特异性小分子RNA沉默转录因子E2F-1表达对小鼠精原干细胞凋亡的影响

Inhibitory effects of E2F transcription factor 1 small interfering RNA on apoptosis of mouse sper- matogoniai stem cells

目的 观察小分子干扰RNA（siRNA）沉默转录因子E2F-1表达对小鼠精原干细胞（SSCs）凋亡的影响。方法 采用Percoll液密度梯度离心分离和流式细胞仪分选纯化SSCs进行体外培养,以脂质体法转染pSilencer4.1-E2F1至SSCs,并用实时定量逆转录聚合酶链反应（RT-qPCR）和Western blot检测E2F-1的表达水平,流式细胞仪检测siRNA干扰E2F-1表达后对SSCs凋亡的影响。结果 RT-qPCR结果实验组mRNA的2ΔΔCt值（0.32）明显低于未转染组（0.10）和阴性对照组（1.04）,Western blot显示实验组条带灰度低于未转染组和阴性对照组,流式细胞术检测显示实验组凋亡率（7.69%）低于未转染组（23.07%）和阴性对照组（24.19%）,差异有统计学意义（P〈0.05）。结论 siRNA靶向干扰转录因子E2F-1能显著降低小鼠SSCs的凋亡率。

Objective To observe inhibitory effects of E2F transcription factor 1 （ E2F-1 ） small interfering RNA （siRNA） on apoptosis of mouse spermatogonial stem cells （SSCs）. Methods Percoll density gradient centrifugation and flow cytometry was used to isolate, enrich and purify SSCs. Real-time reverse transcriptase-polymerase chain reaction （RT-qPCR） and Western blotting were carried out to investigate the expression of E2F-1, and flow cytometry was ued to analyze the apoptosis rate. Results Compared to non-transfected group and control group, the expression of E2F-1 in experimental group was re- markably down-regulated at mRNA [ 2 -（ΔΔCt） , 0. 32 VS. 0. 10 and 1.04, P 〈 0. 05 ] and protein levels （P 〈0. 05）. Tthe apoptosis rate in experimental group was reduced remarkably （7.69% vs 23.07% and 24. 19% ;P 〈 0. 05 ）. Conclusion E2F-1 siRNA could remarkably suppress the apoptasis of SSCs in vitro.