RSPO2对卵巢颗粒细胞氧化应激和凋亡的影响
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华南农业大学动物科学学院,广东省农业动物基因组学与分子育种重点实验室,国家生猪种业工程技术研究中心

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国家生猪产业技术体系,广东省基础与应用基础研究基金


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

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the earmarked fund for China Agriculture Research System ,the National Natural Science Foundation of Guangdong Province

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    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(RT-PCR)、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,RT-PCR、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。

    Abstract:

    Objective Utilizing human ovarian granulosa tumor cells as the cellular model, we tried to investigate the effect of roofplate-specific spondin 2 (RSPO2) on the oxidative stress and apoptosis level of ovarian granulosa cells. Methods We performed transient transfection of RSPO2 overexpression plasmid and small interfering RNA (siRNA) in COV434 cells. Real-time Quantitative PCR (RT-PCR) and Western blot were used to detect efficiency of overexpression and interference. The reactive oxygen species assay kit was used to determine the amount of intracellular reactive oxygen species. The apoptosis level was detected by flow cytometry. RT-PCR and Western blot were used to determine the expression levels of oxidative stress and apoptosis-related genes. Co-immunoprecipitation was used to detect interacting proteins. Results The overexpression and interference of RSPO2 were successfully realized. Overexpression of RSPO2 significantly inhibited the level of reactive oxygen species in COV434 cells (P < 0.05) and extremely significant inhibited the early apoptosis of granulosa cells (P < 0.01), interference of RSPO2 obtained the opposite results. Overexpression of RSPO2 significantly upregulated the mRNA and protein levels of SOD1, SOD2, CAT (P < 0.05), and significant downregulated the levels of caspase 3 and caspase 8 (P < 0.05). On the contrary, interference of RSPO2 significantly downregulated the levels of SOD1 and CAT (P < 0.05), and significantly upregulated the level of caspase 3 and caspase 8 (P < 0.05). We found the protein interactions between RSPO2, SOD1, CAT and caspase 3. Conclusion Interfering with RSPO2 promotes the accumulation of reactive oxygen species within cells, 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.

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  • 收稿日期:2024-07-24
  • 最后修改日期:2024-08-16
  • 录用日期:2024-10-30
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