AAPH体外诱导小鼠睾丸间质细胞氧化应激模型建立与评价

目的:利用偶氮二异丁脒盐酸盐(AAPH)作为刺激源,建立体外小鼠睾丸间质细胞(LCs)氧化应激损伤模型,并进行生理功能评价. 方法:实验一将小鼠睾丸间质细胞(TM3)随机分为6组,分别加入0、1、5、10、50和100 mmol/L AAPH,分别作用4、8、24 h.测定TM3细胞活性(MTS法)、衰老情况(β-半乳...

Establishment and evaluation of an AAPH-induced oxidative stress model in mouse Leydig cells in vitro

Objective:To establish a model of oxidative stress(OS)injury in mouse Leydig cells usingα-α′-azodiisobutyramidine hydrochloride(AAPH)and evaluate the physiological function.Methods:In Experiment 1,we treated mouse TM3 Leydig cells with AAPH at 0,1,5,10,50 and 100 mmol/L for 4,8 and 24 h respectively and measured the activity of the cells using MTS,their aging byβ-galactoside staining,mitochondrial membrane potential by JC-1 fluorescence and mitochondrial DNA copy number by qPCR.In Experiment 2,we treated the TM3 cells selected in Experiment 1 according to the AAPH concentration range(≤10 mmol/L)with AAPH at 0,0.1,0.5,1,2,4,6,8 and 10 mmol/L for 24,48 and 72 h respectively,detected the activity and aging of the cells and the ROS positive rate,and determined the optimal concentration and time of AAPH in inducing OS injury in the TM3 cells.Results:Experiment 1 showed that the survival rate of the TM3 cells was≥50%in the 4-h 50 mmol/L,8-h 10 mmol/L and 24-h 5 mmol/L AAPH groups,the initial concentration of AAPH was≤10 mmol/L,with the action time of≥24 h.Experiment 2 manifested that in the 24-h 6 mmol/L AAPH group,the survival rate of the TM3 cells was≥70%,with an ROS positive rate of 56.88%,normal mitochondrial membrane potential,increased number of mtDNA copies,but no senescence.Conclusion:Treatment with AAPH at the concentration of 6 mmol/L for 24 hours is suitable for induction of OS injury in the TM3 cells.