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-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HeLa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-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30 min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);2 h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);24 h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30 min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24 h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4 h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.

abstractsObjective To investigate the biology of HeLa cells upon inhibition of human telomerase catalytic subuait (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 both groups 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 silencing of hTERT gene can down-regulate the telomerase activity, leading to an inhibition of the malignant phenotype of HeLa cells, including decreased ability of cell spreading and adhesion, reduction of cell migration, and declined invasive ability through Matrige assay.