Page 15 - 南京医科大学自然版

第44卷第12期 南京医科大学学报（自然科学版）
2024年12月 Journal of Nanjing Medical University（Natural Sciences） ·1629 ·

·基础研究·

4⁃羟基过氧环磷酰胺通过靶向P53损伤人卵巢颗粒细胞线粒体

自噬功能的研究

赵辰希 ，徐 聪 ，谢思远 ，高 超 ，覃莲菊 ，吴 畏 1，2*
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南京医科大学生殖医学与子代健康全国重点实验室，江苏 南京 211166；南京医科大学第一附属医院生殖医学中心，江
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苏 南京 210029；南京中医药大学医学院·整合医学学院，江苏 南京 210023
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［摘 要］ 目的：探讨环磷酰胺体外活化物4⁃羟基过氧环磷酰胺（4⁃hydroperoxy cyclophosphamide，4⁃HC）导致人卵巢颗粒细胞
系SVOG功能损伤的效应及机制。方法：以0.2、2.0、10.0 μmol/L的4⁃HC处理SVOG细胞24、48、72 h，CCK⁃8法检测各组细胞的
细胞活力变化，选择构建损伤模型的时间和浓度；Western blot、RT⁃qPCR法检测各组线粒体自噬通量的变化；透射电镜观察正
常组和4⁃HC损伤组线粒体的变化；RT⁃qPCR检测P53相关基因的表达；免疫荧光法检测各组P53蛋白，Parkin蛋白以及线粒体
外膜蛋白（translocase of outer mitochondrial membrane 20，TOMM20）的表达水平。结果：在体外建立了 2.0 μmol/L 4⁃HC 处理
SVOG 细胞48 h诱导的损伤模型。4⁃HC损伤的SVOG细胞中，线粒体自噬通量受到抑制且线粒体形态异常，受损线粒体明显
增加；P53的表达水平明显增加；且存在胞质中P53和Parkin蛋白结合增加，而线粒体外膜蛋白TOMM20和Parkin蛋白的结合
受到抑制。结论：在体外，4⁃HC可能是通过P53⁃Parkin通路抑制受损线粒体自噬导致人卵巢颗粒细胞受损。
［关键词］ 4⁃羟基过氧环磷酰胺；P53；线粒体自噬
［中图分类号］ R321.1 ［文献标志码］ A ［文章编号］ 1007⁃4368（2024）12⁃1629⁃09
doi：10.7655/NYDXBNSN240993

Study on 4⁃Hydroperoxy cyclophosphamide targeting P53 to impair mitophagy function in
human ovarian granulosa cells
1 2 3 2 2 1，2*
ZHAO Chenxi ，XU Cong ，XIE Siyuan ，GAO Chao ，QIN Lianju ，WU Wei
1 National Key Laboratory of Reproductive Medicine and Offspring Health，Nanjing Medical University，Nanjing
211166；Reproductive Center，the First Affiliated Hospital of Nanjing Medical University，Nanjing 210029；School of
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Medicine & Holistic Intergrative Medicine，Nanjing University of Chinese Medicine，Nanjing 210023，China

［Abstract］ Objective：To investigate the effect of the in vitro activation product of cyclophosphamide，4 ⁃ hydroperoxy
cyclophosphamide（4⁃HC），on the functional impairment of the human ovarian granulosa cell line SVOG，and the potential underlying
mechanisms. Methods：SVOG cells were treated with 0.2，2.0，and 10.0 μmol/L of 4⁃HC for 24，48，and 72 h. The cell viability in each
group was measured using the CCK⁃8 assay to determine the optimal time and concentration for constructing an injury model. Western
blot and RT⁃qPCR were used to detect changes in mitochondrial autophagy flux. Transmission electron microscopy was employed to
observe mitochondrial changes in both normal and 4⁃HC⁃injured cells. RT⁃qPCR was used to assess the expression of P53⁃related
genes，and immunofluorescence was applied to detect the expression levels of P53，Parkin，and the translocase of the outer
mitochondrial membrane 20（TOMM20）proteins. Results：A model of SVOG cell injury induced by 2.0 μmol/L 4⁃HC for 48 h was
established in vitro. Mitochondrial autophagy flux was inhibited，and mitochondrial morphology was abnormal in 4⁃HC⁃injured SVOG
cells，with a significant increase in damaged mitochondria. The expression level of P53 was significantly increased in 4⁃HC⁃injured
SVOG cells. An increase in the cytoplasmic interaction between P53 and Parkin protein was observed，while the binding of TOMM20
and Parkin protein was inhibited in 4⁃HC⁃injured SVOG cells. Conclusion：In vitro，4⁃HC may induce damage to human ovarian
granulosa cells by inhibiting mitochondrial autophagy through the P53⁃Parkin pathway.
［Key words］ 4⁃hydroperoxy cyclophosphamide；P53；mitophagy
［J Nanjing Med Univ，2024，44（12）：1629⁃1637］
［基金项目］ 国家重点研发计划（2022YFC2703203）；江苏省生殖医学创新中心（CXZX202207）
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通信作者（Corresponding author），E⁃mail：weiwu77@163.com