反义寡核苷酸及RNA干扰靶向人类端粒酶反转录酶的效果比较  

Comparison of telomerase inhibition by antisense-oligodeoxynucleotide and RNAi

作　　者：方美云[1] 李梅凤[1] 应晓阳[1] 王一[1] 王业伟[1] 

机构地区：[1]大连医科大学附属第一医院血液科,116011

出　　处：《白血病．淋巴瘤》2010年第4期196-199,206,共5页Journal of Leukemia & Lymphoma

基　　金：辽宁省自然科学基金资助（0052173）

摘　　要：目的观察反义寡核苷酸（ASODN）、RNAf扰（RNAi）两种方法对端粒酶的抑制效应。方法设计合成寡核苷酸及siRNA链，两者均在脂质体介导下转染K562细胞，RNA干扰采用直接转染后筛选高效链，质粒载体构建，再转染后分析效果。结果合成3条siRNA链，直接转染后，检测hTERTmRNA表达，发现第一、第二条链有效，第三条链无效，人端粒酶反转录酶（hTERT）mRNA表达抑制仅维持48h，72h即恢复。筛选两条链构建质粒载体，转染后P．1组作用48h后hTERTmRNA为0．39±0．13，72h为0．57±0．32，P-2组48h为0．554-0．20，72h为0．884-0．23，端粒酶相对活性P-1组48h为0．424-0．07，72h为0．314-0．08，P-2组48h为0．49±0．27，72h为0．394-0．03。siRNA的最佳作用浓度为：100μmol／L。反义寡核苷酸以0．6μmol／L浓度组抑制作用最佳，24h后hTERTmRNA为0．42±0．16。48h为0．71±0．18。端粒酶相对活性24h为0．52±0．002，48h为0．482±0．018。结论靶向hTERT的反义寡核苷酸和RNA干扰均可以抑制hTERTmRNA表达，从而抑制端粒酶活性，抑制效果与靶位点选择密切相关。RNA干扰效果优于反义寡核苷酸，前者不仅表现为抑制效率高，且持续时间长。质粒载体构建后的RNA干扰优于化学合成后直接转染的RNA干扰。Objective To select an efficient method to inhibit telomerase activity, antisenseoligodeoxynucleotide and plasmid-vector mediated RNAi against hTERT were used to inhibit telomerase activity. The inhibiting effects of the two methods were compared. Methods Against hTERT mRNA, siRNA and oligodeoxynucleotide were designed and transfected into K562 cells by liposome. Effective and specific siRNA strands were selected and then plasmid was constructed and transfected into K562 cells; followed by analysis of the results. Results hTERT mRNA were detected after the three chemo-synthesized strands were transfected. It was found that si-bTERT-1 and si-hTERT-2 were effective, but si-hTERT-3 had no effect. The inhibiting effect of hTERT mRNA lasted only 48 h and disappeared at 72 h. Two siRNA strands were sieved and plasmids were constructed and transfected into K562 cells. In the P-1 group, hTERT mRNA was 0.39±0.13 at 48 b, 0.57±0.32 at 72 h. In the P-2 group, hTERTmRNA was 0.55±0.20 at 48 h, 0.88±0.23 at 72 h. In the P-1 group, the relative telomerase activity was 0.42±0.07 at 48 h, 0.31±0.08 at 72 h. In the P-2 group was 0.49±0.27 at 48 h, 0.39±0.03 at 72 h. The best concentration of siRNA was 100 μmol /L. The best concentration of ASODN was 0.6μmol/L. hTERTmRNA was 0.42±0.16 at 24 h, 0.71±0.18 at 48 h. Relative telomerase activity was 0.52±0.002 at 24 h, 0.482±0.018 at 48 b. Conclusion Both ASODN and RNAi targeting hTERT can inhibit the expression of hTERT mRNA, and then inhibit telomerase activity. The inhibiting effect is closely relative to the targeting site. The inhibiting effect of RNAi is better than that of ASODN. RNAi has better efficiency and lasts for a longer time. Plasmid mediated RNAi has better inhibiting effect than the chemo-synthesized siRNA.

关 键 词：端粒酶 反转录酶 RNAI 反义寡核苷酸 

分 类 号：R730.2[医药卫生—肿瘤]