cmyc脱氧核酶对白血病细胞端粒酶活性抑制作用.docVIP

cmyc脱氧核酶对白血病细胞端粒酶活性抑制作用 　 作者：杨展 ，赵家明 ，王旭光, 吴平，何惠娟，杨勤 【摘要】 目的： 探讨cmyc脱氧核酶的切割作用及对白血病细胞端粒酶活性的影响. 方法： 合成针对cmyc mRNA的“10～23”型脱氧核酶DzMycI及其类似物DzMycI提取总RNA在细胞外切割cmyc mRNA；转染白血病细胞系HL60后，用RTPCR扩增cmyc基因片段和端粒酶蛋白亚基hTERT的片段，用PCRELISA法测定端粒酶活性. 结果： 脱氧核酶DzMycI在细胞外和细胞内均能够有效地切割cmyc mRNA；在转染HL60后，DzMycI显著抑制白血病细胞生长(Plt;0.05)，下调端粒酶蛋白亚基hTERT的表达，显著降低HL60细胞端粒酶活性(Plt;0.05). DzMycI催化中心的一个碱基被替换后形成的脱氧寡核苷酸DzMycI在胞外和胞内均不表现对cmyc mRNA的切割作用. 结论： 脱氧核酶DzMycI确实能够高效特异地切割cmyc mRNA，降低白血病细胞端粒酶活性，抑制白血病细胞生长，有可能作为cmyc抑制剂用于白血病的基因治疗领域中. 【关键词】 催化性DNA；原癌基因蛋白质cmyc；端粒酶；白血病 　　【Abstract】AIM： To explore the cleavage of cmyc mRNA by a DNAzyme and its effect on telomerase activity in a human leukemia cell line HL60.METHODS： A “1023” DNAzyme(DzMycI) targeted against cmyc mRNA and its analogues(DzMycI) were synthesized and used to cleave cmyc mRNA.The cleaving efficiency and the expression of human telomerase reverse transcriptase(hTERT) was assessed by RTPCR and telomerase activity was assayed by PCRELISA. RESULTS： DzMycI exhibited effective cleaving ability and after transfection into HL60 cells, downregulated the hTERT expression and telomerase activity(Plt;0.01), and inhibited the growth of the cells(Plt;0.05). DzMycI, which was derived from DzMycI with a base displacement in the conserved catalytic motif, did not exhibit notable cleaving effect on cmyc mRNA either outside the cells or in cells. CONCLUSION： The synthesized DNAzyme DzMycI can cleave cmyc mRNA with effectiveness and specificity, decrease the telomerase activity and induce the apoptosis of HL60 cells. As newly found cmyc inhibitors, DNAzymes will probably play an important role in the gene therapy of leukemia. 　　【Keywords】 DNA, catalytic; cmyc; telomerase; leukemia 　　0 引言 　　脱氧核酶(deoxyribozyme，DNAzyme)是具有酶活性的单链小DNA分子，能够特异切割靶RNA分子［1］. cmyc基因的异常扩增和表达、端粒酶活性的升高与许多恶性肿瘤的发生有关，抑制这两个基因的表达能够促进恶性肿瘤的凋亡［2-6］. 本文通过合成针对人cmyc基因的脱氧核酶，在白血病细胞内和细胞外切割cmyc mRNA，并观察其对细胞生长和端粒酶活性的影响，研究利用脱氧核酶抑制cmyc表达来进行白血病基因治疗的可能性. 　　1 材料和方法 　　1.1 材料 　　1.1.1