反义抑制大鼠精原细胞端粒酶活性及其对细胞因子反应性的研究

Effect of Antisense Oligonucleotide Against Telomerase in Rat Spermatogonia and Its Reactivity to Cytokine

目的 :探讨精原细胞在封闭其端粒酶RNA(mTR)后的变化情况 ,及其对细胞因子 (SCF、TGF β1 )的反应性。 方法 :以脂质体为载体将端粒酶RNA的反义寡核苷酸 (PS ASON)导入体外增殖的精原细胞后 ,加入SCF或TGF β1 ,四甲基偶氮唑盐 (MTT)比色法检测抑制前后细胞增殖情况 ,端粒重复扩增法 (TRAP)检测端粒酶活性变化。 结果 :1μmol L终浓度的PS ASON明显下调精原细胞端粒酶活性 ,抑制细胞增殖 ,而抑制结束后 ,端粒酶活性有一定程度的恢复 (P <0 .0 1)。细胞因子对抑制后的精原细胞有调控作用 ,SCF提高端粒酶活性 ,并促进细胞增殖。相反 ,TGF β1 则抑制端粒酶活性的恢复。 结论 :反义抑制精原细胞端粒酶活性能有效抑制细胞增殖 ,抑制结束后有较好的可逆性 ,并受细胞因子的调控 。

Objectives: To study the change of telomerase activity in rat spermatogonia when the telomerase RNA was enclosed, and reactivity of the change to cytokine(SCF, TGF β 1 ). Methods: The antisense oligonucleotides(PS ASON) of telomerase was transfected into proliferating spermatogonia in vitro with the liposomes as the vector.Then the cytokine stem cell factor (SCF) or transforming growth factor β 1 (TGF β 1 ), was added . The proliferative activity of the spermatogonia was determined before and after the inhibition by MTT [3 (4,5 dimethylthiazol 2 yl) 2,5 diphenyltetrazolium bromide]assay. The change of telomerase activity was detected by telomeric repeat amplification protocol (TRAP). Results: 1 μmol/L PS ASON obviously downregulated the telomerase activity and inhibited spermatogonia proliferation. When the inhibition was over, the activity recovered to some extent( P < 0.01 ) . Growth factors can regulate the spermatogonia after inhibition, SCF may improve the activity of telomerase and the proliferation of spermatogonia. Adversely, TGF β 1 may inhibit the recovery of telomerase activity. Conclusions: To inhibit spermatogonia telomerase activity antisensely can limit the proliferation of spermatogonia efficiently, which was regulated by cytokine. This method might be a new and efficient way in male birth control.