摘要目的 观察磷脂酰肌醇-3激酶(P13K)/AKT信号转导通路特异性抑制剂LY294002对人官颈癌细胞株(HeLa细胞)端粒酶活性的影响,同时观察其对细胞生物学行为的改变,探讨肿瘤细胞内端粒酶活性可能的调节机制.方法 ccK.8法测定LY294002作用于HeLa细胞的Ic50值;Western blot检测总AKT以及磷酸化AKT(P-AKT);端粒重复序列扩增-酶联免疫吸附测定法(TRAP-ELISA)测定细胞的端粒酶活性;生长曲线、流式细胞术和Hoechst33258染色分别检测细胞增殖、细胞周期和细胞的凋亡;最后用划痕实验检测细胞的迁移能力.结果 LY294002作用于HeLa细胞的Ic50值为1.73 mg/L;Western blot结果显示LY294002作用使细胞在总AKT蛋白表达相同的情况下明显抑制了P-AKT,此时细胞内端粒酶活性由对照组的98.61%显著降低至36.72%.LY294002降低了细胞内端粒酶活性同时,使细胞的生长增殖能力明显降低,处于Go/G.期(静止期)的细胞由对照组的47.36%增加到实验组的66.88%,凋亡细胞的比例则由2.4%增加至14.9%,同时细胞的运动迁移明显降低,迁移距离由对照组62.57%明显降低为24.6%.结论 LY294002抑制P13K/AKT信号转导通路能明显改变HeLa细胞内端粒酶活性,同时引起细胞生物学行为的改变,其细胞内端粒酶活性的调节可能与P13K/AKT信号转导通路密切相关.

abstractsObjective To investigate the telomerase activity and to document biological behaviors of HeLa cells upon treatment with specific P13K/AKT signaling pathway inhibitor. LY294002. Methods CCK-8 assay Was used to determine IC50 of LY94002. The expressions of total AKT and phosphorylation AKT(P-AKT)were determined using Western blot. Telomerase activity of cell was measured by TRAPEUSA assay. Cell growth curve. flow cytometry technique and Hoeehst33258 stain were used to evaluate the cell growth, cell cycle and apoptosis respectively. Cell migration Was determined by cell wound healing assay. Results ICm valne of LY294002 of treated HeLa cells Was 1. 73 mg/L. Western blot showed that LY294002 enabled to decrease P. AKT activitv in the presence of same total AKT protein. The cell telomerase activity Was decreased to 36. 72%jn contrast to 98. 61%of the contr01. LY294002 decreased the telomerase activity in HeLa cells. and tlle growth capacity of the cells Was significantly suppressed. The number of cells at Go/Gl phases increased to 66. 88%compared with that of the control cells(47. 36%). The apoptosis rate alSO increased from 2. 4%to 14. 9%. The relative migration distance decreased to 24. 6%compared with that of control(62. 57%). Conclusion LY294002 inhibition of P13K/AKT signaling pathway leads to alteration of telomerase activity along with changes of the biological behaviors of the HeLa cells suggesting that regulation of telomerase activity may be closely related to P13 K/AKT signaling pmhway.