RSPO2对卵巢颗粒细胞氧化应激和凋亡的影响  

Effects of RSPO2 on oxidative stress and apoptosis in ovarian granulosa cells

作　　者：李硕 李念 周小枫 袁晓龙[1] LI Shuo;LI Nian;ZHOU Xiaofeng;YUAN Xiaolong(State Key Laboratory of Swine and Poultry Breeding Industry,National Engineering Research Center for Breeding Swine Industry,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding,College of Animal Science,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]猪禽种业全国重点实验室,国家生猪种业工程技术研究中心,广东省农业动物基因组学与分子育种重点实验室,华南农业大学动物科学学院,广州510642

出　　处：《中国比较医学杂志》2024年第12期59-69,共11页Chinese Journal of Comparative Medicine

基　　金：广东省基础与应用基础研究基金(2024A1515012999,2023A1515010364,2022A1515012490);国家生猪产业技术体系(CARS-35)。

摘　　要：目的以人卵巢颗粒肿瘤细胞为模型,探究特异性顶部盘状底板反应蛋白2(RSPO2)对卵巢颗粒细胞氧化应激和凋亡的影响。方法在COV434细胞中瞬时转染RSPO2过表达质粒和小干扰RNA(siRNA),实时荧光定量PCR(qPCR)、蛋白免疫印迹法(Western blot)检测过表达和干扰效率,利用活性氧检测试剂盒测定细胞内活性氧含量,流式细胞术检测凋亡水平,qPCR、Western blot测定氧化应激和凋亡相关基因表达量,免疫共沉淀检测互作蛋白。结果成功实现RSPO2的过表达和干扰。过表达RSPO2显著抑制颗粒细胞内活性氧的水平(P<0.05),极显著抑制颗粒细胞早期凋亡(P<0.01),干扰RSPO2可得到相反的试验结果。过表达RSPO2能显著上调SOD1、SOD2、CAT mRNA和蛋白表达水平(P<0.05),显著下调Caspase 3、Caspase 8表达水平(P<0.05)。相反,干扰RSPO2显著下调SOD1、CAT表达水平(P<0.05),显著上调Caspase 3、Caspase 8表达水平(P<0.05),并发现RSPO2和SOD1、CAT、Caspase 3存在明显的蛋白互作现象。结论干扰RSPO2促进细胞内活性氧的积累,下调SOD1和CAT的表达,从而促进氧化应激;干扰RSPO2上调Caspase 3和Caspase 8的表达,促进细胞的凋亡,提示RSPO2在卵巢颗粒细胞中发挥着重要的作用。Objective We aimed to investigate the effect of roofplate-specific spondin 2(RSPO2)on oxidative stress and apoptosis in ovarian granulosa cells,using human ovarian granulosa tumor cells as a cellular model.Methods We transiently transfected COV434 human granulosa cells with RSPO2 overexpression plasmid and small interfering RNA(siRNA).The efficiencies of the overexpression and interference were detected by quantitative real-time polymerase chain reaction(qPCR)and Western blot.Intracellular reactive oxygen species(ROS)were detected using a ROS assay kit,and apoptosis was detected by flow cytometry.Expression levels of oxidative stress-and apoptosis-related genes were determined by qPCR and Western blot,and interacting proteins were detected by co-immunoprecipitation.Results RSP02 overexpression and interference were successfully realized.Overexpression of RSPO2 significantly inhibited ROS levels in COV434 cells(P<0.05)and the early apoptosis of granulosa cells(P<0.01),while RSP02 interference had the opposite result.Overexpression of RSPO2 significantly upregulated the mRNA and protein levels of superoxide dismutase(SOD)1,SOD2,and catalase(CAT)(P<0.05),and significantly downregulated the levels of Caspase 3 and Caspase 8(P<0.05).In contrast,RSPO2 interference significantly downregulated SOD1 and CAT(P<0.05)andsignificantly upregulated Caspase 3 and Caspase 8(P<0.05).We found protein interactions between RSPO2,SOD1,CAT,and Caspase 3.Conclusions Interfering with RSPO2 promotes the accumulation of ROS within cells and downregulates the expression of SOD1 and CAT,thereby enhancing oxidative stress.Additionally,interfering with RSPO2 upregulates the expression of Caspase 3 and Caspase 8,promoting cell apoptosis.These findings suggest that RSPO2 plays a crucial role in ovarian granulosa cells.

关 键 词：RSPO2 卵巢颗粒细胞 氧化应激 凋亡 

分 类 号：R-33[医药卫生]