牛肝细胞中脂滴大小和融合的评估
Summary
本协议描述了如何使用油红O染色脂滴（LDs），计算脂肪酸诱导的脂肪肝细胞模型中LDs的大小和数量，并使用BODIPY 493 / 503观察小LDs通过活细胞成像融合成大LD的过程。
Abstract
脂滴（LD）是在细胞中的脂质代谢和中性脂质储存中起重要作用的细胞器。它们与多种代谢疾病有关，如肥胖、脂肪肝和糖尿病。在肝细胞中，LD的大小和数量是脂肪肝疾病的征兆。此外，氧化应激反应、细胞自噬和细胞凋亡通常伴随着LDs的大小和数量的变化。因此，LDs的尺寸和数量是目前LD生物发生机制研究的基础。在这里，在脂肪酸诱导的牛肝细胞中，我们描述了如何使用油红O来染色LD并研究LD的大小和数量。对LDs的大小分布进行统计分析。活细胞成像系统也观察到小LD融合成大LD的过程。本工作为直接观察不同生理条件下LDs的大小变化趋势提供了一种途径。
Introduction
脂滴（LD）在肝细胞中的积累是非酒精性脂肪性肝病（NAFLD）的典型特征，可进展为肝纤维化和肝细胞癌。已经发现脂肪肝疾病的最早表现是脂肪变性，其特征是LD在肝细胞1的细胞质中蓄积。肝脂肪变性总是与LDs的数量增加和/或扩大有关2。LD被认为是由内质网（ER）产生的，由甘油三酯（TG）作为核心组成，并被蛋白质和磷脂包围3。作为负责TG储存的亚细胞器，LDs在大小，数量，脂质组成，蛋白质以及与其他细胞器的相互作用方面表现出不同的特征，所有这些都会影响细胞能量稳态4。TG水平与LDs的大小呈正相关，较高的细胞内TG含量可以形成更大的LDs5。LD通过TG的局部合成，ER中的脂质掺入以及多个LD的融合而增加尺寸6。含有大LD的细胞（脂肪细胞，肝细胞等）具有通过LD融合有效增加脂质储存的特殊机制。LDs的动态变化反映了细胞不同的能量代谢状态。开发能够观察和分析健康和异常细胞中各种肝LD的方法至关重要。
LDs的主要非荧光染料是苏丹黑B和油红O.苏丹黑B染色中性脂质，磷脂和类固醇7。油红O主要用于骨骼肌、心肌细胞、肝组织、脂肪细胞等LD染色8.，被认为是定量检测小鼠和人类肝脂肪变性的标准工具9。LDs的动态变化主要通过荧光染色进行。尼罗红和BODIPY都是常用的荧光脂质染料10,11。与尼罗河红相比，BODIPY具有更强的组织通透性，与LDs12结合更好。BODIPY标记的LD可用于活细胞染色和与其他细胞器共定位13。
反刍动物脂肪肝的发病率明显高于单胃动物14。在过渡期间，奶牛会经历负能量平衡状态3.牛肝细胞中大量非酯化脂肪酸（棕榈酸、油酸、亚油酸等）合成TGs，导致肝功能异常，大大降低奶制品品质和生产效率15。本研究旨在提供一种协议来分析LD的大小和数量，以及监测LD融合动力学。通过在肝细胞16 中添加不同浓度的亚油酸（LA）构建LD形成模型，并通过用油红O染色LD观察LD在此过程中大小和数量的变化。此外，通过用BODIPY 493/503染色也观察到LDs快速融合的过程。
Protocol
所有程序均按照河南农业大学（中国河南省）动物护理委员会的道德标准进行批准和执行。 1.牛肝细胞培养 解冻原代肝细胞17 ，并在室温下离心400× g4 分钟。注意：原代肝细胞按照先前发表的报告17进行培养和维持。 用移液管丢弃冷冻储存溶液，并用含有 10% 胎牛血清 （FBS） 和 Dulbecco 改良 Eagle 培养基…
Representative Results
细胞LD的染色如图 1所示。红点反映细胞LD，蓝点反映细胞核。可以看出，在LA的处理下，每张图片中LD的大小和数量是不同的。 随着LA剂量的增加，LD的平均直径和数量显示出显着增加的趋势，这取决于LA浓度（图2）。如图2A所示，每个细胞的LD数量与不同浓度的 LA呈负相关。100 μmol/L LA处理组每个细胞的中位LD?…
Discussion
根据病理状态，肝LD的大小和数量会发生巨大变化。LD广泛存在于肝细胞中，在肝脏健康和疾病中起关键作用18。LDs的数量和大小是目前LDs19生物发生研究的基础。细胞和组织LD的大小和数量反映了它们储存和释放能量的能力。LDs的动态变化维持脂质代谢活动的稳定性20,21。LDs的异常积累发生在各种病理条件下，可能是?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究由国家自然科学基金（U1904116）共同支持。
Materials
| 0.25% trypsin | Gibco | 25200072 | reagent |
| 4% paraformaldehyde | Solarbio | P1110 | reagent |
| BODIPY 493/503 | invitrogen | 2295015 | reagent |
| Cedar oil | Solarbio | C7140 | reagent |
| cell counting chamber | equipment | ||
| cell culture dish | Corning | 353002 | material |
| cell sens software | Olympus IX73 | software | |
| Centrifuge | Eppendorf | equipment | |
| DMEM | HyClone | SH30022.01 | reagent |
| Fetal Bovine Serum | Gibco | 2492319 | reagent |
| hematoxylin | DingGuo | AR0712 | reagent |
| Image view | image analysis sodtware | ||
| linoleic acid | Solarbio | SL8520 | reagent |
| Live Cell Station | Nikon A1 HD25 | equipment | |
| NIS-Elements | Nikon | software | |
| oil red O | Solarbio | G1260 | reagent |
| optical microscope | Olympus IX73 | equipment | |
| Penicillin & Streptomycin 100× | NCM Biotech | CLOOC5 | reagent |
| Phosphate Buffered Saline | HyClone | SH30258.01 | reagent |
| Pipette | Eppendorf | equipment | |
| Sealing agent | Solarbio | S2150 | reagent |
References
- Fujimoto, T., Parton, R. G. Not just fat: the structure and function of the lipid droplet. Cold Spring Harbor Perspectives in Biology. 3 (3), 004838 (2011).
- Grasselli, E., et al. Models of non-alcoholic fatty liver disease and potential translational value: The effects of 3,5-L-diiodothyronine. Annals of Hepatology. 16 (5), 707-719 (2017).
- Herdt, T. H. Ruminant adaptation to negative energy balance: Influences on the etiology of ketosis and fatty liver. Veterinary Clinics of North America: Food Animal Practice. 16 (2), 215-230 (2000).
- Pino-de la Fuente, F., et al. Exercise regulation of hepatic lipid droplet metabolism. Life Sciences. 298, 120522 (2022).
- O’Connor, D., Byrne, A., Berselli, G. B., Long, C., Keyes, T. E. Mega-stokes pyrene ceramide conjugates for STED imaging of lipid droplets in live cells. Analyst. 144 (5), 1608-1621 (2019).
- Gao, G., et al. Control of lipid droplet fusion and growth by CIDE family proteins. Biochimica et Biophysica Acta (BBA). Molecular and Cell Biology of Lipids. 1862 (10), 1197-1204 (2017).
- Tütüncü Konyar, S. Dynamic changes in insoluble polysaccharides and neutral lipids in the developing anthers of an endangered plant species, Pancratium maritimum. Plant Systematics and Evolution. 304, 397-414 (2018).
- Spangenburg, E. E., Pratt, S. J. P., Wohlers, L. M., Lovering, R. M. Use of BODIPY (493/503) to visualize intramuscular lipid droplets in skeletal muscle. Journal of Biomedicine and Biotechnology. 2011, 598358 (2011).
- Mehlem, A., Hagberg, C. E., Muhl, L., Eriksson, U., Falkevall, A. Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nature Protocols. 8 (6), 1149-1154 (2013).
- Diaz, G., Melis, M., Batetta, B., Angius, F., Falchi, A. M. Hydrophobic characterization of intracellular lipids in situ by Nile Red red/yellow emission ratio. Micron. 39 (7), 819-824 (2008).
- Duan, X., et al. The synthesis of polarity-sensitive fluorescent dyes based on the BODIPY chromophore. Dyes and Pigments. 89 (3), 217-222 (2011).
- Rumin, J., et al. The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae. Biotechnology for Biofuels. 8, 42 (2015).
- Fam, T. K., Klymchenko, A. S., Collot, M. Recent advances in fluorescent probes for lipid droplets. Materials. 11 (9), 1768 (2018).
- Raboisson, D., Mounié, M., Maigné, &. #. 2. 0. 1. ;. Diseases, reproductive performance, and changes in milk production associated with subclinical ketosis in dairy cows: A meta-analysis and review. Journal of Dairy Science. 97 (12), 7547-7563 (2014).
- Ospina, P. A., Nydam, D. V., Stokol, T., Overton, T. R. Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States. Journal of Dairy Science. 93 (4), 1596-1603 (2010).
- Campos-Espinosa, A., Guzmán, C. A model of experimental steatosis in vitro: hepatocyte cell culture in lipid overload-conditioned medium. Journal of Visualized Experiments. (171), e62543 (2021).
- Liu, L., et al. Effects of nonesterified fatty acids on the synthesis and assembly of very low density lipoprotein in bovine hepatocytes in vitro. Journal of Dairy Science. 97 (3), 1328-1335 (2014).
- Wang, L., Liu, J. Y., Miao, Z. J., Pan, Q. W., Cao, W. L. Lipid droplets and their interactions with other organelles in liver diseases. The International Journal of Biochemistry & Cell Biology. 133, 105937 (2021).
- Sanjabi, B., et al. Lipid droplets hypertrophy: a crucial determining factor in insulin regulation by adipocytes. Scientific Reports. 5, 8816 (2015).
- Saponaro, C., Gaggini, M., Carli, F., Gastaldelli, A. The subtle balance between lipolysis and lipogenesis: a critical point in metabolic homeostasis. Nutrients. 7 (11), 9453-9474 (2015).
- Yang, A., Mottillo, E. P. Adipocyte lipolysis: from molecular mechanisms of regulation to disease and therapeutics. Biochemical Journal. 477 (5), 985-1008 (2020).
- Gluchowski, N. L., Becuwe, M., Walther, T. C., Farese, R. V. Lipid droplets and liver disease: from basic biology to clinical implications. Nature Reviews Gastroenterology & Hepatology. 14 (6), 343-355 (2017).
- Meex, R. C. R., Schrauwen, P., Hesselink, M. K. C. Modulation of myocellular fat stores: lipid droplet dynamics in health and disease. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 297 (4), 913-924 (2009).
- Sarnyai, F., et al. Effect of cis-and trans-monounsaturated fatty acids on palmitate toxicity and on palmitate-induced accumulation of ceramides and diglycerides. International Journal of Molecular Sciences. 21 (7), 2626 (2020).
- Ricchi, M., et al. Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes. Journal of Gastroenterology and Hepatology. 24 (5), 830-840 (2009).
- Fei, W., et al. A role for phosphatidic acid in the formation of "supersized" lipid droplets. PLoS Genetics. 7 (7), e1002201 (2011).
- Kowada, T., Maeda, H., Kikuchi, K. BODIPY-based probes for the fluorescence imaging of biomolecules in living cells. Chemical Society Reviews. 44 (14), 4953-4972 (2015).
- Wang, J., et al. Application of the fluorescent dye BODIPY in the study of lipid dynamics of the rice blast fungus Magnaporthe oryzae. Molecules. 23 (7), 1594 (2018).
Cite This Article
Yang, J., Kang, F., Wei, A., Lu, W., Zhang, X., Han, L. Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells. J. Vis. Exp. (193), e65234, doi:10.3791/65234 (2023).