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

doi:10.19586/j.2095-2341.2023.0057

生物技术进展 ›› 2023, Vol. 13 ›› Issue (4): 637-644.DOI: 10.19586/j.2095-2341.2023.0057

• 技术与方法 • 上一篇

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

王珍¹(), 黄克让¹, 陈蕾¹, 周敏¹, 薛元夏²

^1.西北农林科技大学，生命科学大型仪器共享平台，陕西杨凌 712100
^2.杨凌职业技术学院生物工程学院，陕西杨凌 712100

收稿日期:2023-04-18 接受日期:2023-05-18 出版日期:2023-07-25 发布日期:2023-08-03
作者简介:王珍E-mail: 1477164660@qq.com
基金资助:
国家自然科学基金青年基金项目(32100578)

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

Zhen WANG¹(), Kerang HUANG¹, Lei CHEN¹, Min ZHOU¹, Yuanxia XUE²

^1.Life Science Research Core Services，Northwest A&F University，Shaanxi Yangling 712100，China
^2.College of Biological Engineering，Yangling Vocational & Technical College，Shaanxi Yangling 712100，China

Received:2023-04-18 Accepted:2023-05-18 Online:2023-07-25 Published:2023-08-03

摘要/Abstract

摘要：

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。

关键词: 铁死亡, Erastin, 高压冷冻-冷冻替代, 超微结构, 颗粒细胞

Abstract:

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.

Key words: ferroptosis, Erastin, high pressure freezing-freezing substitution, ultrastructure, granulosa cell

中图分类号:

Q-336

王珍, 黄克让, 陈蕾, 周敏, 薛元夏. 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[J]. 生物技术进展, 2023, 13(4): 637-644.

Zhen WANG, Kerang HUANG, Lei CHEN, Min ZHOU, Yuanxia XUE. Effect of Erastin on the Ultrastructure of Granular Cells by High Pressure Freezing-freezing Substitution Technology[J]. Current Biotechnology, 2023, 13(4): 637-644.

图/表 6

表1 Erastin处理对颗粒细胞增殖的影响

Table 1 Effect of Erastin treatment on proliferation of granulosa cells

组别	24 h	48 h
CK-N	2.30±0.14 a	3.18±0.12 a
T-N	2.16±0.05 a	2.73±0.08 b
CK-Z	2.94±0.07 a	4.50±0.14 a
T-Z	2.41±0.11 b	3.29±0.05 b

表2 Erastin处理对颗粒细胞ATP和ROS水平的影响

Table 2 Effects of Erastin treatment on ATP and ROS levels in granulosa cells

组别

牛颗粒细胞ATP含量/(μmol·L^-1)

猪颗粒细胞ATP含量/(μmol·L^-1)

牛颗粒细胞ROS水平

(荧光强度)

猪颗粒细胞ROS水平

(荧光强度)

图1 不同固定方法对牛颗粒细胞超微结构的影响注：M—线粒体；L—脂滴；G—高尔基体；Np—核孔；Nuc—细胞核；E—内质网。

Fig. 1 Effect of different fixation methods on the ultrastructure of bovine granulosa cells

图2 Erastin处理对牛颗粒细胞线粒体的影响注：M—线粒体；L—脂滴；Nuc—细胞核；E—内质网。

Fig. 2 Effect of Erastin treatment on the mitochondria of bovine granulosa cells

图3 不同固定方法对猪颗粒细胞超微结构的影响注：M—线粒体；L—脂滴；G—高尔基体；Np—核孔；Nuc—细胞核；RE—粗面内质网；SE—滑面内质网。

Fig. 3 Effect of different fixation methods on ultrastructure of porcine granulosa cells

图4 Erastin处理对猪颗粒细胞线粒体的影响注：M—线粒体；L—脂滴；Nuc—细胞核；RE—粗面内质网；SE—滑面内质网;G—高尔基体。

Fig. 4 Effect of Erastin treatment on the mitochondria of porcine granulosa cells

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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

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