Fas expression and mediated activation of an apoptosis programme in bovine follicular granulosa cells in vitro.
Fas expression and mediated activation of an apoptosis programme in bovine follicular granulosa cells in vitro.
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DOI：
10.1111/j.1439-0531.2011.01914.x
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发表时间：
2012-08
期刊：
影响因子：
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通讯作者：
R. Yang;J. Li;Z. Zhao;X. Gao;H. Gao;S. Z. Xu
中科院分区：
文献类型：
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作者：
R. Yang;J. Li;Z. Zhao;X. Gao;H. Gao;S. Z. Xu
Fas抗原是一种跨膜受体，可以触发多种体细胞的凋亡。卵巢卵泡闭锁和黄体溶解被认为是通过细胞凋亡发生的。为了揭示牛卵巢卵泡发育过程中细胞内信号转导分子的作用，本研究采用RT-PCR技术扩增了不含终止密码子的牛卵巢Fas基因，并将其直接克隆到真核表达载体pAcGFP-N1中。将重组质粒pAcGFP-bFas转染牛卵泡颗粒细胞。荧光显微镜下观察AcGFP的表达，RT-PCR和western blot检测Fas的转录和翻译。采用四甲基偶氮唑蓝（MTT）比色法、Hoechst 33342染色法和DNA梯状条带法检测细胞生长抑制和凋亡情况。结果显示，转染后24 h即可检测到GFP的表达。RT-PCR检测到Fas mRNA的994 bp片段，Westernblot检测到Fas融合蛋白的64.5 kD条带，证实Fas融合基因在颗粒细胞中成功表达。转染后72 h，颗粒细胞活力明显下降，pAcGFP-Fas转染组细胞凋亡率明显高于对照组。Fas转染组细胞呈凋亡特征的梯形图，细胞核皱缩、深染或碎裂，提示Fas过表达可抑制牛卵泡颗粒细胞增殖并诱导细胞凋亡。本研究有助于进一步了解Fas对牛卵母细胞形成和发育的调控机制。
The Fas antigen is a transmembrane receptor that can trigger apoptosis in a variety of somatic cells. Ovarian follicular atresia and luteolysis are thought to occur by apoptosis. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, Fas gene without the stop codon was amplified in the present study using RT-PCR and directly cloned into the eukaryotic expression vector pAcGFP-N1. The resultant recombinant plasmid pAcGFP-bFas was then transfected into bovine follicular granulosa cells. Expression of AcGFP was observed under fluorescent microscopy, and the transcription and translation of Fas were detected by RT-PCR and western blot analysis. The methyl-tetrazolium (MTT) assay, Hoechst33342 staining and DNA ladder method were performed to determine the growth inhibition and apoptosis of the cells. The results showed that GFP expression was detected as early as 24 h after transfection. The Fas fusion gene was successfully expressed in granulosa cells as evidenced by the detection of a 994-bp fragment corresponding to the Fas mRNA by RT-PCR and a 64.5-kD band corresponding to the Fas fusion protein by western blot. Granulosa cell viability decreased significantly at 72 h after transfection, and the apoptosis rate of the cells transfected with pAcGFP-Fas was significantly higher than that of the control group. Cells in the Fas transfection group showed ladder patterns characteristic of apoptosis, and the nuclei were shrunken and densely hyperchromatic or fragmented, suggesting that Fas is capable of inhibiting the proliferation of bovine follicular granulosa cells and inducing cell apoptosis when over-expressed. This study will aid in further understanding the mechanism of regulation of Fas on bovine oocyte formation and development.