RNA干扰端粒酶活性对HeLa细胞生物学行为的影响  

Inhibition of malignant phenotype of HeLa cells by RNA interference of the telomerase activity

作　　者：石英爱[1] 赵强[2] 张丽红[1] 王光兰[1] 于晓霞[1] 李玉林[1] 吴珊[1] 

机构地区：[1]吉林大学病理生物学教育部重点实验室,长春130021 [2]吉林大学第一临床医院

出　　处：《中华病理学杂志》2010年第3期187-191,共5页Chinese Journal of Pathology

基　　金：基金项目：教育部高校优秀青年教师奖励计划（2000134）;吉林省科技发展计划项目（200705369）;吉林省卫生厅重点实验室科研项目

摘　　要：目的观察RNA干扰（RNAi）体外培养的HeLa细胞端粒酶催化亚单位（hTERT）对HeLa细胞生物学行为的影响，进一步探讨端粒酶活性与肿瘤细胞恶性生物学行为的相关性。方法体外转录法设计合成4条针对hTERT基因的shRNA，脂质体转染HeLa细胞后经免疫荧光染色和端粒重复序列扩增一酶联免疫法（TRAP—ELISA）测定端粒酶活性，筛选出端粒酶沉默效果最佳片段即B链shRNA。以B链shRNA转染HeI。a细胞为实验组，转染无关siRNA的HeLa细胞为对照组，显微镜下观察细胞在纤维黏连蛋白（FN）上的铺展；CCK一8细胞计数试剂盒检测细胞在FN上黏附；划痕实验评价细胞迁移；Boyden小室侵袭实验检测细胞侵袭能力。结果铺展实验显示细胞接种到FN上30min时，对照组铺展细胞的比率为（31．3±7．9）％，而实验组铺展细胞的比率仅为（5．6±2．3）％，两组差异有统计学意义（P〈0．01）；2h后对照组和实验组铺展细胞的比率分别为（79．4±4．8）％和（26．3±6．1）％，两组差异仍有统计学意义（P〈0．01）；24h后两组所有细胞几乎均呈铺展状态。细胞黏附实验显示细胞在FN上黏附30rain时对照组细胞黏附率为（83．7±5．4）％，而实验组的细胞黏附率为（67．2±2．8）％，明显低于对照组（P〈0．05）。划痕实验检测细胞的迁移能力显示实验组细胞24h迁移率为（27．1±6．2）％，明显低于对照组（58．7±15．0）％。Boyden小室侵袭实验显示在Matrigel胶上培养4h后，实验组和对照组侵袭的细胞数分别为75．7±14．5和165．1±11．0，差异有统计学意义（P〈0．05）。结论降低体外培养HeLa细胞的端粒酶活性使细胞的生物学特性发生改变，表现为减低了HeLa细胞的恶性生物学行为。Objective To investigate the biology of HeLa cells upon inhibition of human telomerase catalytic subunit （hTERT） gene by RNA interference in vitro. Methods Four shRNAs （A, B, C and D） targeting hTERT gene were designed and prepared by in-vitro transcription. The expression of hTERT gene was evaluated by immunofluorescent staining and telomeric repeat amplification protocol （TRAP） ELISA （TRAP-ELISA） , after transient transfection of shRNAs by lipid formulation. Through the initial selection, shRNA （B） was noticed as the most efficient one in down-regulating hTERT gene and therefore was chosen as the ultimate shRNA used in the experimental groups. Those transfected by non-silencing RNAi were chosen as the control groups. Cell spreading and migration were studied by microscopy and cell adhesion to fibronectin （FN） was assayed by cell counting kit-8 （CCK-8）. Cell invasion was assessed by Boyden chamber assay. Results Cell spreading study revealed that the rates of spreading cells in the experimental groups were （5.6 ± 2.3） % at 30 min, and （26. 3 ± 6. 1 ） % 2 h after the inoculation, respectively, whereas the rates of spreading cells in the control groups were （31.3 ±7.9）% and （79.4 ±4. 8）% , respectively. There were significant differences between the two groups（P 〈 0. 01 ）. However, most of the cells in bothgroups became spreading after 24 h. Cell adhesion assay demonstrated that the rate of adhesion cells on FN in experimental groups was （67. 2 ± 2. 8 ） %, less than that in control groups （ 83.7 ± 5.4 ） % （ P 〈 0. 05 ）. The relative migration distance was （27. 1 ± 6. 2） % in the experimental group, lower than that of the control group（58. 7 ± 15.0 ）%. The invasion assay revealed that the invading cells were 75.7± 14. 5 in the experimental group, in contrast to 165. 1 ± 11.0 in the control group after 4 h incubation on matrigel. The difference between these two groups was significant （P 〈 0. 05）. Conclusion In vitro shRNA silenc

关 键 词：HELA细胞 RNA 小分子干扰 催化域 

分 类 号：R730.2[医药卫生—肿瘤]