敲减自噬相关基因Beclin 1可降低TM3小鼠睾丸间质细胞的睾酮合成功能  

Knockdown autophagy related gene Beclin 1 might lead to decline of steroidogenesis in TM 3 cells

作　　者：李维仁[1,2] 侯垒[2] 崔功静 常智杰[3] 贾金铭[1] 辛钟成[4] 

机构地区：[1]中国中医科学院广安门医院男科北京,100053 [2]北京航天总医院泌尿外科 [3]清华大学医学院生物化学系 [4]北京大学第一医院男科中心

出　　处：《中国男科学杂志》2013年第12期7-11,16,共6页Chinese Journal of Andrology

摘　　要：目的：初步探讨自噬活性降低对TM3小鼠睾丸间质细胞系睾酮合成功能的影响。方法 TM3小鼠睾丸间质细胞系中，通过siRNA敲减TM3细胞的自噬相关基因Beclin1，一部分细胞转染GFP-LC3质粒48h后共聚焦显微镜观察GFP颗粒分布情况，一部分细胞用电镜观察其超微结构变化，观察自噬相关结构的变化，另一部分细胞用黄体生成素（LH）刺激后，提取细胞总蛋白，用Western blot比较青年组和老年组睾丸间质细胞中自噬相关蛋白LC3蛋白表达水平的差异，ELISA法检测细胞上清睾酮浓度，分光光度法检测活性氧物质（ROS）。结果利用siRNA敲减自噬相关基因Beclin 1后，TM3细胞中LC3-Ⅱ的蛋白表达降低（P ＜0.01）（图1A），电镜下观察并分析发现，TM3细胞中自噬小体所占面积比减少（P ＜0.01）（图1B），共聚焦显微镜下观察发现，GFP- LC3的点状荧光所占面积比显著减少（P＜0.01）（图1C）。TM3细胞中敲减Beclin 1导致LH诱导的StAR蛋白表达降低（P＜0.01）（图2A），睾酮合成水平下降（P＜0.01）（图2B），ROS水平升高（P＜0.01）（图3）。结论 TM3小鼠睾丸间质细胞系中，利用siRNA敲减自噬相关基因Beclin 1后，细胞自噬受到抑制，ROS水平显著升高，LH诱导的StAR蛋白表达及睾酮合成显著降低。Objective To study the effects of autophagic change on steroidogenesis in TM3 Leydig cells. Methods Autophagy related gene Beclin 1 was knocked down in TM3 mouse Leydig cells using siRNA. Some cells were transfected with GFP-LC3 plasmid, and GFP particals were observed under confocal microscope; Cell ultrastructure was observed under electron microscopy（EM）; and the other cells were collected for detection of protein expression by western blot, the concentration of testosterone in cell culture medium supernatant was detected by ELISA and reactive oxygen species （ROS） level was measured by spectrophotometry. Results Decrease of LC3-Ⅱexpression in the treated TM3 cells was confirmed by western blot（P〈0.01） （fig 1A）, less autophagic structure and less GFP partical in TM3 cells with knockdown of Bcelin 1 was observed under EM （fig 1B） under confocal microscopy （fig 1C）, respectively. The expression of StAR and testosterone production were decreased （P〈0.01, respectively） and ROS level was increased （P〈0.01） in TM3 cells with knockdown of Beclin 1. Conclusion Knockdown of Beclin 1 in TM3 cells might result in inhibition of autophagy, increase of ROS level and decrease of testosterone production.

关 键 词：衰老 自噬 莱迪希细胞 

分 类 号：R339.38[医药卫生—人体生理学] R339.21[医药卫生—基础医学]