反义hTERT对K562细胞端粒酶活性及细胞增生的影响

EFFECT OF TELOMERASE ANTISENSE hTERT ON TELOMERASE ACTIVITY AND PROLIFERATION OF K562 CELLS

目的 :观察反义 h TERT表达质粒转染 K5 6 2细胞后是否能够抑制端粒酶活性及K5 6 2细胞的增生。方法 :质粒的构建与转染 :将 2 90 bp h TERT c DNA片段 ,反向连接于 p L NCX- neo质粒上得到反义 h TERT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细胞中 ,以G4 18进行筛选得到阳性克隆 ;以 MTT法和形态学法检测反义 h TERT表达质粒对 K5 6 2细胞增生的抑制作用 ;以 TRAP- PCR EL ISA法来研究反义 h TERT基因对 K5 6 2细胞的端粒酶活性的抑制作用。结果 :转染反义 h TERT表达质粒组与对照组 (空白对照及空质粒组 )相比较 ,生长速率明显变慢 ,部分细胞出现凋亡等形态学改变。反义 h TERT对 K5 6 2细胞的端粒酶活性具有明显的抑制作用。结论 :研究构建的端粒酶反义 h TERT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h TERT- m RNA的表达 ,在抑制端粒酶活性的同时 ,减慢了 K5 6 2细胞的生长速度。

Objective:To construct a plasmid,antisense hTERT plasmid then transferred it into K562 cells,and to see if it can inhibit the activity of telomerase and the proliferation of K562 cells.Methods:Plasmid construction and transfection.An antisense plasmid was constructed by reverse design of PCR primers was cloned to the plasmid pLNCX neo,G418 was added into the medium after the plamid was successfully introduced into K562 cells by using lipofectactin mediated DNA transfection.Analysis the effects of antisense hTERT on K562 cells.The effects of telomerase inhibition on the proliferation of K562 cells were analyzed by means of MTT and morphology.The telomerase activity of K562 cells was studied by TRAP PCR ELISA methods.Results:The growth rate of antisense hTERT gene transfected K562 cells (K562 as cell) was significantly slower than those of the controls,and apoptosis appear in part of K562 as cells the telomerase activity of antisense hTERT gene transfected cells was significantly inhibited than those of controls.Conclusion:The expression of a partial compliment antisense sequence to the mRNA sequence of the protein subunit of telomerase can inhibit the activity of telomerase,slow the growth of K562 cells.