【摘要】 目的:寻找大鼠精子发生相关蛋白,研究β-肌动蛋白在大鼠睾丸组织的表达和分布。方法:用牛血清白蛋白梯度沉降(STAPUT)法从9日龄雄性SD大鼠睾丸中分离出A型精原细胞,从成年雄性大鼠睾丸中分离出粗线期精母细胞、圆形精子细胞;分别提取这3种细胞的总蛋白,进行双向电泳;对所得到的双向电泳图谱用Im ageM aster2D E lite图像分析软件分析,找出差异蛋白,对挑选出的差异蛋白做质谱分析。进一步用β-肌动蛋白抗体做免疫组化的睾丸组织定位研究。结果:在双向电泳图谱中,β-肌动蛋白在A型精原细胞、粗线期精母细胞中表达量较高,在圆形精子细胞中则表达量极少。免疫组化研究发现:A型精原细胞、粗线期精母细胞有阳性颗粒反应,圆形精子细胞则无阳性颗粒反应;在接近成熟的精子细胞中,呈现极强的阳性颗粒反应,并且越接近排放期的精子细胞,阳性反应越强。在接近成熟的精子头部,阳性颗粒反应最强。β-肌动蛋白主要在精原细胞和精母细胞的细胞质中表达;在接近成熟的精子细胞中主要表达在细胞核。结论:β-肌动蛋白在精子发生过程中有明显的阶段差异表达,推测其对精子发生起重要调节作用。更多还原

【Abstract】 Objective: To screen the stage-specific expression proteins during rats spermatogenesis,and to investigate the β-actin expression and localization in the tissues of rat testicular.Methods: Highly enriched type A spermatogonia,pachytene spermatocytes and round spermatids were isolated by STAPUT method(sedimentation velocity at unit gravity,with 2%～4% BSA gradient in DMEM/F12 medium) respectively to get the total proteins.The difference of protein expression between the three kinds of cells was analyzed by two-dimensional electrophoresis.Then the distribution of β-actin in rat testicular tissues was investigated using specific anti-β-actin antibodies by immunohistochemical method.Results: β-actin was identified as a stage-specific expression protein by two-dimensional electrophoresis.β-actin protein was more strongly expressed in type A spermatogonia and pachytene spermatocytes,but not in round spermatids.The immunohistochemical results showed that β-actin was mainly located in the cytoplasm of type A spermatogonia and pachytene spermatocytes and in the nuclei of nearly mature spermatids.Conclusion: β-actin protein is a stage-specific expressed protein and may play an important role in spermatogenesis. Natl J Androl,2005,11(10):755-760更多还原