CAJ | 학술논문

目的：通过研究丝氨酸／精氨酸蛋白特异激酶2（ serine／arginine-rich protein specific kinase 2， SRPK2）基因mRNA及其编码蛋白产物在小鼠睾丸组织中的表达特征，探讨该基因在精子发生过程中的作用及意义。方法分别采用半定量逆转录聚合酶链反应（ RT-PCR）和蛋白免疫印迹杂交（ Western blotting）分析该基因mRNA及蛋白产物在小鼠多种组织中的表达；利用实时定量PCR（ real-time quantitative PCR）分析SRPK2 mRNA在不同发育阶段小鼠睾丸组织中的差异表达；应用免疫组织化学染色和间接免疫荧光技术观察SRPK2蛋白在小鼠曲精小管中的细胞定位和生精细胞内的亚细胞定位。结果半定量RT-PCR和Western blotting分析显示SRPK2 mR-NA和蛋白在小鼠睾丸组织中均大量表达；实时定量PCR分析发现SRPK2 mRNA在5周及8周龄雄性小鼠睾丸组织中显著表达，具有明显的阶段特异性表达特征。免疫组织化学染色结果表明SRPK2蛋白阳性着色主要位于曲精小管中的长形精子细胞核；间接免疫荧光分析显示SRPK2蛋白定位于长形精子细胞核表面。结论 SRPK2基因在小鼠睾丸组织中大量表达，并且具有显著的阶段特异性表达特征和明确的细胞核定位，极有可能在小鼠精子发生的变态成形期参与mRNA前体分子的剪接过程，其作用机制值得进一步深入研究。
목적：통과연구사안산／정안산단백특이격매2（ serine／arginine-rich protein specific kinase 2， SRPK2）기인mRNA급기편마단백산물재소서고환조직중적표체특정，탐토해기인재정자발생과정중적작용급의의。방법분별채용반정량역전록취합매련반응（ RT-PCR）화단백면역인적잡교（ Western blotting）분석해기인mRNA급단백산물재소서다충조직중적표체；이용실시정량PCR（ real-time quantitative PCR）분석SRPK2 mRNA재불동발육계단소서고환조직중적차이표체；응용면역조직화학염색화간접면역형광기술관찰SRPK2단백재소서곡정소관중적세포정위화생정세포내적아세포정위。결과반정량RT-PCR화Western blotting분석현시SRPK2 mR-NA화단백재소서고환조직중균대량표체；실시정량PCR분석발현SRPK2 mRNA재5주급8주령웅성소서고환조직중현저표체，구유명현적계단특이성표체특정。면역조직화학염색결과표명SRPK2단백양성착색주요위우곡정소관중적장형정자세포핵；간접면역형광분석현시SRPK2단백정위우장형정자세포핵표면。결론 SRPK2기인재소서고환조직중대량표체，병차구유현저적계단특이성표체특정화명학적세포핵정위，겁유가능재소서정자발생적변태성형기삼여mRNA전체분자적전접과정，기작용궤제치득진일보심입연구。
Objective To analyze the expression characteristics of SRPK2 ( serine /arginine-rich protein specific kinase 2 mRNA and its encoded protein products in mouse testis, and to reveal its molecular role during spermatogenesis. Methods Using semi-quantitative reverse transcription polymerase chain reaction ( RT-PCR) and Western blotting to an-alyze the expression of SRPK2 mRNA and its protein products in several mouse tissues.The stage-specific expression pat-tern of SRPK2 mRNA in mouse testis was examined by real-time quantitative PCR.Immunohistochemical staining was ap-plied to identify the SRPK2 protein distribution in mouse testicular seminiferous tubules and indirect immunofluorescence staining was used to detect the subcellular localization of SRPK2 protein in spermatic cells.Results Semi-quantitative RT-PCR and Western blotting analysis revealed that SRPK2 was highly expressed in mice testis.Real-time quantitative PCR analysis showed that SRPK2 mRNA was expressed abundantly at the stage of 5-and 8-week old mouse testes, suggesting that SRPK2 gene has stage-specific expression patterns during mouse spermatogenesis.Immunohistochemical and indirect immu-nofluorescence staining demonstrated that SRPK2 protein was located around the surface of elongated sperm nucleus. Conclusions SRPK2 is highly expressed in mouse testis and has significant stage-specific expression characteristics with distinct nuclear localization.These results indicate that SRPK2 may participate in precursor mRNA splicing during mouse spermiogenesis.The molecular mechanism of SRPK2 is yet to be further studied.