氧化应激在三邻甲苯磷酸酯诱导小鼠睾丸间质细胞自噬中的作用

发布时间：2018-03-25 11:55

本文选题：三邻甲苯磷酸酯　切入点：小鼠睾丸间质细胞　出处：《南昌大学》2016年硕士论文

【摘要】：目的:三邻甲苯磷酸酯(TOCP)是三甲苯磷酸酯(TCP)的3种异构体之一,普遍用在工业上及日常生活上。其除了可以引起机体出现神经毒性症状之外,还可导致生殖毒性。本课题主要通过观察TOCP对体外培养的小鼠睾丸间质TM3细胞的影响,探究氧化应激在TOCP诱导小鼠睾丸间质细胞毒性中的作用及调节机制。方法:MTT法分别检测TOCP和H2O2对小鼠睾丸间质细胞活性的影响;ELISA法检测TOCP对小鼠睾丸间质细胞内睾酮含量的影响;分别用RT-PCR和流式细胞仪检测TOCP对小鼠睾丸间质细胞周期的影响;分别用Annexin V-FITC/PI双染色法以及Hoechst染色法检测TOCP对小鼠睾丸间质细胞凋亡的影响;分别用Western blot法和电镜检测TOCP和H2O2对小鼠睾丸间质细胞自噬的影响;用活性氧试剂检测盒分别检测TOCP对小鼠睾丸间质细胞氧化应激的影响。结果:用不同浓度的TOCP(0,0.125,0.25,0.5 m M)作用于小鼠睾丸间质细胞TM348 h,小鼠睾丸间质细胞活性显著下降(P0.05),其睾酮含量也显著下降(P0.05);但细胞周期和凋亡并无明显影响;Western blot结果显示,细胞内自噬蛋白LC3-II/LC3-I的比例明显增加,Atg5和Beclin1的蛋白表达量也显著增加;电镜结果显示,0.5 mM TOCP可显著增加TM3细胞质内自噬小泡的数量;这些结果提示,TOCP可诱导小鼠睾丸间质TM3细胞自噬。在用不同浓度的TOCP(0,0.125,0.25,0.5 m M)处理小鼠睾丸间质TM3细胞48 h后,小鼠睾丸间质细胞内MDA的含量显著增加(P0.05),而SOD、GSH和GSH-PX活性显著下降(P0.05),提示TOCP可诱导小鼠睾丸间质TM3细胞氧化应激。用不同浓度的H2O2(0,100,200μM)作用于小鼠睾丸间质细胞48 h后,TM3细胞活性显著下降,细胞自噬显著增加,提示氧化应激可诱导TM3细胞自噬。而氧化应激抑制剂N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)可缓解TOCP对TM3细胞活性的抑制及自噬的诱导。结论:TOCP可抑制小鼠睾丸间质TM3细胞活性并诱导其自噬,但对其细胞周期和凋亡无明显影响;TOCP可诱导小鼠睾丸间质TM3细胞氧化应激;TOCP通过氧化应激诱导小鼠睾丸间质TM3细胞自噬。
[Abstract]:Objective: Trio-toluene phosphate (TTP) is one of the three isomers of TCPs, which is widely used in industry and daily life. In this study, we observed the effect of TOCP on the TM3 cells of mouse testis stromal cells cultured in vitro. To explore the role of oxidative stress in TOCP induced mouse testicular interstitial cytotoxicity and its regulatory mechanism methods the effects of TOCP and H2O2 on the activity of mouse testicular stromal cells were detected by TOCP method. The effect of testosterone content; RT-PCR and flow cytometry were used to detect the effect of TOCP on the interstitial cell cycle of mouse testis, and Annexin V-FITC/PI double staining and Hoechst staining were used to detect the effect of TOCP on the apoptosis of mouse testicular interstitial cells. The effects of TOCP and H2O2 on mouse testicular interstitial cell autophagy were detected by Western blot and electron microscope. The effects of TOCP on oxidative stress of mouse testicular interstitial cells were detected with reactive oxygen species test kit. Results: the activity of interstitial cells of mouse testis decreased significantly when different concentrations of TOC 0. 125 and 0. 25 0. 5 mm M were added to TM348 hs of mouse testicular interstitial cells. The testosterone content of P0.05 was also significantly decreased, but the cell cycle and apoptosis were not significantly affected by Western blot. The percentage of intracellular autophagy protein (LC3-II/LC3-I) increased significantly, and the protein expression of Atg5 and Beclin1 increased significantly, and the results of electron microscopy showed that 0.5 mm TOCP could significantly increase the number of autophagy vesicles in the cytoplasm of TM3. These results suggested that TOC could induce autophagy of mouse testis interstitial TM3 cells. After 48 hours of treatment with different concentrations of TOCP0 / 0.125 ~ 0.250.50mm / ml, the TM3 cells of mouse testis stromal cells were treated with TOC for 48 h. The content of MDA in the interstitial cells of mouse testis increased significantly, while the activities of GSH-PX and GSH decreased significantly, suggesting that TOCP could induce oxidative stress in the interstitial TM3 cells of mice testis. After 48 h of treatment with different concentrations of H2O2(0100200 渭 M, TOCP could induce oxidative stress in the interstitial cells of mice testis. The activity of TM3 cells decreased significantly. Autophagy increased significantly, These results suggest that oxidative stress can induce autophagy of TM3 cells, while N-acetyl-L-cysteine (NACN), an inhibitor of oxidative stress, can alleviate the inhibition of TM3 cell activity and autophagy induced by TOCP. Conclusion: TOC can inhibit the activity of TM3 cells and induce autophagy in mouse testis interstitial TM3 cells. However, there was no significant effect on the cell cycle and apoptosis. TOC could induce oxidative stress of TM3 cells in mouse testis. TOC induced autophagy of TM3 cells in mouse testis by oxidative stress.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R114

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