2-脱氧-D-葡萄糖逆转七氟醚引起胚胎干细胞自我更新能力下降的机制研究

2-Deoxy-D-Glucose rescues the self-renewal of embryonic stem cells inhibited by sevoflurane

目的探讨2-脱氧-D-葡萄糖在逆转七氟醚引起胚胎干细胞自我更新能力下降的机制。方法采用鼠胚胎干细胞(m ESCs)作为研究七氟醚对孕早期胚胎毒性的模型,随机分成4组:对照组(暴露于5%CO2及21%O2的环境中)、七氟醚组(4.1%七氟醚的密封塑料盒中处理6 h)、2-DG组(10 mmol/L 2-DG处理6 h)、2-DG+七氟醚组(4.1%七氟醚+10 mmol/L 2-DG处理6 h)。各组干预6 h后,通过从形态学观测细胞宏观改变,荧光测定细胞内活性氧自由基(ROS)水平,qRT-PCR检测干性基因的mRNA表达情况和免疫萤光染色直接观察干性基因的水平四个方面比较七氟醚组与对照组在ROS水平和自我更新能力方面的差异。结果与对照组相比,七氟醚组细胞呈现高ROS水平,干性基因及其表达也有显著降低(P<0.05);对照组和2-DG组上述指标则无显著差异(P>0.05);2-DG+七氟醚组较七氟醚组ROS水平低,干性基因及其表达显著升高(P<0.05)。结论七氟醚通过诱导细胞内ROS产生从而导致了m ESCs的自我更新能力的下降。2-DG缓解其引起的高水平ROS从而逆转其对胚胎干细胞自我更新能力的抑制。

Objective To assess the effects of sevoflurane on self-renewal of ESCs and determine whether the effects of sevoflurane on ROS level could be rescued by 2-DG. Methods In this study,murine embryonic stem cells（ m ESCs） were used as a model to study the toxicity of sevoflurane to the first trimester embryo. m ESCs were randomly divided into 4 groups： control group,sevoflurane group（ 4. 1% sevoflurane in a sealed plastic box for 6 hours）,2-DG group（ 10 mmol/L 2-DG for 6 hours）,2-DG ＋ sevoflurane group（ 4. 1% sevoflurane ＋ 10 mmol/L2-DG for 6 hours）. After 6 hours of treatment with SQF in 4. 1% sevoflurane group,the intracellular ROS level was measured by fluorescence microscopy,the mRNA expression of the embryonic stem cell related gene was detected by qRT-PCR and immunofluorescence staining. Results Compared with the control group,the ROS level in the sevoflurane group was significantly higher（ P〈0. 05）. The expression of embryonic stem cell related genes were decreased（ P〈0. 05）. Compared with the sevoflurane group,the ROS level in the sevoflurane ＋ 2-DG group was lower（ P〈0. 05）. The expression of embryonic stem cell related genes were increased（ P〈0. 05）. Conclusion Sevoflurane induces a decrease in the self-renewal capacity of m ESCs by inducing intracellular ROS production. 2-DG attenuates the high levels of ROS and reverses its ability to inhibit the self-renewal of embryonic stem cells. Our study has provided new insights into the toxic effects and mechanisms of sevoflurane-induced fetal development in the fetus,as well as in the prevention and treatment of embryotoxicity associated with inhalation anesthesia.