睾丸扭转复位后生精上皮细胞核转录因子表达的改变及其凋亡

目的探讨睾丸扭转2h复位后第3天睾丸生精上皮细胞核转录因子(NF-κB)表达的改变及其凋亡的关系。方法用24只雄性SD大鼠建立左侧睾丸扭转复位模型。分为Ⅰ、Ⅱ和Ⅲ组,每组8只。Ⅰ组动物睾丸扭转复位后用柳氮磺胺吡啶灌胃;Ⅱ组动物睾丸扭转复位后用等量的生理盐水灌胃;Ⅲ组动物假手术后用等量的生理盐水灌胃。分别用Western蛋白印迹和免疫组织化学原位检测睾丸生精上皮细胞中NF-κB的表达情况;TUNEL法检测生精上皮细胞凋亡情况。结果Ⅱ组动物睾丸扭转复位后第3天扭转侧睾丸生精上皮细胞胞质NF-κB蛋白质(9·4±2·68)表达与Ⅰ、Ⅲ组扭转侧睾丸生精上皮细胞胞质NF-κB表达(分别为:12±2·2;11·1±3)相比下降水平差异无统计学意义,胞核NF-κB表达升高水平差异有统计学意义(3组分别为8·4±3·1;21·1±3·6;6·0±2·3)。免疫组织化学原位检测Ⅰ和Ⅲ组扭转侧睾丸生精上皮细胞中NF-κB的表达以胞质为主,Ⅱ组扭转侧睾丸生精上皮细胞中NF-κB的表达以细胞核为主,而且NF-κB阳性细胞比例较其他两组阳性率显著上升(三组分别为15·6%±2·6%,66·1%±3·8%,10·8%±2·7%)。Ⅰ组(7·7%±2·0%)和Ⅲ组(5·9%±1·7%)扭转侧睾丸生精上皮细胞凋亡指数差异不明显;Ⅱ组扭转侧睾丸生精上皮细胞凋亡水平(37·2%±3·3%)跟以上两组相比升高有显著性差异。结论睾丸扭转2h复位后第3d,睾丸生精上皮细胞NF-κB蛋白已被激活,从胞质转移至核内,启动生精上皮细胞凋亡过程。NF-κB蛋白质的激活是导致睾丸扭转复位后生精上皮细胞特别是精原细胞和各级精母细胞凋亡增加的重要环节。

Changes of nuclear factor-kappa gene binding expression in and apoptosis of spermatogenic epithelial cells in the restored testis after torsion: experiment with rats

OBJECTIVE: To investigate the changes of nuclear factor-kappa gene binding (NF-kappaB) expression in and apoptosis of spermatogenic epithelial cells in the restored testis after torsion and analyze the relationship between them. METHODS: Sixteen male SD rats underwent torsion of the left testis clockwise at an angle of 720 degrees for 2 hours and then the testis was restored to the original position and fixed. Then the 16 rats were randomly divided into 2 equal group: Group I in which salicylazosulfapyridium (SASP) suspension was infused intra-gastrically 5 h after operation and then once a day for 4 times, and Group II in which normal saline (NS) was infused in the same manner. Eight rats (Group III) underwent sham operation and then infused with NS in the same manner as that of Group II. Three days after operation the rats were killed and the samples of the testes at the torsion side were taken out and the seminiferous tubules were isolated. Western blotting was used to detect the NF-kappaB expression in the cytoplasm and nucleus of spermatogenic epithelial cells. Immunohistochemistry was used to detect the in situ expression of NF-kappaB. The apoptosis of the spermatogenic epithelial cells was examined by TUNEL method. RESULTS: Western blotting showed that the NF-kappaB expression in the cytoplasm of spermatogenic epithelial cells of Group II was 9.4 +/- 2.68, somewhat lower, but not significantly, than those of Group I and III (12 +/- 2.2 and 11.1 +/- 3 respectively, both P > 0.05). The NF-kappaB expression in the nucleus of spermatogenic epithelial cells of Group II was 21.1 +/- 3.6, significantly higher than those of Group I and III (8.4 +/- 3.1 and 6.0 +/- 2.3 respectively, both P < 0.05). However, there were no significant differences in the NF-kappaB expression in the cytoplasm and nucleus of spermatogenic epithelial cells between Groups I and III. The NF-kappaB activity coefficient of spermatogenic epithelial cells of Group II was 2.32 +/- 0.4, significantly higher than those of Groups I and III (0.68 +/- 0.3 and 0.52 +/- 0.1 respectively, both P < 0.01). However, there was no significant difference in the NF-kappaB activity coefficient of spermatogenic epithelial cells between Groups I and III (P > 0.05). The NF-kappaB positive cell rate of Group II was 66.1% +/- 3.8%, significantly higher than those of Groups I and III (15.6% +/- 2.6% and 10.8% +/- 2.7%, both P < 0.01). The apoptotic cell rate of Group II was 37.2% +/- 3.3%, significantly higher than those of Groups I and III (7.7% +/- 2.0% and 5.9% +/- 1.7%, both P < 0.01). CONCLUSION: After the torsion of testis, NF-kappaB was activated and released from the nucleus into the cytoplasm, thus initiating the apoptosis of spermatogenic epithelial cells.