.29±1.59)%, LY294002(74.57+0.59)% and RAD001+LY294002(82.28+3.01)%.And all were higher than that of control group (67.15±l.49)%and the differences were statistically significant (P<0.01). The union group RAD001+LY294002 was higher than other unique groups (P<0.05).By WB,the expressions of protein p-Akt (0.75±0.04),p-ERK (0.54±0.08)and p-eIF4E (1.50±0.34)after a 24h pretreatment of RAD001 was higher than control group p-Akt F(0.43±0.08),p-ERK (0.27± 0.04

关 键 词：血管瘤 细胞 培养的 PI3K/Akt/mTOR信号通路 PBK抑制剂 MTOR抑制剂 

分 类 号：R979.5[医药卫生—药品]