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王婕, 刘志成, 张彩凤, 郝焱, 游卉, 尤昭玲, 王慧颖, 金波.丹参酮ⅡA对玻璃化冷冻小鼠卵母细胞保护机制的研究[J].湖南中医药大学学报,2026,46(1):42-50[点击复制] |
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| 丹参酮ⅡA对玻璃化冷冻小鼠卵母细胞保护机制的研究 |
| 王婕,刘志成,张彩凤,郝焱,游卉,尤昭玲,王慧颖,金波 |
| (北京中医药大学深圳医院(龙岗)妇科, 广东 深圳 518000;银丰低温医学科技有限公司, 山东 济南 250000;山东银丰生命科学研究院, 山东 济南 250000;湖南中医药大学第一附属医院, 湖南 长沙 410208) |
| 摘要: |
| 目的 探讨丹参酮ⅡA对小鼠卵母细胞玻璃化冷冻的保护机制。方法 选取4~8 周龄SPF级ICR雌性小鼠腹腔注射5 IU孕马血清促性腺激素（PMSG），48 h后从卵巢中收集形态正常的未成熟（GV期）卵母细胞。将GV期卵母细胞分为对照组及丹参酮ⅡA组进行16~18 h过夜培养到MⅡ阶段，形态正常的MⅡ卵母细胞进行体外受精，观察其各组的受精、卵裂和囊胚形成情况。通过免疫荧光技术检测MII卵母细胞对照组及丹参酮ⅡA组中细胞膜水通道蛋白3（AQP3）的蛋白表达水平。将GV期卵母细胞分为对照组（未冷冻）、丹参酮ⅡA组（未冷冻）、对照组（玻璃化冷冻）、丹参酮ⅡA组（玻璃化冷冻）进行培养至成熟卵母细胞，观察分析四组卵母细胞的受精率、卵裂率、囊胚形成率。采用低温显微镜观察丹参酮ⅡA在MⅡ卵母细胞冷冻保存过程中对其细胞内冰晶形成的影响。结果 与对照组相比，丹参酮ⅡA组卵母细胞成熟率、受精率、卵裂率和囊胚形成率均提高（P＜0.05），其中添加0.5 μg/mL丹参酮ⅡA组的囊胚率最高。丹参酮ⅡA组中AQP3的蛋白表达水平明显高于对照组（P＜0.000 1）。与对照组（未冷冻）相比，丹参酮ⅡA组（未冷冻）的受精率、卵裂率、囊胚形成率均提高（P＜0.05）。与对照组（玻璃化冷冻）相比，丹参酮ⅡA组（玻璃化冷冻）的受精率、卵裂率、囊胚形成率均提高（P＜0.05）。在丹参酮ⅡA组，细胞内冰晶形成的温度更低及温度域的分布更集中，且低温冰晶形成概率更少。结论 丹参酮ⅡA通过上调卵母细胞细胞膜上AQP3的表达，并降低冷冻保存过程中细胞内冰晶形成的温度，进而提高玻璃化冻存卵母细胞的成活率及后续发育潜能。 |
| 关键词: 卵母细胞 玻璃化冷冻 丹参酮ⅡA 水通道蛋白3 细胞内冰晶 渗透性 |
| DOI：10.3969/j.issn.1674-070X.2026.01.006 |
| 投稿时间：2025-11-04 |
| 基金项目:育龙计划-三龙人才项目（2022-BUCMSZYLRC05）；深圳市龙岗区科技创新专项（LGWJ2024-37）；深圳市“医疗卫生三名工程”项目（SZZYSM202311019）；北京中医药大学深圳医院（龙岗）联合银丰低温医学科技有限公司合作课题（KJK-HX-2023-001）；国家中医药管理局“尤昭玲全国名中医传承工作室”建设项目（国中医药办人教函〔2022〕5号）。 |
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| Protective mechanism of Tanshinone ⅡA in vitrified mouse oocytes |
| WANG Jie, LIU Zhicheng, ZHANG Caifeng, HAO Yan, YOU Hui, YOU Zhaoling, WANG Huiying, JIN Bo |
| (Department of Gynaecology, Shenzhen Hospital (Longgang), Beijing University of Chinese Medicine, Shenzhen, Guangdong 518000, China;Yinfeng Cryomedicine Technology Co., Ltd, Jinan, Shandong 250000, China;Shandong Yinfeng Life Science Research Institute, Jinan, Shandong 250000, China;The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To investigate the protective mechanism of Tanshinone ⅡA against vitrification-induced injury in mouse oocytes. Methods Female ICR mice aged 4-8 weeks (SPF grade) were intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin (PMSG). After 48 hours, morphologically normal immature oocytes at the germinal vesicle (GV) stage were collected from the ovaries. The GV-stage oocytes were divided into control group and Tanshinone ⅡA group and cultured overnight for 16-18 hours to reach the metaphase Ⅱ(MⅡ) stage. Morphologically normal MⅡ oocytes were then subjected to in vitro fertilization, and fertilization, cleavage, and blastocyst formation were observed in each group. The protein expression level of aquaporin 3 (AQP3) on the cell membrane was detected in MII oocytes from the control group and the Tanshinone ⅡA group using immunofluorescence technique. In another experiment, GV-stage oocytes were divided into four groups for culture until maturation: a control group (non-vitrified), a Tanshinone ⅡA group (non-vitrified), a control group (vitrified), and a Tanshinone ⅡA group (vitrified). Fertilization rate, cleavage rate, and blastocyst formation rate were observed and analyzed in the four groups. A cryomicroscope was employed to observe the effect of Tanshinone ⅡA on intracellular ice formation (IIF) during the cryopreservation of MⅡ oocytes. Results Compared with the control group, the Tanshinone ⅡA group showed increased rates of oocyte maturation, fertilization, cleavage, and blastocyst formation（P＜0.05）, with the highest blastocyst rate observed when treated with 0.5 μg/mL Tanshinone ⅡA. The protein expression level of AQP3 was significantly higher in the Tanshinone ⅡA group than in the control group (P＜0.000 1). Compared with the control group (non-vitrified), the Tanshinone ⅡA group (non-vitrified) showed significantly higher fertilization, cleavage, and blastocyst formation rates (P＜0.05). Similarly, compared with the control group (vitrified), the Tanshinone ⅡA group (vitrified) also exhibited significantly increased fertilization, cleavage, and blastocyst formation rates (P＜0.05). Additionally, IIF occurred at a lower temperature with a more concentrated temperature range, resulting in a lower probability of ice formation during cooling. Conclusion Tanshinone ⅡA improves the survival rate and developmental potential of vitrified oocytes by upregulating the expression of AQP3 on the oocyte membrane and reducing the temperature of IIF during cryopreservation. |
| Key words: oocyte vitrification Tanshinone ⅡA AQP3 intracellular ice crystals permeability |
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