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林鹏飞, 郝元斌, 郭慧利, 刘红林, 芮 荣. 2010: 猪分离卵泡体外培养过程中Fas/FasL对颗粒细胞凋亡的作用. 动物学研究, 31(3): 268-274. DOI: 10.3724/SP.J.1141.2010.03268
引用本文: 林鹏飞, 郝元斌, 郭慧利, 刘红林, 芮 荣. 2010: 猪分离卵泡体外培养过程中Fas/FasL对颗粒细胞凋亡的作用. 动物学研究, 31(3): 268-274. DOI: 10.3724/SP.J.1141.2010.03268
LIN Peng-Fei, HAO Yuan-Bing, GUO Hui-Li, LIU Hong-Lin, RUI Rong. 2010: Role of Fas/FasL on Apoptosis of Porcine Follicular Granulose Cells Derived from Isolated Follicles During Culture in vitro. Zoological Research, 31(3): 268-274. DOI: 10.3724/SP.J.1141.2010.03268
Citation: LIN Peng-Fei, HAO Yuan-Bing, GUO Hui-Li, LIU Hong-Lin, RUI Rong. 2010: Role of Fas/FasL on Apoptosis of Porcine Follicular Granulose Cells Derived from Isolated Follicles During Culture in vitro. Zoological Research, 31(3): 268-274. DOI: 10.3724/SP.J.1141.2010.03268

猪分离卵泡体外培养过程中Fas/FasL对颗粒细胞凋亡的作用

Role of Fas/FasL on Apoptosis of Porcine Follicular Granulose Cells Derived from Isolated Follicles During Culture in vitro

  • 摘要: 从猪卵巢分离完整有腔卵泡,按质量分为3类:健康卵泡、早期闭锁卵泡和晚期闭锁卵泡。猪分离卵泡经眼观检查后再行石蜡切片和HE染色,形态学研究表明,眼观检查对于健康卵泡的判定准确率为92%。取健康卵泡按直径大小分为3组:直径>5 mm大卵泡组、3~5 mm中卵泡组和≤3 mm小卵泡组。卵泡培养8、16和24 h,以Annexin-V FITC/PI双染流式细胞仪检测壁层颗粒细胞凋亡情况,结果发现培养卵泡颗粒细胞的总凋亡率(早期凋亡+晚期凋亡)在8 h时就已达到70%以上,至24 h则为81.1%~94.6%。收集无血清培养0、8、16、24、48和72 h的卵泡颗粒细胞,用real time PCR SYBRgreen法检测各组卵泡颗粒细胞FasL和Fas mRNA相对表达量。各级卵泡颗粒细胞中FasL mRNA水平随培养时间显著增加,培养至24 h达最大值(P<0.05);小卵泡颗粒细胞FasL mRNA水平均高于大、中卵泡组。各级卵泡颗粒细胞Fas mRNA相对表达量在培养前(0 h)差异不显著,8 h时显著增加,48 h达最大值。该实验表明,所用无血清卵泡培养体系可有效诱导卵泡颗粒细胞的凋亡,细胞凋亡是卵泡闭锁的主要诱因,但卵泡闭锁程度可因卵泡大小而异,小卵泡似乎更容易发生闭锁。

     

    Abstract: The whole antral follicles were isolated from porcine ovaries and classified as follows: healthy follicles (HF), early atretic follicles (EF) and progressed atretic follicles (PF). The isolated porcine follicles were used for routine histological section and HE staining after examination by eyesight. Morphological research shows that the accuracy rate of eyesight examination for HF is 92%. Healthy follicles were chosen for further experiment and divided into 3 groups: large follicle (Æ>5 mm), medium follicles (3−5 mm) and small follicles (≤3 mm). All follicles were cultured for 8, 16 and 24 h, respectively and the apoptosis of of their granulose cells were examined by Annexin V-FITC/PI double-labeling. It showed that the total apoptotic rate of granulose cells derived from cultured follicles could reach over 70% at 8 h after culture and be 81.1%-94.6% at 24 h after culture. Granulosa cells from groups were collected at 0, 8, 16, 24, 48 and 72 h after culture without serum and used for the examination of expression of FasL and Fas mRNA with real time PCR SYBRgreen method. The expression level of FasL mRNA of granulose cells from different size of follicles increased with culture time and reached the highest level at 24 h after culture (P<0.05). Expression level of FasL mRNA of granulose cells from small follicles was higher than those from large and medium follicles. There exists no difference for expression level of Fas mRNA of granulose cells among groups before culture but significantly increased at 8 h after culture and reached the highest level at 48 h after culture. It showed in the present experiment that the follicular culture system without serum used could effectively induce the apoptosis of follicular granulose cells. Cell apoptosis is the main cause of follicular atresia, the degree of which varied with the size of follicles. Small follicles seemed to be easier atretic than medium and large follicles.

     

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