中国实验动物学报
中國實驗動物學報
중국실험동물학보
ACTA LABORATORIUM ANIMALIS SCIENTIA SINICA
2015年
4期
359-364
,共6页
Rab13%血睾屏障%蛋白激酶A
Rab13%血睪屏障%蛋白激酶A
Rab13%혈고병장%단백격매A
Rab13%Blood-testis barrier%Protein kinase A%Rats
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。
目的：探討Rab13-PKA通路對大鼠血睪屏障功能的調節。方法首先構建靶嚮大鼠Rab13的shRNA載體，通過大鼠睪汍內註射轉染Rab13 shRNA，Western印跡檢測Rab13體內沉默效果及沉默後BTB相關連接蛋白錶達改變，放射自顯影檢測睪汍組織內PKA活性變化，睪汍組織冰凍切片免疫熒光染色及鬼筆環肽染色分彆觀察occludin及肌動蛋白絲在生精上皮的分佈。結果 Rab13 shRNA睪汍內轉染後Rab13錶達量與對照shRNA轉染相比下降約70％（ P＜0.01），而BTB相關連接蛋白錶達無變化；Rab13沉默後PKA活性與對照組相比有明顯升高（P＜0.01）；免疫熒光結果錶明，Rab13沉默後occludin在VIII期生精上皮的分佈顯著高于對照組，而在其他期彆生精上皮中則無顯著變化；鬼筆環肽染色顯示Rab13沉默後F-actin在BTB處的分佈明顯增彊；此外，occludin及F-actin的分佈變化可被PKA抑製劑H89所拮抗。結論 Rab13可通過影響PKA活性調節大鼠BTB緊密連接蛋白occludin及F-actin的分佈，從而參與調節BTB功能。
목적：탐토Rab13-PKA통로대대서혈고병장공능적조절。방법수선구건파향대서Rab13적shRNA재체，통과대서고환내주사전염Rab13 shRNA，Western인적검측Rab13체내침묵효과급침묵후BTB상관련접단백표체개변，방사자현영검측고환조직내PKA활성변화，고환조직빙동절편면역형광염색급귀필배태염색분별관찰occludin급기동단백사재생정상피적분포。결과 Rab13 shRNA고환내전염후Rab13표체량여대조shRNA전염상비하강약70％（ P＜0.01），이BTB상관련접단백표체무변화；Rab13침묵후PKA활성여대조조상비유명현승고（P＜0.01）；면역형광결과표명，Rab13침묵후occludin재VIII기생정상피적분포현저고우대조조，이재기타기별생정상피중칙무현저변화；귀필배태염색현시Rab13침묵후F-actin재BTB처적분포명현증강；차외，occludin급F-actin적분포변화가피PKA억제제H89소길항。결론 Rab13가통과영향PKA활성조절대서BTB긴밀련접단백occludin급F-actin적분포，종이삼여조절BTB공능。
Objective To investigate the regulation of blood-testis barrier by Rab13-PKA pathway in rats.Meth-od 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, immunoflu-orescence 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 occlu-din at BTB was remarkably increased after Rab13 RNAi silencing around stage VIII but not at other stages of the seminifer-ous epithelial cycle.The assembly of F-actin at BTB was also intensified in Rab13-silenced testis.Both the changes of dis-tribution 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 regu-lating PKA activity, which may participate in the regulation of BTB function.
