应用高压冷冻-冷冻替代技术研究Erastin对颗粒细胞超微结构的影响

Effect of Erastin on the Ultrastructure of Granular Cells by High Pressure Freezing-freezing Substitution Technology

为了探究铁死亡对颗粒细胞超微结构的影响,采用高压冷冻-冷冻替代法(high pressure freezing-freezing substitution,HPF-FS)和常规化学固定法(chemical fixation,CF)对Erastin诱导处理的牛颗粒细胞和猪颗粒细胞超微结构进行研究。结果发现,与对照组相比,经Erastin诱导处理48 h的牛颗粒细胞和猪颗粒细胞的增殖受到显著抑制,ATP含量显著降低,ROS水平显著升高,指示颗粒细胞出现了铁死亡;电镜观察发现两个处理组的细胞均出现空泡化,线粒体变小,嵴断裂甚至消失,且对照组猪颗粒细胞的内质网比处理组的结构更丰富。与CF处理相比,HPF-FS技术对细胞超微结构的保存具有显著优势,胞质均匀、细胞膜界限清晰,线粒体、高尔基复合体、自噬泡、细胞核等结构清晰完整,更加接近于细胞瞬时生理状态。结果表明,利用HPF-FS技术能更加真实、全面地展现细胞的超微结构,为进一步研究颗粒细胞的凋亡调控机制及介导卵泡发育等相关研究提供参考。

In order to investigate the effect of ferroptosis on the ultrastructure of granulosa cells,the high pressure freezing-freezing substitution method(HPF-FS)and conventional chemical fixation method(CF)were used to study the ultrastructures of bovine and porcine granulosa cells induced by Erastin.The results showed that,after induction by Erastin for 48 h,the proliferation of both bovine granulosa cells and porcine granulosa cells were significantly inhibited,the ATP contents were significantly decreased,and the ROS levels were significantly increased when compared with the control group,all of these indicated ferroptosis in granulosa cells.Electron microscopy observation indicated that vacuoles appeared in the cells,mitochondria size were significantly decreased,cristae broken or even disappeared of granulosa cells in both treatment groups;the endoplasmic reticulum of the control porcine granulosa cells were more abundant than the treatment group.The maintenance of the ultrastructure of the cells by HPF-FS were better than CF in both cell morphology and membrane boundary.Compared with CF,the structures of mitochondria,golgi apparatus,autophagy and nucleus were clear and intact,which were closer to the transient physiological state of cells.Therefore,the technology of HPF-FS can more realistically and comprehensively display the ultrastructure of cells,which can providing more ideas and references for further research on the apoptosis regulation mechanism of granulosa cells or the related research on follicle development.