肿瘤转移抑制因子(Kisspeptin)对牛卵巢颗粒细胞凋亡的影响

摘要

肿瘤转移抑制因子(Kisspeptin)在颗粒细胞上有表达,但其对颗粒细胞凋亡的作用尚不清楚,本试验旨在利用流式细胞术和荧光定量PCR的技术研究Kisspeptin对牛卵巢颗粒细胞凋亡的影响.向DMEM-F12培养液中分别添加0、100 nmol/L的Kp-10培养牛颗粒细胞,24 h后收集细胞,用流式细胞技术检测细胞凋亡和细胞周期,用荧光定量PCR技术检测Caspase-3、Bcl-2、Fas和Fasl的mRNA表达.结果表明:Kisspeptin可显著促进牛颗粒细胞凋亡,且伴随着Caspase-3、Fas和Fasl mRNA表达上调及Bcl-2 mRNA表达下调(P＜0.01);流式细胞分析表明,Kisspeptin可使牛颗粒细胞在G1期增加、S期下降(P＜0.05).研究结果说明,Kisspeptin可促进牛颗粒细胞凋亡,这一作用可能通过上调促凋亡基因Caspase-3、Fas、Fasl和下调抗凋亡基因Bcl-2的mRNA表达实现.%Previous studies showed that Kisspeptin expresses in granulosa cells,however,its effect on apoptosis of bovine granulosa cells is still unknown.In the present study,flow cytometry analysis and q Real Time quantitative-PCR were used to study effect of Kisspeptin on apoptosis of bovine granulosa cells.Bovine granulosa cells were cultured with DMEM-F12 solution added with 0 or 100 nmol/L of Kp-10 for 24 h,then flow cytometry was used to study apoptosis and cell cycle of the bovine granulosa cells,and Real Time quantitative-PCR was used to study mRNA expression of Caspase-3,Bcl-2,Fas and Fasl.The results showed that Kisspeptin could promote apoptosis of bovine granulosa cells,accompanied with the decreased mRNA levels of Bcl-2 and the increased mRNA levels of Caspase-3,Fas and Fasl (P＜0.01).Additionally,results of flow cytometry analysis showed that Kisspeptin could significantly increase accumulation of cells in the G1 phase,decrease accumulation of cells in the S phase,and promote apoptosis of bovine granulosa cells (P＜ 0.05).Thus,our findings demonstrated for the first time that Kisspeptin promoted apoptosis of bovine ovarian granulosa cells,which may be achieved through regulation of Caspase-3,Fas,Fasl and Bcl-2 mRNA expression.