RNA干扰抑制端粒酶反转录酶对晶状体上皮细胞增殖的影响

Impact of telomerase reverse transcriptase RNA interference on the proliferation of human lens epithelial cells

目的探讨RNA干扰(RNAi)抑制端粒酶反转录酶(TERT)对晶状体上皮细胞(LEC)增殖的影响。方法收集培养的LEC和视网膜色素上皮细胞(RPEC),用半定量反转录聚合酶链反应(RT-PCR)检测二者内源性TERT的表达差异;人工合成TERT小分子干扰RNA(siRNA)片段,用LipofectamineTM2000转染LEC,实时PCR检测TERT基因表达的变化。LipofectamineTM2000TERTsiRNAi持续转染LEC和RPEC96h,细胞计数试剂盒8(CCK-8)检测TERTsiRNAi对LEC和RPEC增殖的影响。结果 RPEC中未见TERT表达,LEC中有低水平TERT表达。TERTsiRNA转染LEC48h后,siRNA组TERT相对表达水平(0.20±0.01)与阴性对照组(1.00±0.03)相比,差异有统计学意义(P<0.05)。TERTsiRNA转染LEC和RPEC96h后,CCK-8试剂盒检测细胞的增殖抑制情况显示:LECsiRNA组LEC活性(0.55±0.04)与LEC阴性对照组(0.79±0.02)相比,差异有统计学意义(P<0.05);转染的RPECsiRNA组RPEC活性(1.13±0.04)与阴性对照组RPEC活性(1.16±0.01)相比,差异无统计学意义(P>0.05)。结论较长时间RNAiTERT能有效抑制LEC增殖,但对RPEC无明显抑制作用。

Objective To explore the effect of RNA interference （RNAi） targeting telomerase reverse transcriptase（TERT） on the proliferation of lens epithelial cells （LEC）. Methods TERT expression of LEC and retinal pigment epithelial cell （RPEC） was evaluated using semi-quantitative reverse transcriptional polymerase chain reaction （RT-PCR）. LEC was transfected with artificially synthesized TERT small interfering RNA （siRNA） by lipofectamineTM 2000 and the TERT gene expression change of LEC was detected with real-time PCR. LEC and RPEC were transfected continually with TERT siRNA by lipofectamineTM 2000 for 96 hours and the cellular activity of LEC and RPEC was detected by using cell counting kit-8. Results There was low expression of TERT in LEC but non-existing in RPEC. Com- pared with the LEC negative control group（ 1.00 ±0.03 ）, the expression of TERT in LEC siRNA group （ 0.20 ± 0.01 ） was lower after being transfected with TERT siRNA for 48 hours （ P 〈 0.05 ）. There was significant difference in the LEC viability between LEC siRNA group and LEC negative control group after being transfected with TERT siRNA for 96 hours （P 〈 0. 05）. However, no significant difference was observed in the RPEC viability between RPEC siRNA group and RPEC negative control group （P 〉 0.05 ）. Conclusions Long-time RNAi targeting TERT could inhibit the proliferation of LEC but not the RPEC proliferation.