摘要
目的探讨β-谷甾醇对人卵巢颗粒细胞系(KGN)增殖、凋亡及P-PI3KCA、P-AKT和Caspase-3蛋白表达的影响。方法体外培养KGN细胞,分为对照组和β-谷甾醇处理组,CCK-8测定添加10、20、40和80μmol·L^(-1)β-谷甾醇的KGN细胞的增殖情况以及20μmol·L^(-1)β-谷甾醇的KGN细胞在12、24、36和48 h细胞的增殖情况;流式细胞术检测细胞凋亡及周期分布,细胞免疫荧光检测KGN细胞内P-PI3KCA、P-AKT和凋亡家族Caspase-3蛋白定位和分布,Western blot检测PI3K/AKT/mTOR通路关键蛋白P-PI3KCA、PI3K、AKT、P-AKT和凋亡家族Caspase-3蛋白表达。结果CCK-8结果显示,与对照组相比较,添加20μmol·L^(-1)β-谷甾醇作用于KGN细胞,在24 h细胞分裂增殖能力增强(P<0.01);细胞周期结果显示,添加20μmol·L^(-1)β-谷甾醇处理24 h后KGN细胞周期显示G0/G1期细胞所占比例减少,S+G2/M期细胞所占比例增加;细胞凋亡结果表明,β-谷甾醇处理组KGN细胞总凋亡率低于对照组(P<0.05);细胞免疫荧光显示,P-PI3KCA、P-AKT和Caspase-3在KGN细胞胞核和胞质有表达,Western blot结果显示,β-谷甾醇处理组KGN细胞中P-PI3KCA、P-AKT蛋白相对表达量上调,Caspase-3蛋白表达下调(P均<0.05)。结论β-谷甾醇可促进颗粒细胞增殖,抑制凋亡,其机制与PI3K/AKT信号通路有关。
Objective To investigate the effects ofβ-sitosterol on proliferation,apoptosis and protein expression of P-PI3KCA,P-AKT and Caspase-3 in human ovarian granulosa cell line(KGN).Methods KGN cells were cultured in vitro and divided into control group andβ-sitosterol treated group.CCK-8 assay was performed to determine the proliferation of KGN cells supplemented with 10,20,40 and 80μmol·L^(-1)β-sitosterol and that of KGN cells supplemented with 20μmol·L^(-1)β-sitosterol at 12,24,36 and 48 h.Apoptosis rate and cell cycle distribution were detected by flow cytometry and P-PI3KCA,P-AKT and Caspase-3 protein localization and distribution were determined by cell immunofluorescence in KGN.The protein expressions of key proteins of the PI3K/AKT/mTOR pathway,P-PI3KCA,PI3K,AKT and P-AKT and apoptotic family protein Caspase-3 were determined by Western blot.Results CCK-8 results showed that KGN cells treated with 20μmol·L^(-1)β-sitosterol exhibited a significant increase(P<0.01)in their ability to divide and proliferate at 24 h compared with the control cells.The cell cycle results of KGN cells treated with 20μmol·L^(-1)β-sitosterol for 24 h showed thatβ-sitosterol could decrease the proportion of cells in the G0/G1 phase and increase the proportion of cells in the S+G2/M phase.The results of apoptosis showed that the total apoptosis rate of granulosa cells in the group treated with 20μmol·L^(-1)β-sitosterol at 24 h was significantly decreased compared with that in the control group(P<0.05).Cell immunofluorescence showed that P-PI3KCA,P-AKT and Caspase-3 were expressed in the nucleus and cytosol of KGN cells,and Western blot analysis showed that the protein expression of P-PI3KCA,P-AKT was up-regulated and Caspase-3 was down regulated in KGN cells after 20μmol·L^(-1)β-sitosterol treated for 24 h(P all<0.05).Conclusionβ-sitosterol can promote granulosa cell proliferation and inhibit apoptosis,and the mechanism is related to the PI3K/AKT signaling pathway.
作者
赵帅
陈冬梅
虎娜
杨张杰
王宇欣
马会明
ZHAO Shuai;CHEN Dongmei;HU Na;YANG Zhangjie;WANG Yuxin;MA Huiming(Key Laboratory of Fertility Maintenance of Ministry of Education,Ningxia Medical University,Yinchuan 750004,China;Institute of Stem Cell Research,General Hospital of Ningxia Medical University,Yinchuan 750004,China)
出处
《宁夏医科大学学报》
2021年第4期339-344,共6页
Journal of Ningxia Medical University
基金
国家自然科学基金(81660813)
宁夏回族自治区重点研发项目(2020BEG03042)。
