高内涵脂滴三维影像定量分析研究细胞内的脂质储存  

High Content Three-dimensional Imaging and Analysis of Lipid Droplets for the Study of Lipid Storage in Cells

作　　者：张少燕 梁燕[1] 印叶盛 马成新 陈丰荣 ZHANG Shao-yan;LIANG Yan;YIN Ye-sheng;MA Cheng-xin;CHEN Feng-jung(Institute of Metabolism and Integrative Biology,Fudan University,Shanghai,200438,China)

机构地区：[1]复旦大学代谢与整合生物学研究院,上海200438

出　　处：《现代生物医学进展》2021年第20期3801-3808,共8页Progress in Modern Biomedicine

基　　金：国家自然科学基金委重大研究计划培育项目(91857103)。

摘　　要：目的:研究细胞内脂滴含量的变化对肥胖、糖尿病等代谢性疾病发生发展的影响。方法:建立高内涵脂滴三维成像和定量分析系统,获得脂滴三维动态表型参数,例如细胞内脂滴的总体积量、脂滴平均体积、单一细胞内脂滴平均数量等指标。选择HeLa、AML-12、COS-7和3T3-L1四种细胞系进行油酸、基因沉默、酶活性抑制剂的处理,量化处理后四种细胞内的脂滴数量与大小的表型差异。结果:在加入油酸情况下,细胞随油酸浓度增加而生成更多、更大的脂滴,但AML-12细胞只有展现增加脂滴数量的变化表型;在HeLa细胞中进行19种中性脂合成通路上关键基因的转录表达沉默,发现需要同时双敲降两种甘油三酯合成酶DGAT1和DGAT2才能显着降低细胞内脂滴总体积储存量,但在COS-7细胞中只需要单敲降DGAT1即可降低脂滴存量;进一步使用了DGAT1/2抑制剂处理四种细胞后,发现对抑制剂响应可区分为两类细胞分组(HeLa、AML-12与COS-7、3T3-L1)的脂滴存量表型差异,其原因是DGAT1和DGAT2的转录表达谱在这两类细胞分组中的不同。结论:建立了高内涵脂滴三维成像和定量分析系统,量化了四种细胞系的脂滴数量与大小的表型差异,揭示了细胞的脂滴脂储存方式与蛋白酶表达谱的关系。Objective: To study the effect of lipid storage level in cellular lipid droplets(LDs) on the development of metabolic diseases such as obesity and diabetes. Methods: We built up a high content LD imaging and quantitative analysis system to characterize cellular LDs as three-dimensional dynamic parameters such as the total volume of cellular LDs in a cell, the number of total LDs in a cell,and the average volume of LDs. We selected four types of cells including HeLa, AML-12, COS-7, and 3 T3-L1 pre-adipocytes as our cell models. Next, we characterized the various phenotypes of cellular LDs in the four types of cells including LD number and LD volume,under different treatments by oleic acids, gene knockdown using RNA interference, and inhibitors for enzymes. Results: After treatment of cells by using oleic acids, more LDs but not larger LDs were produced in AML-12 cells, while the other three types of cells exhibited more and larger cellular LDs as the concentration of oleic acids increased. After knocking down the transcriptional expressions of 19 critical genes in a neutral lipid synthesis pathway in HeLa cells by using RNA interference, we found that double knockdown of DGAT1 and DGAT2 decreased lipid storage in cellular LDs. However, in COS-7 cells, only DGAT1 knockdown was sufficient to reduce the lipid storage of cellular LDs. Interestingly, after treatment of cells by using DGAT1 and DGAT2 inhibitors, we further found that the four types of cells were classified into two groups, HeLa and AML-12 versus COS-7 and 3 T3-L1, based on the different resultant phenotypes of LDs. We measured and confirmed that the two different treated responses of cells resulted from the different transcriptional profiles of DGAT1 and DGAT2. Conclusions: In this study, we built up a high content LD imaging and quantitative analysis system to characterize the number and size of cellular LDs in the four types of cell lines. We found that the various phenotypes of LDs in the four types of cells and these results uncover a relationship b

关 键 词：脂滴 脂滴数量 脂滴总体积 中性脂合成通路 

分 类 号：R-33[医药卫生] Q26[生物学—细胞生物学]