CAJ | 학술논문

目的观察反义寡核苷酸(ASODN)、RNA干扰(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48 h,72 h即恢复.筛选两条链构建质粒载体,转染后P-1组作用48 h后hTERT mRNA为0.39±0.13,72 h为0.57±0.32,P-2组48 h为0.55±0.20,72 h为0.88±0.23,端粒酶相对活性P-1组48 h为0.42±0.07,72 h为0.31±0.08,P-2组48 h为0.49±0.27,72 h为0.39±0.03.siRNA的最佳作用浓度为:100μmol/L.反义寡核苷酸以0.6 μmol/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24 h为0.52±0.002,48 h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.
목적 관찰반의과핵감산(ASODN)、RNA간우(RNAi)량충방법대단립매적억제효응.방법 설계합성과핵감산급siRNA련,량자균재지질체개도하전염K562세포,RNA간우채용직접전염후사선고효련,질립재체구건,재전염후분석효과.결과 합성3조siRNA련,직접전염후,검측hTERTmRNA표체,발현제일、제이조련유효,제삼조련무효,인단립매반전록매(hTERT)mRNA표체억제부유지48 h,72 h즉회복.사선량조련구건질립재체,전염후P-1조작용48 h후hTERT mRNA위0.39±0.13,72 h위0.57±0.32,P-2조48 h위0.55±0.20,72 h위0.88±0.23,단립매상대활성P-1조48 h위0.42±0.07,72 h위0.31±0.08,P-2조48 h위0.49±0.27,72 h위0.39±0.03.siRNA적최가작용농도위:100μmol/L.반의과핵감산이0.6 μmol/L농도조억제작용최가,24h후hTERTmRNA위0.42±0.16.48h위0.71±0.18.단립매상대활성24 h위0.52±0.002,48 h위0.482±0.018.결론 파향hTERT적반의과핵감산화RNA간우균가이억제hTERTmRNA표체,종이억제단립매활성,억제효과여파위점선택밀절상관.RNA간우효과우우반의과핵감산,전자불부표현위억제효솔고,차지속시간장.질립재체구건후적RNA간우우우화학합성후직접전염적RNA간우.
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-hTERT-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 h, 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 0.6 μ mol/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 h. 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.