一种小鼠睾丸细胞涂片的快速制备方法  

A fast method for preparing the mouse germ cell slides

作　　者：杨梦[1,2] 左笑丛[1,2] 李艳[3] 黄利华[3] 邢晓为[3] 

机构地区：[1]中南大学湘雅三医院药学部,湖南长沙410013 [2]中南大学药学院,湖南长沙410013 [3]中南大学湘雅三医院医学实验中心,湖南长沙410013

出　　处：《细胞与分子免疫学杂志》2014年第2期202-205,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81170615);湖南省科技厅项目(S2010R1040)

摘　　要：目的建立一种用于免疫荧光实验的小鼠睾丸细胞涂片的快速制备新方法。方法分别给出生后16、21、28 d和120 d的小鼠腹腔注射秋水仙素,3 h后取睾丸组织剔除白膜,分离曲精小管进行剪碎,加入冷PBS重悬细胞,过滤,并用冷PBS洗涤细胞3次,然后低渗处理,最后经多聚甲醛固定后铺片。对细胞核进行DAPI染色,并以21 d小鼠睾丸涂片为例进行免疫荧光实验,检测所制备的细胞涂片的质量。结果所制备的小鼠睾丸细胞涂片经DAPI染色,每张细胞涂片均分布大量生殖细胞,背景清晰,根据细胞核的大小与着色,可初步判断生殖细胞类型;用不同发育阶段小鼠所制备的睾丸细胞涂片,细胞分布类型有很大差异,其中28 d小鼠制备的睾丸细胞涂片中生殖细胞类型最多;以21 d小鼠睾丸细胞涂片为例进行免疫荧光实验,红色和绿色荧光标记的目标蛋白在精母细胞中表达清晰,背景干净,说明所制备的细胞涂片适合于免疫荧光检测。结论成功建立了一种快速制备小鼠睾丸细胞涂片的新方法。Objective To establish a new method of fast preparation of mouse germ cell slides for immunofluorescence assay. Methods Mice of different developing stages （at postnatal days 16, 21,28 and 120） were treated with colchicine by intraperitoneal injection respectively, and 3 hours later, the testes were taken out and the tunica albuginea were removed. The seminiferous tubules were separated and minced into small pieces, resuspended with ice-cold PBS, and the cells were filtered and collected by centrifugation. After washed with ice-cold PBS for three times, the cells were treated with a hypotonic solution, fixed with 4% paraformaldehyde, and finally mounted onto the slides. To test the slide quality, the cells mounted on the slides were stained with DAPI, and immunofluorescence staining was performed using 21-day-old mouse slides. Results Stained with DAPI, the distribution of various germ cells was observed on each slide by the new method and the background was clear. According to the size and color of the cell nucleus, the types of germ cells could be recognized preliminarily. The types of germ cells on the slides were very different among 16-, 2t-, 28- and t20-day-old mice, of which the slides from 28- day-old mice had the most types of germ cells. Immunofluorescence staining using 2J-day-old mice slides revealed that the red-and green-fluorescence labeled target proteins were observed in spermatocytes, and the background was clear, suggesting the slides were suitable for immunofluorescence detection. Conclusion A new method of fast preparing the mouse germ cell slides was successfully established, providing a foundation for the study on the process of spermatogenesis.

关 键 词：小鼠睾丸 细胞涂片 低渗处理 免疫荧光技术 精子发生 

分 类 号：R392-33[医药卫生—免疫学] R321.1[医药卫生—基础医学]