小鼠棕色脂肪透射电子显微镜样品固定方法的改良  

An improved fixation method for preparing mouse brown adipose tissue for transmission electron microscopy

作　　者：魏纯纯 王平[2] 林方兴 麻献华 章卫平 谢志芳 WEI Chun-chun;WANG Ping;LIN Fang-xing;MA Xian-hua;ZHANG Wei-ping;XIE Zhi-fang(Department of Pathophysiology,Naval Medical University,Shanghai 200433,China;NHC Key Laboratory of Hormones and Development(Tianjin Medical University),Tianjin Key Laboratory of Metabolic Diseases,Tianjin Medical University Chu Hsien-I Memorial Hospital&Tianjin Institute of Endocrinology,Tianjin Medical University,Tianjin 300134,China;Department of Biophysics,Naval Medical University,Shanghai 200433,China;Ministry of Education-Shanghai Key Laboratory of Children’s Environmental Health,Xinhua Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200092,China)

机构地区：[1]海军军医大学病理生理学教研室,上海200433 [2]国家卫生健康委员会激素与发育重点实验室(天津医科大学),天津市代谢性疾病重点实验室、天津医科大学朱宪彝纪念医院&天津市内分泌研究所,天津300134 [3]海军军医大学生物物理学教研室,上海200433 [4]上海交通大学医学院附属新华医院环境与儿童健康重点实验室,上海200092

出　　处：《解剖学报》2023年第6期738-742,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金青年基金(32100929)。

摘　　要：目的改良小鼠棕色脂肪组织透射电子显微镜样品固定方法,以获得更好的线粒体和脂滴的超微结构。方法对小鼠棕色脂肪组织的固定方法进行优化,主要包括将前固定剂中多聚甲醛的浓度从2%提高到4%;将后固定剂四氧化锇的缓冲液换成咪唑缓冲液;同时采用心脏灌注和浸泡结合的固定方式。比较两种方法制备的细胞超微结构(每组3只小鼠);并将改良后的方法应用于线粒体和脂滴的定量分析实验。结果与改良前相比,用改良后的方法制备的棕色脂肪细胞的线粒体嵴及其他膜性细胞器的结构更完整;脂滴呈现为高电子密度的圆形结构。用该方法观察到了冷适应后棕色脂肪细胞线粒体嵴密度升高和脂滴增多现象。结论改良后的方法能更好地保存棕色脂肪细胞中的细胞器特别是线粒体和脂滴的超微结构,可应用于研究该类型细胞在不同生理或病理条件下发生的超微结构变化。Objective To improve the fixation method of the transmission electron microscope for better morphological preservation of mitochondria and lipid droplets in mouse brown adipose tissue. Methods The fixation method for mouse brown adipose tissue was optimized, mainly including an increased concentration of paraformaldehyde from 2% to 4% in the pre-fixative, employment of transcardial perfusion followed by immersion fixation in pre-fixation, and using imidazole-buffered osmium tetroxide as the post-fixative. The ultrastructures of brown adipocytes prepared by the improved method were observed and compared with those of a known standard protocol(3 mice in each group). The improved method was further validated in the quantitative analysis of mitochondrial cristae density and lipid droplets. Results The mitochondrial cristae and membrane structure of other organelles of brown adipocytes were better preserved using the optimized method compared with those of the standard method. Lipid droplets were presented as round structures with high electron density instead of vacuolated appearances. Using this method, we observed that the density of mitochondrial cristae and the content of lipid droplets increased in brown adipocytes after cold adaptation. Conclusion The optimized method can better preserve the ultrastructure of organelles in brown adipocytes, especially mitochondria and lipid droplets, and may be applicable for studying the ultrastructures remodeling of brown adipose tissue under different physiological or pathological conditions.

关 键 词：棕色脂肪细胞 超微结构 固定方法 线粒体 脂滴 组织学 透射电子显微术 小鼠 

分 类 号：Q24[生物学—细胞生物学]