CAJ | 학술논문

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。
목적：초보탐토자서활성강저대TM3소서고환간질세포계고동합성공능적영향。방법 TM3소서고환간질세포계중，통과siRNA고감TM3세포적자서상관기인Beclin1，일부분세포전염GFP-LC3질립48h후공취초현미경관찰GFP과립분포정황，일부분세포용전경관찰기초미결구변화，관찰자서상관결구적변화，령일부분세포용황체생성소（LH）자격후，제취세포총단백，용Western blot비교청년조화노년조고환간질세포중자서상관단백LC3단백표체수평적차이，ELISA법검측세포상청고동농도，분광광도법검측활성양물질（ROS）。결과이용siRNA고감자서상관기인Beclin 1후，TM3세포중LC3-Ⅱ적단백표체강저（P ＜0.01）（도1A），전경하관찰병분석발현，TM3세포중자서소체소점면적비감소（P ＜0.01）（도1B），공취초현미경하관찰발현，GFP- LC3적점상형광소점면적비현저감소（P＜0.01）（도1C）。TM3세포중고감Beclin 1도치LH유도적StAR단백표체강저（P＜0.01）（도2A），고동합성수평하강（P＜0.01）（도2B），ROS수평승고（P＜0.01）（도3）。결론 TM3소서고환간질세포계중，이용siRNA고감자서상관기인Beclin 1후，세포자서수도억제，ROS수평현저승고，LH유도적StAR단백표체급고동합성현저강저。
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.