mTR反义寡核苷酸在体外培养的A型精原细胞中的分布特点及稳定性研究

Study of the stability and intracellular distribution of antisense oligodeoxynucleotide in type A spermatogonia in vitro

目的 :探讨鼠端粒酶RNA (mousetelomeraseRNA ,mTR)基因的反义寡核苷酸 (antisenseoligodeoxynucleotide,ASODN)在体外培养的A型精原细胞中的分布特点及稳定性。 方法 :以脂质体Lipofec tAMINE 2 0 0 0 (LF 2 0 0 0 )介导或直接将硫代磷酸修饰的端粒酶mTR ASODN转染体外分离纯化的Sprague dawley(SD)鼠A型精原细胞 ,在荧光显微镜下对各实验组进行连续观察、摄片、分析。结果 :在脂质体介导下 ,荧光细胞数目显著增加 ,1h后绝大多数细胞迅速荧光积聚于细胞核 ,1～ 4h后 ,细胞核内荧光强度进一步增强 ,4h后细胞内荧光减弱直至消失。而非修饰型mTR ASODN在直接转染或脂质体介导下均发现荧光在 3h后减弱甚至消失。结论 :LF 2 0 0 0可增加mTR ASODN转染A型精原细胞的数量 ,同时使其在细胞核内分布聚集明显。硫代修饰型mTR ASODN在精原细胞内具有更好的稳定性。

Purpose:To explore the stability and distribution of mouse telomerase RNA (mTR) antisense oligodeoxynucleotide(ASODN) in type A spermatogonia in vitro. Methods:Phosphorothioate mTR antisence ODN was encapsulated by LipofectAMINE 2000(LF 2000) and transfected into Sprague dawley (SD) rat type A spermatogonia in vitro.The stability and distribution of mTR ASODN in type A spermatogonia was observed successively with fluorescence microscope. Results:In the absence of LF 2000,the oligonucleotide residing in discrete structures in the cytoplasm of the cell,presented as a punctate fluoresence pattern.In the presence of LF 2000,cellular fluoresence remarkablely increased and the oligonucleotide localized in the nucleus,as well as in discrete structures in the cytoplasm. The fluorescence of unmodified mTR ASODN disappeared after 3h in the absence or presense of LF 2000. Conclusions:Liposome(LF 2000) could help more mTR ASODN to conjugate with spermatogonia and enter into the nucleus.Being compared with unmodified mTR ASODN, phosphorothioate mTR ASODN were more stable in the spermatogonia.