p53在环磷酰胺代谢物丙烯醛致未成熟睾丸支持细胞凋亡中的作用  

Effect of p53 on apoptosis of Sertoli cells caused by acrolein

作　　者：邓常开 刘丰[2] 李旭良[2] 林涛[2] 何大维[2] 魏光辉[2] 刘星[2] 华燚 张德迎[2] 吴胜德[2] 

机构地区：[1]成都市妇女儿童中心医院小儿外科,610091 [2]重庆医科大学附属儿童医院泌尿外科儿童发育疾病研究省部共建教育部重点实验室儿童发育重大疾病国家国际科技合作基地儿科学重庆市重点实验室,400014

出　　处：《中华小儿外科杂志》2016年第8期621-626,共6页Chinese Journal of Pediatric Surgery

基　　金：重庆市自然科学基金项目（cstc2013jcyjA10057）;国家临床重点专科建设项目基金（国卫办医函[2013]544号）

摘　　要：目的探讨p53在环磷酰胺（CP）代谢产物丙烯醛（ACR）导致未成熟睾丸支持细胞线粒体功能紊乱、细胞凋亡中的作用及机制。方法建立未成熟SD大鼠支持细胞原代培养模型，实验组给予100μmol／L浓度的ACR溶液，对照组给予PBS溶液。ACR处理后10min、30min及1h时用原位免疫荧光检测p53的迁移定位，并在ACR处理1h、3h和12h后分别做如下处理：①Western blot检测细胞内p53及Bax的表达；②细胞行JC-1染色，流式细胞仪检测线粒体膜电位的变化；③流式细胞仪检测细胞凋亡情况。结果①支持细胞分离培养成功，见细胞贴壁生长，长梭形，2～3个突起；②在ACR处理10min时线粒体中可见少许p53分布，30min时线粒体中p53蛋白明显增加，之后维持在这一水平未见明显增加；③ACR处理1h后，细胞内的p53和Bax的表达均明显高于对照组（P〈0．05）；ACR处理3h后细胞内p53的表达明显高于对照组（P〈0．05），但与12h组比较未见明显差异（P〉0．05）；而3h后Bax的表达明显高于对照组（P〈0．05），并随时间延长表达明显增多；④ACR处理1h后，实验组线粒体膜电位为（87．43±0．76）％，与对照组（98．07±0．67）％相比，下降了约11％；处理3h后，线粒体膜电位为（72．0±1．73）％，与对照组相比下降约26％；在处理12h后，线粒体膜电位为（51．53±1．93）％，与对照组相比下降约47％。提示线粒体功能受损；⑤ACR处理细胞1h、3h及12h后，细胞凋亡率分别为：（3．25±0．18）％、（8．74±0．64）％及（36．87±0．61）％。与对照组凋亡率（0．43±0．13）％比较，差异有统计学意义（P〈0．05）。实验组不同时间点之间凋亡率比较也有统计学意义（P〈0．05）。结论CP及其代谢产物ACR可能通过激活p53信号网络，损害细胞线粒体功能，激活内源性细胞凋亡途径及非转录凋亡途径，导致支持细胞凋亡�Objective To explore the effect and mechanism of p53 in mitochondrial dysfunction and apoptosis of immature Sertoli cell. Methods The Sertoli cells were harvested from 14-day-old Sprague-Dawley rats＇ testes. The experimental group received acrolein, a major toxicant metabolite of cyclophosphamide （CP） while the control group had phosphate buffered solution. At 10, 30 and 60 rain, the cells were stained with fluorescent antibody for detecting the migration of p53. At 1, 3 and 12 h, the expressions of p53 and Bax were detected by Western blot and mitochondrial membrane potential and cell apoptosis by flow cytometry. Results As compared with controls, there were a few p53 in mitochondria at 10 rain. However, p53 protein increased significantly at 30 min and then stabilized up to 1 h. In experimental group, the expressions of p53 and Bax increased and mitochondrial membrane potentials were （87.43 ±0. 76）%, （72. 0 ± 1.73）% and （51.53 ± 1.93）% respectively. All decreased significantly as compared with control group （98. 07 ± 0. 67） % and the rates of cellular apoptosis were （3.25± 0.18）%, （8.74 ± 0.64）% and （36.87 ± 0.61）% respectively. All increased significantly as compared with control group （0. 43 ± 0. 13）%. Conclusions Acrolein may induce an apoptosis of Sertoli cells through activating p53 signaling network and damaging mitochondrial functions. It will provide new conceptual and experimental rationales for further elucidating the reproductive toxicity mechanism of CP and designing a more effective protective strategy.

关 键 词：塞尔托利细胞 环磷酰胺 丙烯醛 肿瘤抑制蛋白质P53 细胞凋亡 

分 类 号：R96[医药卫生—药理学]