温和冷应激及复温对牦牛卵巢颗粒细胞的影响

Effects of Mild Cold Shock Followed by Warming Up at 37 ℃ on the Ovarian Granulosa Cellular Stress Response of the Yak

细胞冷应激反应是一个复杂的过程,它包括低温本身的应激阶段和复温后的应激阶段。本研究以牦牛卵巢颗粒细胞为研究对象,利用实时荧光定量PCR、Western blot技术,分析温和冷处理1、5d及复温1、2、4、8及24h条件下,细胞内CIRBP、HSP70及P53变化规律。结果显示,细胞在25℃培养5d,然后通过复温至37℃,会影响细胞生理状态以及诱导细胞应激反应。CIRBP的表达量在25℃温和冷处理第1天开始上升,并且在复温后24h左右恢复到正常水平。HSP70的表达量在37℃复温培养后逐步上升,复温8h出现峰值,随后下降。P53mRNA表达水平在复温培养4h出现峰值,随后下降,并且其在CIRBP及HSP70mRNA高表达时,均有明显的下降。结果表明,低温会诱导细胞CIRBP mRNA和蛋白表达水平的上升;HSP70能够被冷应激后的复温诱导;CIRBP和HSP70都具有抗应激损害和细胞凋亡的作用。这些数据将为亚低温和复温在各种研究和治疗领域的应用提供参考。

The cellular effects of hypothermia has been recognized a complex process including a stress response to cold and subsequent rewarming.In this study,real-time fluorescence quantitative PCR and Western blot techniques were used to observe the dynamic expression of CIRBP,HSP70 and P53in the yak ovarian granulosa cells.Cells were preincubated at 25℃for 1and 5d,and rewarmed at 37 ℃ for 1,2,4,8and 24 h.The results showed that hypothermia and subsequent rewarming affected cell biology and induced a cellular stress response.The expression of CIRBP was increased at 25 ℃ already at day 1and returned to basal level upon rewarming after24 h.While the expression of HSP70 was gradually increased upon rewarming at 37 ℃,maximal expression was occurred at 8hafter rewarming and followed by decline.Moreover the maximal expression of P53 was appeared at 4hafter rewarming and followed by a decline.Additionally,the observation that P53 mRNA was significantly decreased,aprocess that accompanied a maximal expression of CIRBPand HSP70 mRNA,suggested that the expression of CIRBP mRNA and protein was increased upon exposure to hypothermia,and HSP70 was induced upon rewarming.Overall,the CIRBPand HSP70 were shown to play a role in protecting cells from the deleterious effects of stress and apoptosis.These findings would bring new insights into the potential beneficial effects of mild hypothermia and rewarming used in various research and therapeuticalfields.