铁过载损伤小鼠睾丸支持细胞

Iron overload injures Sertoli cells of mouse

目的探讨铁过载对体外原代培养的小鼠睾丸支持细胞(SC)的影响。方法取原代分离培养小鼠睾丸SC纯化并鉴定,加入不同浓度(50、100、200μmol/L)右旋糖酐铁注射液,培养至24、48和72 h后收集细胞,光学显微镜与流式细胞术鉴定铁过载,流式细胞术检测细胞活性氧(ROS)水平,试剂盒检测丙二醛(MDA)、谷胱甘肽(GSH)和一氧化氮(NO)的含量及超氧化物歧化酶(SOD)、一氧化氮合酶(NOS)含量和谷胱甘肽过氧化物酶(GSH-Px)的活性,Western blot与免疫细胞化学检测闭锁蛋白(occludin)、雄激素结合蛋白(ABP)、转铁蛋白(TRF)、抑制素(INH)和波形蛋白(VIM)的表达。结果与对照组相比,铁过载组细胞内ROS、MDA、NO与NOS的含量显著增高(P<0.01),GSH含量、SOD和GSH-Px的活性显著降低(P<0.01),occludin、ABP、TRF、INH和VIM蛋白的表达明显降低(P<0.05)。结论铁过载可导致体外培养的睾丸支持细胞氧化性损伤并降低其功能。

Objective To investigate the effect of iron overload on Sertoli cells. Methods Sertoli cells were isolated and cultured by adding iron dextran at different concentrations （50,100,200 μmoL/L） for different lengths of time （24,48,72 h）. Iron overload was observed by microscopy and flow cytometry. ROS production was observed by flow cytometry. GSH and GSH-Px were determined by nitrobenzoic acid method. MDA content was measured by thiobarbi- turic acid way. NO and NOS contents were detected by nitrate reductase way. SOD content was examined by xanthine oxidase method. The expression level of occluding, ABP, TRF, INH and VIM protein was observed by Western blot and immunocytochemistry. Results Compared with control group, the level of intracellular ROS, the contents of MDA, NO and NOS all significantly increased, with consequent decrease in the contents of SOD, GSH, GSH-Px （P 〈 0. 01 ）. The expression of occluding, ABP, TRF, INH and VIM also significantly decreased （ P 〈 0. 05 ）. Conclusions Iron overload may induce oxidative damage of Sertoli cells and weaken its function.