A型精原细胞体外分离纯化及其端粒酶活性抑制后的形态学变化

目的 :探讨分离纯化、体外培养的A型精原细胞在端粒酶活性受到抑制后细胞形态学的改变。方法 :以脂质体LipofectAMINE 2 0 0 0介导将硫代磷酸修饰的端粒酶mTR反义寡核苷酸 (ASODN)转染体外分离纯化的 (SD)大鼠精原细胞 ,采用生物发光技术检测精原细胞中端粒酶活性的改变 ,光镜及电镜观测端粒酶活性抑制后A型精原细胞的形态学变化。结果 :mTR ASODN可明显抑制精原细胞端粒酶活性 (P <0 .0 5 )。ASODN转染 4 8～ 72h后 ,光镜下可见细胞生长缓慢 ,染色质浓缩 ,胞质出现颗粒样物质 ,72h后死细胞数较空白对照组明显增多。电镜下可见部分细胞核异染色质浓聚集、浓缩和边集 ,胞质胞核内均可见空泡 ;部分细胞核完全固缩。单纯脂质体组及正义寡核苷酸对照组形态学均无显著变化。结论 :mTR ASODN可明显抑制体外培养的A型精原细胞端粒酶活性 ,并在转染 4 8～ 72h后使细胞形态学出现明显改变

Purification of type A spermatogonia in vitro and its morphological changes after inhibition of telomerase activity

Purpose:To explore the morphological changes of type A spermatogonia whose telomerase activity were repressed by antisense oligodeoxynucleotide(ASODN).Methods:Phosphorothioate mTR antisence ODNs was encapsulated by liposomes LipofectAMINE 2000(LF 2000) and transfected to Sprague Dawley (SD) rat spermatosonia. The inhibition of telomerase activity was detected aided by Bioluminescence technique. The morphological changes of type A spermatogonia was studied with microscope and electron microscope.Results:mTR ASODN conjugated with LipofectAMINE could significantly inhibit telomerase activity of spermatgonia(P< 0.05). Significant morphological changes could be observed in some spermatgonia with the aid of microscope and electron microscope. No morphological change of telomerase activity were observed in the control term either Liposome or SODN.Conclusions:mTR ASODN could significantly inhibit telomerase activity of spermatgonia. Significant morphological changes were observed in some spermatgonia when telomerase activity was repressed.