bFGF对3-硝基丙酸处理的小鼠受精卵体外发育能力的影响

doi:10.16431/j.cnki.1671-7236.2022.02.022

摘要/Abstract

摘要： 【目的】研究在体外培养液中添加碱性成纤维细胞生长因子(bFGF)对3-硝基丙酸(3-NPA)处理的小鼠受精卵体外发育能力的影响, 为提高氧化应激早期胚胎体外发育质量提供参考。【方法】在小鼠受精卵体外培养液中添加0、20、50、100和150 ng/mL bFGF, 培养24、48和96 h, 统计2-细胞率、4-细胞率和囊胚率, 筛选最佳bFGF处理浓度。经腹腔注射12.5 mg/kg 3-NPA生产氧化应激体内受精卵, 正常组小鼠腹腔注射等体积生理盐水, 将获得的受精卵分为添加或不添加bFGF组, 即3-NPA和3-NPA+bFGF组及对照组(C)和bFGF组, 培养到囊胚后, 用DCFH-DA检测胚胎内活性氧(reactive oxygen species, ROS)水平, CMF2HC检测谷胱甘肽(glutathione, GSH)水平, JC-1检测早期胚胎线粒体膜电位强度。【结果】 0、20、50、100和150 ng/mL bFGF组2-细胞率均无显著差异(P>0.05), 100 ng/mL bFGF组囊胚率显著高于其他各组(P<0.05), 150 ng/mL bFGF组4-细胞率和囊胚率均显著低于其他各组(P<0.05), 因此后续试验选用100 ng/mL bFGF。3-NPA+bFGF组4-细胞率显著高于3-NPA组(P<0.05), bFGF组囊胚率显著高于其他组(P<0.05)。bFGF+3-NPA组囊胚率显著高于3-NPA组(P<0.05);与对照组相比, 3-NPA组ROS水平显著升高(P<0.05), bFGF组ROS水平显著降低(P<0.05);3-NPA组GSH和线粒体膜电位水平均显著降低(P<0.05), bFGF组GSH和线粒体膜电位水平均显著增加(P<0.05)。bFGF+3-NPA组ROS水平显著低于3-NPA组(P<0.05), GSH和线粒体膜电位水平均显著高于3-NPA组(P<0.05)。【结论】在体外培养液中添加100 ng/mL bFGF可减少3-NPA诱导的胚胎氧化应激、改善胚胎线粒体功能, 从而提高小鼠受精卵体外发育的能力。

关键词: 小鼠; 胚胎发育; 碱性成纤维细胞生长因子(bFGF); 3-硝基丙酸(3-NPA); 活性氧

Abstract: 【Objective】 This experiment was to study the effect of adding basic fibroblast growth factor (bFGF) to the in vitro development medium of mouse fertilized eggs treated with 3-nitropropionic acid (3-NPA), in order to provide reference for improving the in vitro development quality of early embryos under oxidative stress.【Method】 0, 20, 50, 100 and 150 ng/mL bFGF were added to the in vitro culture medium of mouse zygotes, cultured for 24, 48 and 96 h, and the 2-cell rate, 4-cell rate and blastocyst rate were counted to screen the best bFGF treatment concentration.In vivo zygotes were produced by intraperitoneal injection of 12.5 mg/kg 3-NPA, and the same volume of normal saline was injected intraperitoneally as the control, the fertilized eggs were divided into 3-NPA and 3-NPA+bFGF groups, control group (C) and bFGF group.After blastocysts were obtained, the level of reactive oxygen species (ROS) in embryos was detected by DCFH-DA, the level of glutathione (GSH) was detected by CMF2HC, and the mitochondrial membrane potential intensity of early embryos was detected by JC-1.【Results】 There was no significant difference in 2-cell rate among 0, 20, 50, 100 and 150 ng/mL bFGF groups (P > 0.05), and the blastocyst rate in 100 ng/mL bFGF group was significantly higher than that of the other groups (P < 0.05), the 4-cell rate and blastocyst rate in 150 ng/mL bFGF group were significantly lower than those in the other groups (P < 0.05), so 100 ng/mL bFGF was used in subsequent experiments.The 4-cell rate in 3-NPA+bFGF group was significantly higher than that in 3-NPA group (P < 0.05), the blastocyst rate in bFGF group was significantly higher than that of the other groups (P < 0.05).The blastocyst rate in bFGF+3-NPA group was significantly higher than that in 3-NPA group (P < 0.05). Compared with control group, the level of ROS in 3-NPA group was increased significantly (P < 0.05), and the level of ROS in bFGF group was decreased significantly (P < 0.05). GSH and mitochondrial membrane potential were decreased significantly in 3-NPA group (P < 0.05), and were increased significantly in bFGF group (P < 0.05). The level of ROS in bFGF+3-NPA group was significantly lower than that in 3-NPA group (P < 0.05), and the levels of GSH and mitochondrial membrane potential were significantly higher than those in 3-NPA group (P < 0.05).【Conclusion】 The addition of 100 ng/mL bFGF to the in vitro culture medium could reduce the embryonic oxidative stress induced by 3-NPA and improve the function of embryonic mitochondria, so as to improve the in vitro development ability of mouse zygotes.

Key words: mice; embryonic development; basic fibroblast growth factor (bFGF); 3-nitroponic acid (3-NPA); reactive oxygen species

中图分类号:

S813.3

田微, 方妍雅, 柳海星, 李钟淑. bFGF对3-硝基丙酸处理的小鼠受精卵体外发育能力的影响[J]. 中国畜牧兽医, 2022, 49(2): 608-614.

TIAN Wei, FANG Yanya, LIU Haixing, LI Zhongshu. Effect of bFGF on in vitro Development of Mouse Zygotes Treated with 3-nitropropionic Acid[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 608-614.

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