硫代修饰型反义寡核苷酸抑制肺癌细胞系端粒酶活性及增殖的研究

Inhibition of hTR activity for suppression of lung cancer cell proliferation using antisense phosphorothioate oligonucleotides

目的 了解人肺癌细胞系的端粒酶活性情况。探讨硫代修饰型端粒酶RNA反义寡核苷酸封阻端粒酶RNA ,对癌细胞代谢、生长的抑制作用。方法 采用端粒酶PCRELISA方法检测 8种体外培养人肺癌细胞系端粒酶活性。合成端粒酶RNA的反义硫代寡核苷酸 (6聚体、9聚体、1 2聚体 )和随机序列寡核苷酸 ,分别导入LTEP a 2细胞系 ,作用 72h。采用端粒酶PCRELISA方法检测端粒酶活性、四甲基偶氮唑盐 (MTT)光吸收法检测琥珀酸脱氢酶 (SDH)活性并做细胞增殖计数。结果 8种人肺癌细胞系端粒酶活性均呈不同程度阳性表达。 3种端粒酶RNA反义寡核苷酸对LTEP a 2细胞端粒酶活性、SDH活性及生长增殖均有抑制作用并随浓度升高而增强。 3种反义寡核苷酸 5～ 40μmol/L对细胞端粒酶活性、SDH活性和增殖的抑制率分别为 2 5 7%～ 84 0 %、1 9.4%～ 74.7%和1 4 8%～ 72 .5 %。硫代修饰随机序列寡核苷酸对细胞端粒酶活性、SDH活性及生长增殖无明显抑制作用 ,与 3种反义寡核苷酸抑制作用比较差异有非常显著性意义 (P <0 .0 1 )。结论 人肺癌细胞系呈端粒酶阳性表达 ,反义寡核苷酸可抑制端粒酶活性 。

Objective Telomerase activity was measured in 8 lung tumor cell lines and transfected antisense phosphorothiate oligodeoxynucleotides (anti PS ODN) of hTR as therapeutic agents into target cell LTEP a 2 to investigate the inhibitory effect on telomerase activity and tumor cell growth. Methods 1. Telomerase activity assay in 8 human lung tumor cell lines using telomerase PCR ELISA. 2. Synthesized anti PS ODNs of hTR and random PS ODN transfected with Clonfectin into LTEP a 2 cell lines for 72 hr. 3. Measure telomerase activity by telomerase PCR ELISA , SDH activity by MTT assay and cell growth. Results Eight cell lines showed positive expression of telomerase activity. Various anti PS ODNs could inhibit telomerase activity , SDH activity and cell growth. The inhibition became more marked with the increase of anti PS ODNs concentrations. Concentrations of 5～40 μmol/L anti PS ODNs of hTR specifically reduced the telomerase activity by 25.7%～84.0%. PS ODNs of anti hTR were also able to reduce SDH activity by 19.4%～74.7% at 5～40 μmol/L. The dose of 5～40 μmol/L PS ODNs of anti hTR had the ability to inhibit cell growth by 14.8%～72.5%. The results indicate that random sequence of PS ODN 9 failed to inhibit telomerase, SDH activity and cell growth. A statistically significant difference exists between random PS ODN and three anti PS ODNs ( P <0.01). Conclusions Human lung tumor cell lines express high telomerase activity. PS ODNs of anti hTR had the ability to inhibit telomerase and reduce LTEP a 2 cell growth and metabolism.