目的 哺乳动物精子发生是一种由一系列生精细胞组成的连续细胞增殖和分化并最终产生精子的过程.然而,生精细胞的蛋白稳态分析仍缺乏有效手段.本研究的目的是探索一种在体标记并检测生精细胞新生蛋白合成的方法.方法 利用氨基酰 tRNAs结构类似物——嘌呤霉素(Puromycin,Puro)以 65 mg/kg 小鼠体质量的剂量腹腔注射小鼠,1.5 h后,处死小鼠,分离两侧睾丸,其中一侧睾丸用于总蛋白提取,另一侧睾丸用于石蜡切片制备,随后利用 Puro特异性抗体分别通过 Western blot和免疫荧光检测样品中 Puro 的含量及定位.结果 Western blot结果显示 Puro能够标记总体新生蛋白合成情况;免疫荧光共定位显示 Puro在小鼠睾丸中特异性标记的是精原细胞.结论 Puro能够用于小鼠精原细胞新生蛋白质的在体标记,并可通过相应免疫学技术定量分析新生蛋白合成水平.
A Method for in vivo Labeling Nascent Protein Synthesis of Mouse Spermatogonia
Objective Mammalian spermatogenesis is a process with continuous cell proliferation and differentiation composed of a series of spermatogenic cells,and ultimately to produce sperm.However,the proteostasis analysis of spermatogenic cells still lacks effective method.The aim of this study was to explore a method to label and detect nascent protein synthesis in spermatogenic cells in vivo.Method Puromycin(Puro),an aminoacyl-tRNA structural analog,was intraperitoneally injected into mice at a dose of 65 mg/kg body weight.1.5 hours later,the mice were killed and bilateral testes were isolated.Of these,one of the testes was used for total proteins extraction and the other was used for paraffin sections preparation.Then,the content and localization of Puro in testis were respectively detected by Western blot and immunofluorescence using Puro specific antibody.Result Western blot result showed that Puro could label the overall nascent proteins.Co-immunofluorescence analyses showed that Puro specifically labeled spermatogonia in mouse testis.Conclusion Nascent proteins in mouse spermatogonia can be in vivo labelled by Puro,and their levels can be quantitated through corresponding immunological techniques.
Puromycinspermatogenesisspermatogonianascent protein synthesis
万方数据
维普
