中国农业科学 ›› 2013, Vol. 46 ›› Issue (23): 5037-5043.doi: 10.3864/j.issn.0578-1752.2013.23.021

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

增殖细胞核抗原在水牛腔前卵泡中的表达与卵巢组织定位

 马帆, 周宇, 高亚可, 朱鹏, 雷小灿, 刘庆友, 石德顺   

  1. 广西大学亚热带生物资源保护利用国家重点实验室, 南宁 530005
  • 收稿日期:2013-04-19 出版日期:2013-12-01 发布日期:2013-07-26
  • 通讯作者: 刘庆友,E-mail:qyliu2002@126.com
  • 作者简介:马帆,E-mail:mafan1987@126.com
  • 基金资助:

    国家转基因重大专项(2011ZX08007-003)、国家自然科学基金项目(31160457)

Ovarian Immunohistochemistry Localization and Preantral Follicles Expression Pattern Analysis of Buffalo Proliferating Cell Nuclear Antigen

 MA  Fan, ZHOU  Yu, GAO  Ya-Ke, ZHU  Peng, LEI  Xiao-Can, LIU  Qing-You, SHI  De-Shun   

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bio Resources, Guangxi University, Nanning 530005
  • Received:2013-04-19 Online:2013-12-01 Published:2013-07-26

摘要: 【目的】探讨在水牛中增殖细胞核抗原(PCNA)参与卵泡发生的时期及其在卵巢组织不同时期卵泡中的表达定位;【方法】采用石蜡切片结合免疫组化的方法对PCNA在卵巢组织中进行定位,利用实时荧光定量PCR(QRT-PCR)技术检测PCNA在腔前卵泡中的表达情况。【结果】免疫组化结果显示:原始卵泡中的卵母细胞能检测到PCNA的表达,但颗粒细胞并未观察到PCNA的免疫染色;初级卵泡到次级卵泡,卵母细胞和颗粒细胞中均可检测到PCNA的表达,且在颗粒细胞中的表达有增强的趋势;有腔卵泡中,颗粒细胞、卵泡膜细胞及包围卵母细胞的卵丘细胞中都能观察到很强的免疫染色。定量表达检测结果显示:PCNA在原始卵泡、初级卵泡及次级卵泡中的表达呈上升趋势,并且有显著差异(43.50333±0.138338 vs 1.633804±0.093796 vs 1±0.012219 , P<0.01);【结论】从初级卵泡开始在颗粒细胞中能检测到PCNA的表达,并且其表达随着卵泡的发育有明显的增强趋势,腔前卵泡中PCNA表达的QRT-PCR测定结果与免疫组化结果相一致,研究结果为阐明PCNA参与卵泡发生的分子调控机制奠定了基础。

关键词: 水牛 , PCNA , 卵巢定位 , 定量表达

Abstract: 【Objective】To explore the involvement of nuclear antigen protein (PCNA) duiring different periods of buffalo follicular genesis, the expression pattern and ovary immunohistochemistry localization was performed in the present study.【Method】The localization of PCNA was demonstrated in ovarian tissue through the method of paraffin sections with immunohistochemistry, the mRNA expression level of PCNA in preantral follicles of buffalo was determined by real-time fluorescence quantification PCR(QRT-PCR).【Result】The immunohistochemical results showed that, in primordial follicle, no remarkable staining for PCNA in granulosa cells was observed, except the staining of nuclei in oocyte. In primary to secondary follicle, positive staining in oocytes and in some granulosa cells was observed, and PCNA immunoreactivity in granulosa cells of secondary follicle was obviously enhanced than that of primary follicles.The antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. The results of QRT-PCR showed that,the expression of PCNA in primordial follicles, primary follicles and secondary follicles showed a upregulation trend (43.50333±0.138338 vs 1.633804±0.093796 vs 1±0.012219 , P<0.01).【Conclusion】In buffalo primary follicles, the expression of PCNA could be detected in granulosa cells, and was obviously enhanced following the development of follicle. The expression pattern of PCNA determined by QRT-PCR in preantral follicles was accordant with the results of immunohistochemistry. These results will provide a foundation for clarifing the mechanism of PCNA involving of follicular genesis in Buffalo.

Key words: Buffalo , PCNA , immunohistochemistry , quantitative PCR assay