改良消化富集法提高小鼠精原细胞染色体制备效率的研究及应用  

Study and application of a modified enrichment method to improve the efficiency of chromosome preparation in mouse spermatogonial cells

作　　者：张静[1] 程东[1] 姚文环[1] 杨非[1] 唐慧[1] 孙昌华 颜燕[1] 周雯[1] 李慧[1] ZHANG Jing;CHENG Dong;YAO Wenhuan;YANG Fei;TANG Hui;SUN Changhua;YAN Yan;ZHOU Wen;LI Hui(Shandong Center for Disease Control and Prevention,Jinan 250014,China)

机构地区：[1]山东省疾病预防控制中心,济南250014

出　　处：《中国比较医学杂志》2023年第8期75-79,85,共6页Chinese Journal of Comparative Medicine

基　　金：山东省中医药科技发展计划项目(Z-2022061);山东省中医药科技发展计划项目(2020M055)。

摘　　要：目的建立一种改良的小鼠精原细胞染色体畸变试验方法,以获得大量结构清晰的中期分裂相精原细胞利于阅片,并将改进后的方法应用于玛咖的致突变实验。方法选取雄性ICR小鼠30只,随机分为6组,每组5只,将原染色体制备方法增加了剪碎曲细精管及精原细胞消化富集,采用1.5 mg/mL的胶原酶,35℃消化15 min,每隔3 min搅拌1次,消化富集精原细胞,经低渗、固定、滴片,封片后镜检阅片,同时与国标方法所制备的滴片进行比较阅片。结果改良消化富集法所制备的滴片背景清晰,染色体分散良好,收缩适中,易于观察。分析每只动物100个中期分裂相精原细胞所需观察的高倍视野数(25±3.9)与国标方法组(75±5.6)比较明显减少(P<0.01),精原细胞有丝分裂指数(18.04±2.47)与国标方法组(8.83±2.04)比较明显增加(P<0.01);环磷酰胺处理组染色体畸变率(15.40±2.30)与空白对照组(3.60±1.14)比较显著增加(P<0.01);玛咖各剂量组染色体畸变率与空白对照组比较无统计学意义(P>0.05)。结论改良消化富集法能明显提高精原细胞富集效率及实验成功率,可获得大量分散良好、背景清晰的中期分裂相精原细胞,明显优于国标方法;在本次实验条件下,玛咖对小鼠精原细胞染色体无致突变作用。Objective To establish an improved method for chromosome aberration testing in mouse spermatogonia,obtain large amounts of constant and clear metaphase chromosomes for reading,and apply the improved method to assess the mutagenic effect of Maca.Methods Thirty male ICR mice were randomly divided into six groups with five mice in each group.Seminiferous tubules were minced for spermatogonial cell enrichment.The fragments were treated with a 1.5 mg/mL collagenase solution(in serum-free culture medium at 35℃for 15 minutes with intermittent agitation(3 minute intervals).After routine hypotonic treatment,fixation,and cell smearing,slides were observed under a microscope and compared with those prepared by the conventional method.Results A large number of well-dispersed metaphase phase spermatogonium with a clear background and compact chromosome structure were prepared and observed by our method,and the chromosome slides prepared by this method were easy to read.Compared with the result with the conventional method,the number of high magnification fields required for observation was significantly decreased(P<0.01)and the mitotic index was obviously increased(P<0.01).The chromosomal aberration rate of the cyclophosphamide-positive control group was significantly higher than that of the control group(P<0.01).No significant differences were found in the chromosome aberration rate between the Maca groups of various dosages and the control group(P>0.05).Conclusions The enrichment efficiency of spermatogonial cells and the success rate of experiments were improved by the modified method,and this method was significantly better than conventional method.Maca had no effect on the chromosomes of mouse spermatogonial cells under the experimental conditions.

关 键 词：改良消化富集法 小鼠精原细胞 染色体畸变 玛咖 

分 类 号：R-33[医药卫生]