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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细胞的氧化应激损伤及其机制。方法建立新生SD大鼠Sertoli细胞原代培养模型,实验组分别给予不同浓度的ACR溶液,对照组给予PBS溶液,3h、12h后分别用MTT法检测各组细胞活力,依据CD50分别采用50、100μmol/L浓度的ACR进行干预。免疫细胞化学法检测丙烯醛-蛋白质加合物(ACR-Pro),硫代巴比妥法和Fenton法检测细胞丙二醛(MDA)、羟自由基(OH·)含量,三价铁还原法、黄嘌呤氧化酶法和NADPH还原法测定细胞总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)和谷胱甘肽还原酶(GR)活性。结果①ACR可明显降低Sertoli细胞的活力,呈剂量时间依赖性(P<0.05),3h、12h的CD50分别为105,200μmol/L;②ACR干预后可见细胞内ACR-Pro显著增多(P<0.05);③各实验组中氧化应激损伤的标志物MDA和OH·含量分别较对照组显著升高(P<0.05);①各实验组反映抗氧化能力的指标T-AOC、SOD和GR均较对照组明显降低(P<0.05)。结论体外实验证实ACR对未成熟睾丸Sertoli细胞具有明显的氧化应激损伤,提示CP对未成熟睾丸的损伤很可能即是其代谢产物ACR的氧化应激损伤所致。
Objective To investigate the oxidative stress injury of immature testes Sertoli cells induced by cyclophosphamide (CP) acrolein (ACR) and its mechanism. Methods The primary cultured Sertoli cells of neonatal SD rats were established. The experimental groups were given different concentrations of ACR solution. The control group was given PBS solution. After 3 and 12 hours, the viability of each group was determined by MTT assay. / L concentration of ACR intervention. Immunocytochemistry was used to detect the content of malondialdehyde (MDA) and hydroxyl radical (OH ·) of acrolein-protein adduct (ACR-Pro), thiobarbitol and Fenton method, , Total cellular antioxidant capacity (T-AOC), superoxide dismutase (SOD) and glutathione reductase (GR) activity were determined by xanthine oxidase and NADPH reduction. Results ① ACR significantly decreased the viability of Sertoli cells in a time-and dose-dependent manner (P <0.05). The CD50 values at 3h and 12h were 105 and 200μmol / L, respectively. (2) ACR-Pro was significantly increased after ACR intervention ③ The contents of MDA and OH · in oxidative stress injury in each experimental group were significantly higher than those in the control group (P <0.05). (1) The indexes of T-AOC, SOD and GR Group was significantly lower (P <0.05). Conclusion In vitro experiments showed that ACR had obvious oxidative stress on Sertoli cells in immature testes, suggesting that the damage of CP to immature testes is probably caused by the oxidative stress injury of its metabolite ACR.