二次白消安腹腔注射致小鼠精子再生模型的建立

Establishment of a murine model of spermatogenesis recovery by two doses of Busulfan injection

目的 建立一种小鼠精子再生模型。方法采用不同剂量白消安（单剂10mg／kg、20mg／kg和间隔24d二剂10mg／kg）小鼠腹腔注射，分别于给药后第一、二、三、四、六、八周取材，进行光镜和电镜观察。二次给药组采用免疫组化检测生精细胞Ki-67表达，TUNEL标记法检测生精细胞凋亡。结果间隔24d二剂10mg／kg白消安对生精上皮的损伤效应介于单剂10mg／kg和20mg／kg之间。第二次给药后2周，生精上皮仅基底部残留以单个型精原细胞（As型）为主精原细胞和支持细胞；第三周As型精原细胞增多；第四周分化精原细胞和精母细胞出现；6～8周出现精子发生，生精上皮结构逐步恢复正常。二次给药后1～2周生精细胞凋亡指数显著高于对照组（P〈0．01），以后逐步下降，至第六～八周恢复至对照组水平（P〉O．05）。精原细胞Ki-67阳性率在第一～二周最低，在第三周最高，第四周开始下降，第六～八周与对照组无显著差异。结论间隔24d二次白消安（10mg·kg^-1·次^-1）腹腔注射可建立小鼠精子再生模型，诱导凋亡为白消安致生精细胞损伤的重要机制。二次给药后3周主要为精原干细胞增殖期，4周为分化期，6～8周为精子发生恢复期。

Objective To establish a model of spermatogenesis recovery in male Kunming mice. Methods Adult male Kunming mice were randomly treated with busulfan injection intraperitoneally at different doses or types （A： 10 mg/kg; B： 20 mg/kg; C： two doses of 10 mg/kg, 24 days apart）. Testes were dissected out 1 w,2 w,3 w,4 w,6 w and 8 w after treatment,and normal testes were used as control. The expression of Ki-67 in germ cells was detected by irnmunohistoehemistry and apoptosis was assessed by TUNEL in two doses of busulfan treatment group. Results The damage effect in seminiferous epithelium by two doses of 10mg/kg busulfan injection was between single dose of 10 mg/ kg and 20 mg/kg. In week 2, only fewer dispersed spermatogonia （As type） survived in close contact with the basal portions of adjacent Sertoli ceils. In week 3,the number of As type of spermatogonia increased. In week 4, differentiated spermatogonia and spermatocytes appeared. In week 6-8, spermatogenesis regeneration took place and morphology of seminiferous epithelium got back to normal gradually. The apoptotic indices （AI） of germ cells in 1 and 2 week groups were both higher than those in control group. Later, the AI began to drop in sequence. In week 6 8, it got back to the level of control group. In week 1 and 2, the Ki 67 positive rate of spermatogonia was lower than that in control group. It rebounded rapidly to apex in week 3, descended in week 4 and restored to the control level in week 6 and 8. Conclusions Two doses of busulfan treatment could establish a mice model of recovery of sper matogenesis and apoptosis was possibly an important mechanism of germ cells destroyed by busulfan.