环磷酰胺代谢产物丙烯醛对未成熟睾丸支持细胞骨架的影响及机制研究

Effect of acrolein on cytoskeleton of Sertoli cells

目的 探讨环磷酰胺（cyclophosphamide,CP）代谢产物丙烯醛（acrolein,ACR）对未成熟睾丸Sertoli细胞骨架的损伤及其机制.方法 建立新生SD大鼠Sertoli细胞原代培养模型,实验组给予100 μmol/L浓度的ACR溶液,对照组给予PBS溶液,12 h后分别用超氧化物阴离子荧光探针染色观察Sertoli细胞内超氧化阴离子的变化,透射电子显微镜观察ACR对细胞超微结构的影响,免疫荧光染色观察细胞骨架中F-actin的分布变化,Western blot方法测定ERK和p38的表达水平.结果 ①ACR能增强sertoli细胞内活性氧的水平;②ACR处理后,实验组细胞线粒体发生了肿胀,染色质发生了凝集,出现胞质浓缩、核溢裂及并且空泡化现象,而微丝发生聚集、边缘化和区域化;③ACR干预后实验组ERK和p38MAPK表达活性明显上升.结论 CP的主要毒性代谢产物ACR通过引起未成熟睾丸Sertoli细胞的氧化应激反应损伤支持细胞骨架,从而导致儿童生精功能障碍,而其可能通过活化ERK和p38的表达使MAPK信号途径激活来实现这一损伤过程.

Objective To investigate the mechanism and the effect of Acrolein on cytoskeleton of Sertoli cells. Methods We isolated and identified Sertoli cells from 8-day-old SD rats＇ testes. The experimental groups were treated with Acrolein, The control group were treated with phosphate buffered solution. After 12 hours, the treated cells were dyed with DHE to assay the distribution of superoxide anion, and the ultrastructure of the Sertoli cells was examined with transmission electron microscope, F-actin was stained with fluorescent antibody, and the expression of ERK and p38 in Sertoli cells were assayed by Western blot. Results The photodensity of superoxide anion, the expression of ERK and p38 were increased in the experimental groups. After treatment with Acrolein, the ultrastructure of the Sertoli cells was changed, manisfested as chondriosome swelling; chromatin clumping, condensed endochylema; split nuclei and vacuolization. All of these changes were followed by a F-actin accumulation, marginalization and regionalization. Conclusions Acrolein, the main toxic metabolite of cyclophosphamide, damages the cytoskeleton of immature Sertoli cells through oxidization stress, which might activate the MAPK signaling pathway mainly by increasing the expressions of ERK and p38.