中文摘要：

目的探讨Rab13-PKA通路对大鼠血睾屏障功能的调节。方法首先构建靶向大鼠Rab13的shRNA载体,通过大鼠睾丸内注射转染Rab13 shRNA,Western印迹检测Rab13体内沉默效果及沉默后BTB相关连接蛋白表达改变,放射自显影检测睾丸组织内PKA活性变化,睾丸组织冰冻切片免疫荧光染色及鬼笔环肽染色分别观察occludin及肌动蛋白丝在生精上皮的分布。结果 Rab13 shRNA睾丸内转染后Rab13表达量与对照shRNA转染相比下降约70%（P〈0.01）,而BTB相关连接蛋白表达无变化;Rab13沉默后PKA活性与对照组相比有明显升高（P〈0.01）;免疫荧光结果表明,Rab13沉默后occludin在VIII期生精上皮的分布显著高于对照组,而在其他期别生精上皮中则无显著变化;鬼笔环肽染色显示Rab13沉默后F-actin在BTB处的分布明显增强;此外,occludin及F-actin的分布变化可被PKA抑制剂H89所拮抗。结论 Rab13可通过影响PKA活性调节大鼠BTB紧密连接蛋白occludin及F-actin的分布,从而参与调节BTB功能。

英文摘要：

Objective To investigate the regulation of blood-testis barrier by Rab13-PKA pathway in rats. Method First,shRNA vector targeting at Rab13 was constructed and then the Rab13 shRNA was transfected into the rat testis by injection. Western blot was used to detect the knock-down effect of Rab13 and the expression of blood-testis barrier（ BTB） constituent proteins. PKA activity was detected by autoradiography and scintillation counting. Further,immunofluorescence analysis and phalloidin staining were applied to observe the distribution of occludin and F-actin,respectively.Results The expression level of Rab13 in the testis was reduced by approximately 70% after transfection of Rab13 shRNA as compared with the non-targeted control group（ P〈0. 01）,while the expression of BTB constituent proteins remained unchanged. PKA activity was significantly increased after Rab13 RNAi transfection（ P〈0. 01）. The distribution of occludin at BTB was remarkably increased after Rab13 RNAi silencing around stage VIII but not at other stages of the seminiferous epithelial cycle. The assembly of F-actin at BTB was also intensified in Rab13-silenced testis. Both the changes of distribution of occludin and F-actin induced by Rab13 shRNA were found to be antagonized by the PKA specific inhibitor H89. Conclusions Rab13 can modulate the distribution of occludin and F-actin at the blood-testis barrier in rats by regulating PKA activity,which may participate in the regulation of BTB function.