CAJ | 학술논문

目的研究利用脱氧核酶抑制Bcl-2表达对人肝癌BEL-7402细胞凋亡的影响.方法合成针对Bcl-2基因的“10～23”型脱氧核酶及其类似物；转染入肝癌细胞；RT-PCR检测脱氧核酶细胞内Bcl-2mRNA的切割作用；荧光免疫方法测定脱氧核酶对Bcl-2蛋白表达的影响；流式细胞仪检测脱氧核酶对肝癌细胞凋亡的影响.结果 “10 ～ 23”型脱氧核酶及其类似物成功转染入肝癌；非修饰脱氧核酶(DzT)和修饰的脱氧核酶(DzTi)在胞内能有效地切割Bcl-2 mRNA,DzTi比DzT的切割活性显著；荧光免疫法测的DzT和DzTi能显著地下调细胞内Bcl-2蛋白水平(P＜0.01),抑制肝癌细胞的生长(P＜0.05).流式细胞术结果提示,DzT和DzTi细胞凋亡率明显升高出现凋亡峰,与对照组细胞及脱氧寡核苷酸DzT和 DzTi凋亡率差异有统计学意义(P ＜0.05)；DzT和DzTi组细胞出现细胞周期阻滞,表现为G0/G1细胞所占比例上升,S期细胞所占比例下降.结论脱氧核酶可以有效切割Bcl-2 mRNA,抑制Bcl-2蛋白表达和促进肝癌细胞凋亡.
목적 연구이용탈양핵매억제Bcl-2표체대인간암BEL-7402세포조망적영향.방법 합성침대Bcl-2기인적“10～23”형탈양핵매급기유사물；전염입간암세포；RT-PCR검측탈양핵매세포내Bcl-2mRNA적절할작용；형광면역방법측정탈양핵매대Bcl-2단백표체적영향；류식세포의검측탈양핵매대간암세포조망적영향.결과 “10 ～ 23”형탈양핵매급기유사물성공전염입간암；비수식탈양핵매(DzT)화수식적탈양핵매(DzTi)재포내능유효지절할Bcl-2 mRNA,DzTi비DzT적절할활성현저；형광면역법측적DzT화DzTi능현저지하조세포내Bcl-2단백수평(P＜0.01),억제간암세포적생장(P＜0.05).류식세포술결과제시,DzT화DzTi세포조망솔명현승고출현조망봉,여대조조세포급탈양과핵감산DzT화 DzTi조망솔차이유통계학의의(P ＜0.05)；DzT화DzTi조세포출현세포주기조체,표현위G0/G1세포소점비례상승,S기세포소점비례하강.결론 탈양핵매가이유효절할Bcl-2 mRNA,억제Bcl-2단백표체화촉진간암세포조망.
Objective To study the effects of cleavage of Bcl-2 by two DNAzymes on apoptosis of human hepatoma cell line (HepZ1).Methods Two “10-23” DNAzymes(DzT and DzTi) targeting Bcl-2 mRNA and their analogues(DzT' and DzTi') were synthesized and used to cleave Bcl-2 mRNA in vitro and in BEL-7402 cells.The RT-PCR was performed to assess the cleaving efficiency.Expression of Bcl-2 protein was determined by immunofluorescent method.Cell apoptosis was detected by flow cytometry.Results The unmodified Enzymes DzT,and its modified form DzTi,which had an added 3'-inverted thymidine,could effectively cleave Bcl-2 mRNA in vitro.After transfected into BEL-7402 cells,DzTi exhibited more powerful cleaving ability than DzT,significantly down-regulated the level of Bcl-2 protein(P ＜0.01) and inhibited the cell growth(P ＜0.05).The results of flow cytometry suggested that the apoptosis rate of DzT and DzTi significantly increased,appeared apoptotic peak.Cell cycle was delayed in DzT and DzTi group,proportion of cells in G0/G1 increased,S phase cells decreased.Conclusion The synthesized DNAzymes could effectively cleave Bcl-2 mRNA,decrease the level of Bcl-2 protein and induce hepatoma cells apoptosis.