端粒酶反义hTERT的构建及其对K562细胞的作用  

Construction of antisense telomerase hTERT and its effect on K562 cells

作　　者：孟秀香[1] 苏本利[2] 贾莉[1] 孙宏丹[1] 张卓然[1] 

机构地区：[1]大连医科大学检验医学院,116027 [2]大连医科大学附属第二医院

出　　处：《中华血液学杂志》2003年第5期245-248,共4页Chinese Journal of Hematology

摘　　要：目的　观察反义人类端粒酶逆转录酶 (humantelomerasereversetranscriptase ,hTERT)是否能够抑制端粒酶活性及K5 6 2细胞的增殖。方法　选择hTERT基因上游部分的起始密码子上下 2 90bp长度的核苷酸片段 ,设计引物并进行PCR扩增后 ,反向连接于pLNCX neo质粒上得到反义hTERT基因表达质粒。在体外转染中通过脂质体法将质粒导入K5 6 2细胞。以MTT法和体外集落形成实验检测反义hTERT表达质粒对K5 6 2细胞增殖的抑制作用 ;以TRAP PCRELISA法来研究反义hTERT基因对K5 6 2细胞的端粒酶活性的抑制作用。结果　①转染反义hTERT表达质粒组与对照组 (空白对照及空质粒组 )相比较 ,生长速度明显变慢 ,集落形成能力明显下降 ,空白对照组K5 6 2细胞集落数为 ( 10 0 .33± 7.5 7) / 10 3 细胞 ,空质粒组K5 6 2neo细胞集落数为 ( 92 .6 7± 5 .86 ) / 10 3 细胞 ,转染反义质粒组HL 6 0as集落数为 ( 5 0 .33± 6 .11) / 10 3 细胞 (P <0 .0 1) ;②反义hTERT对K5 6 2细胞的端粒酶活性具有明显的抑制作用 ,K5 6 2neo细胞活性为 1.2 79± 0 .0 73 ,K5 6 2as为 0 .82 6± 0 .0 36 ,两者比较 ,差异有显著性 (P <0 0 1)。结论　该研究构建的端粒酶反义hTERT基因表达质粒转染到K5 6 2细胞中后 ,能够封闭K5 6 2细胞hTERTmRNA的表?Objectives To investigate whether antisense human telomerase reverse transcriptase (hTERT) could inhibit the activity of telomerase and the proliferation of K562 cells. Methods The antisense plasmid was constructed by reverse insertion of hTERT PCR product into plasmid pLNCX-neo. Then the constructed plasmid was introduced into K562 cells by lipofectactin-mediated DNA transfection. The inhibition effects of telomerase on the proliferation of K562 cells were analyzed by MTT and colony formation assay, the telomerase activity of K562 cells by TRAP-PCR ELISA methods. Results The growth rate of antisense hTERT transfected K562 cells was significantly lower than those of the controls,and the colony formation capacity of the transfected cells decreased significantly (P<0.01), the colony number is (100.33±7.57)/103 cells, (92.67±5.86)/103 cells and (50.33±6.11)/103 cells for control K562 cells, K562 neo cells and antisense hTERT transfected HL60 cells, respectively. The telomerase activity of antisense hTERT transfected K562 cells was significantly inhibited. Conclusion The expression of an antisense sequence to the mRNA sequence of telomerase protein subunit can inhibit the activity of telomerase, slow the cell growth and inhibit the capacity of colony formation of K562 cells.

关 键 词：白血病 K562细胞 细胞增殖 反义人类端粒酶逆转录酶 体外集落形成实验 

分 类 号：R733.7[医药卫生—肿瘤]