利用白消安致BALB/c小鼠生殖毒性建立NOA疾病模型

Establishment of NOA Model in BALB/c mice induced by busulfan

目的利用白消安对BALB/c雄性小鼠生殖毒性,建立不同损伤程度的NOA(non-obstructive azoospermia,NOA)动物模型。方法本研究取6~8周龄性成熟的BALB/c雄性小鼠共45只,随机分为3组:0 mg/kg组、30 mg/kg组、40 mg/kg组,每组15只。每2周观察并记录小鼠体重,睾丸和附睾湿重,睾丸体积,及生精细胞损伤程度。通过HE染色,判断生精细胞受损情况;通过免疫组化技术检测受损生精细胞停滞的减数分裂阶段,该法主要借助生精细胞减数分裂过程中的三个标记蛋白:STRA8(Stimulated by Retinoic Acid 8,STRA8)减数分裂启动标记蛋白,SCP3(Synaptonemal Complex Protein 3,SCP3)减数分裂中标记蛋白,TNP1(Transition Protein 1,TNP1)减数分裂后标记蛋白。结果注射白消安后小鼠存活良好,注射白消安2周时,生精细胞开始损伤,30 mg/kg组轻于40 mg/kg组;4周时,40 mg/kg组呈唯支持细胞样变化;6-10周时,生精功能开始恢复,30 mg/kg组恢复明显快于40 mg/kg组;40 mg/kg组STRA8、SCP3、TNP1表达在注射4周最低,第8周开始恢复到正常水平。结论白消安40 mg/kg组单次注射4周时,可建立为唯支持细胞型NOA生精障碍模型;单词注射4-8周期间,可建立损伤程度不同的NOA生精障碍模型,为睾丸组织体外培养提供稳定的研究组织。

Objective To establish a NOA(non-azoospermia)animal model with different degrees of injury by means of busulfan to the reproductive toxicity of BALB/C male mice.Methods A total of 45 BALB/C male mice aged 6-8 weeks were randomly divided into 3 groups:0 mg/kg group,30 mg/kg group and 40 mg/kg group,15 mice in each group.Weight of mice,wet weight of testis and epididymis,testicular volume,and spermatogenic cell damage were observed and recorded every 2 weeks.The damage of spermatogenic cells was determined by HE staining.By immunohistochemical technology to detect damage at the stagnation of sperm cells meiosis stage,it mainly uses three of the born sperm cells meiosis marker Protein:STRA8(Stimulated by Retinoic Acid,8 STRA8)meiosis start marker Protein,SCP3(Synaptonemal Complex Protein 3,SCP3)marker Protein in meiosis,TNP1(Transition Protein 1,TNP1)marker Protein after meiosis.Results The mice survived well after the injection.After 2 weeks of the injection,spermatogenic cells began to damage,and the damage in the 30 mg/kg group was less than that in the 40 mg/kg group.At 4 weeks,the 40 mg/kg group showed only supportive cell changes.At 6-10 weeks,the spermatogenic function began to recover,and the recovery was significantly faster in the 30 mg/kg group than in the 40 mg/kg group.The expression of STRA8,SCP3 and TNP1 in the 40 mg/kg group was lowest at 4 weeks after injection,and returned to normal at 8 weeks.Conclusion After a single injection of 40 mg/kg baishangan for 4 weeks,the model of NOA spermatogenesis disturbance could be established.During 4-8 weeks of injection,NOA spermatogenic disorder models with different damage degrees could be established,providing stable research tissues for testicular tissue culture in vitro.