分化肝细胞中的血管性脂细胞病的诱导和特征
Summary
在这项研究中,我们描述了用脂肪酸盐酸钠酸化酸酸诱导分化肝细胞中肝囊性性性软体炎的详细方案,并采用脂质积累检测和定量的方法,包括相干抗斯托克斯拉曼散射 (CARS) 显微镜、细胞氟分析、油红色 O 染色和 qPCR。
Abstract
肝性性性性性脂肪病是一种代谢功能障碍,由肝细胞中含甘油三酯的脂滴积累产生。过量的脂肪积累导致非酒精性脂肪肝(NAFLD),这是潜在的可逆的,并可能演变为非酒精性脂肪性肝炎(NASH),并最终肝硬化和肝细胞癌(HCC)。将肝细胞中的脂质积累与NASH进展、不可逆转的肝损伤、纤维化、肝硬化,甚至HCC联系起来的分子机制仍不清楚。为此,已经开发出几种体外和体内模型,以阐明引起NAFLD的病理过程。在本研究中,我们描述了一种肝囊性性性骨质化诱导的细胞模型,该模型由用脂肪酸盐酸钠油酸盐处理的DMSO分化的人类肝肝肝糖细胞组成。事实上,经油酸钠处理的HepaRG细胞在细胞质中积累脂液滴,并表现出性增生的典型特征。这种体外人类模型代表了体内小鼠模型以及主要人类肝细胞的宝贵替代品。我们还比较了HepaRG细胞脂肪积累定量和评估的几种方法,包括油红O染色、细胞氟度代谢测量、qPCR代谢基因表达分析、相干抗斯托克斯拉曼散射(CARS)显微镜。CARS 成像将拉曼光谱(一种在材料科学应用中广为人知的化学分析技术)的化学特异性与高速、高分辨率非线性光学显微镜的优点相结合,可实现精确脂质积累和脂滴动力学的定量。建立有效的体外诱导水塞性脂质模型,以及脂质积累定量和表征的准确方法,可以导致NAFLD早期诊断的发展。分子标记的识别,并产生新的治疗策略。
Introduction
肝性性性脂肪分解为肝内脂肪积累,在含甘油三酯的脂滴内,至少为肝脏重量的5%。长期肝脂储存是一个潜在的可逆过程,然而,它可能导致肝代谢功能障碍,炎症和非酒精性脂肪肝疾病(NAFLD)的晚期形式,慢性肝病的主要原因在许多地方世界1,2。NAFLD是一种多因素疾病,可能演变为更具攻击性的非酒精性脂肪性肝炎(NASH),进而进展为肝硬化,在一小部分患者中,可发展到肝细胞癌(HCC)1、3。目前没有批准的治疗是作为NAFLD的一种特定治疗,饮食和生活方式的改变的组合仍然是NAFLD和NASH管理4,5,6的支柱。
导致NAFLD发病机制中肝性性性性性粘突发展的分子机制仍有待阐明。在此背景下,小鼠模型已被开发,以研究人类性脂蛋白病的进展。存在无数种不同的模型,每种模型都有其优缺点,包括结合不同方法的遗传、营养和化学诱导模型。转基因(转基因或淘汰)小鼠自发地发展为肝病。然而,应该指出,这些突变在人类中非常罕见,单个基因(例如,ob/ob mouse)的缺失或过度表达在分子级8、9中可能不能模仿多因素人类疾病的病因。同样,小鼠在饮食或药理学操作后获得的疾病,也不得模仿人类饮食对人类8人NAFLD发展的影响。然而,动物模型促进了对NAFLD的理解的发展,这种方法是目前实验室研究中最常用的策略。然而,在人类身上复制动物模型获得的结果屡屡失败,导致临床翻译不佳。
因此,NAFLD的体外模型在阐明NAFLD进展的分子机制方面可以发挥基础性作用,它们是筛选大量化合物的宝贵工具。原生细胞培养物、永生细胞系和肝脏活检已广泛用于研究目的11。原发性人类肝细胞与人类临床条件非常相似,但捐献者数量有限,由于细胞的变异性,原发性细胞培养表现出较差的可重复性。这些观察,加上伦理和后勤问题,导致人类原发性肝细胞的使用受到限制。因此,肝细胞系代表一种方便的替代方法,与初级培养体具有若干基本优势,因为肝细胞系生长稳定,寿命几乎不受限制,并且具有稳定的表型。此外,细胞系易于访问,肝细胞系的培养条件比原发性肝细胞简单,并且在不同的实验室之间标准化。
在这里,我们详细介绍了一个体外细胞为基础的肝囊性性性骨质化模型,由用脂肪酸酸钠油酸盐治疗的肝分化肝糖细胞表示。HepaRG细胞系是由一名感染丙型肝炎的女性患者和爱德蒙森I级分化良好的肝肿瘤14建立的。HepaRG细胞系是一种人类双能祖细胞系,在暴露于2%二甲基亚硫酸盐(DMSO)时,能够区分两种不同的细胞表型:胆汁状细胞和肝细胞状细胞。分化肝细胞 (dHepaRG) 与成人肝细胞共享一些特征和特性,并具有稳定表达肝脏特定基因的能力,如白蛋白、AldolaseB、细胞色素 P450 2E1 (CYP2E1) 和细胞色素 P450 3A4 (CYP3A4)13(步骤 3)。用脂肪酸盐酸钠(250μM)治疗dHepaRG细胞5天,导致细胞质脂滴的产生,模仿脂肪肝14、15、17、18的影响(步骤 4)。血脂液滴的积累可以很容易地通过油红O染色(步骤5),溶酶脂溶性染料,染色中性甘油三酯和脂质红橙色。为了有效地量化脂肪 dHepaRG 中的脂质,这里我们演示了在染色后使用 4,4-二氟罗-1,3,5,7-四甲基-4-bora-3a,4a-diaza-s-indacene(Bodipy 505/515)(步骤6)的细胞氟度分析,这是一种用于局部的嗜脂荧光探针细胞内脂质体,并已用于标记脂滴19。此外,这里我们展示了如何通过定量聚合酶链反应(qPCR)(步骤7)基因表达解除dHepaRG细胞中几个代谢基因的脂质分质化。为了进一步描述和量化油酸钠处理后脂滴的积累,我们进行了相干抗斯托克斯拉曼散射(CARS)显微镜(步骤8),这是一种创新技术,使脂液滴没有标签20,21。
Protocol
Representative Results
Discussion
该协议描述了如何区分肝糖细胞,以及如何通过油酸钠治疗诱导水泡性骨质化(图1A)。事实上,与其他人类肝细胞癌 (HCC) 细胞系相比,HepaRG 细胞系具有成年人类肝细胞的特征,是外体培养原发人肝细胞13、14 的宝贵替代品 ,15.HepaRG细胞系已广泛应用于肝细胞毒性研究、药物代谢和病毒学研究15、16、2…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢克里斯蒂安·特雷波教授(INSERM U871,法国里昂),他友好地提供了HepaRG细胞。我们感谢丽塔·阿波迪亚提供行政帮助。这项工作得到了MIUR-部长-德尔斯特鲁齐奥内、意大利大学(FIRB 2011-2016 RBAP10XKNC)和罗马萨皮恩扎大学(Prot.)的支持。C26A13T8PS;波特。C26A142MCH;波特。C26A15LSXL)。
Materials
| Hyclone HyClone Fetal Clone II | GE Healthcare | SH30066 | |
| William’s E medium with GlutaMAX | Thermofisher | 32551087 | |
| Penicillin/streptomycin | SIGMA | P4333 | |
| Insulin | SIGMA | I9278 | |
| hydrocortisone hemisuccinate | SIGMA | H2270 | |
| DMSO, dimethyl sulfoxide | SIGMA | D2438 | |
| Sodium Oleate | SIGMA | O7501 | |
| Methanol | SIGMA | 179337 | |
| Isopropanol | SIGMA | 278475 | |
| BODIPY 505/515 | Thermofisher | D3921 | |
| PBS | Thermofisher | 14190-250 | |
| Formaldehyde solution | SIGMA | 252549 | |
| RNAse free DNAseI | Promega | M198A | |
| Glass-bottomed dishes | Willco Wells | GWST-5040 | |
| Oil Red solution | SIGMA | O625 | |
| CellTiter 96 AQueous One Solution | Promega | G3582 | |
| q-PCR oligo name | Sequence | ||
| ACACB FOR | CAAGCCGATCACCAAGAGTAAA | ||
| ACACB REV | CCCTGAGTTATCAGAGGCTGG | ||
| b-actin FOR | GCACTCTTCCAGCCTTCCT | ||
| b-actin REV | AGGTCTTTGCGGATGTCCAC | ||
| ALBUMIN FOR | TGCTTGAATGTGCTGATGACAGG | ||
| ALBUMIN REV | AAGGCAAGTCAGCAGGCATCTCATC | ||
| ALDOB FOR | GCATCTGTCAGCAGAATGGA | ||
| ALDOB REV | TAGACAGCAGCCAGGACCTT | ||
| APOB FOR | CCTCCGTTTTGGTGGTAGAG | ||
| APOB REV | CCTAAAAGCTGGGAAGCTGA | ||
| APOC3 FOR | CTCAGCTTCATGCAGGGTTA | ||
| APOC3 REV | GGTGCTCCAGTAGTCTTTCAG | ||
| CPT1A FOR | TCATCAAGAAATGTCGCACG | ||
| CPT1A REV | GCCTCGTATGTGAGGCAAAA | ||
| CYP2E1 FOR | TTGAAGCCTCTCGTTGACCC | ||
| CYP2E1 REV | CGTGGTGGGATACAGCCAA | ||
| CYP3A4 FOR | CTTCATCCAATGGACTGCATAAAT | ||
| CYP3A4 REV | TCCCAAGTATAACACTCTACACAGACAA | ||
| GAPDH FOR | TGACAACTTTGGTATCGTGGAAGG | ||
| GAPDH REV | AGGGATGATGTTCTGGAGAGCC | ||
| GPAM FOR | TCTTTGGGTTTGCGGAATGTT | ||
| GPAM REV | ATGCACATCTCGCTCTTGAATAA | ||
| IL6 FOR | CCTGAACCTTCCAAAGATGGC | ||
| IL6 REV | ACCTCAAACTCCAAAAGACCAGTG | ||
| PDK4 FOR | ACAGACAGGAAACCCAAGCCAC | ||
| PDK4 REV | TGGAGGTGAGAAGGAACATACACG | ||
| PLIN2 FOR | TTGCAGTTGCCAATACCTATGC | ||
| PLIN2 REV | CCAGTCACAGTAGTCGTCACA | ||
| PLIN4 FOR | AATGAGTTGGAGGGGCTGGGGGACATC | ||
| PLIN4 REV | GGTCACCTAAACGAACGAAGTAGC | ||
| SCD FOR | TCTAGCTCCTATACCACCACCA | ||
| SCD REV | TCGTCTCCAACTTATCTCCTCC | ||
| SLC2A1 FOR | TGCTCATCAACCGCAACGAG | ||
| SLC2A1 REV | CCGACTCTCTTCCTTCATCTCCTG | ||
| 18S FOR | CGCCGCTAGAGGTGAAATTC | ||
| 18S REV | TTGGCAAATGCTTTCGCTC |
References
- Starley, B. Q., Calcagno, C. J., Harrison, S. A. Nonalcoholic fatty liver disease and hepatocellular carcinoma: a weighty connection. Hepatology. 51 (5), 1820-1832 (2010).
- Byrne, C. D., Targher, G. NAFLD: a multisystem disease. Journal of Hepatology. 62 (1), 47-64 (2015).
- Marra, F., Gastaldelli, A., Svegliati Baroni, G., Tell, G., Tiribelli, C. Molecular basis and mechanisms of progression of non- alcoholic steatohepatitis. Trends in Molecular Medicine. 14, 72-81 (2008).
- Chalasani, N., et al. The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases. Hepatology. 67 (1), 328-357 (2018).
- Banini, B. A., Sanyal, A. J. Current and future pharmacologic treatment of nonalcoholic steatohepatitis. Current Opinion in Gastroenterology. 33 (3), 134-141 (2017).
- Takahashi, Y., Sugimoto, K., Inui, H., Fukusato, T. Current pharmacological therapies for nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. World Journal of Gastroenterology. 21 (13), 3777-3785 (2015).
- Younossi, Z., et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nature Reviews Gastroenterology & Hepatology. 15 (1), 11-20 (2018).
- Santhekadur, P. K., Kumar, D. P., Sanyal, A. J. Preclinical models of non-alcoholic fatty liver disease. Journal of Hepatology. 68 (2), 230-237 (2018).
- Nagarajan, P., Mahesh Kumar, M. J., Venkatesan, R., Majundar, S. S., Juyal, R. C. Genetically modified mouse models for the study of nonalcoholic fatty liver disease. World Journal of. Gastroenterology. 18 (11), 1141-1153 (2012).
- Oseini, A. M., Cole, B. K., Issa, D., Feaver, R. E., Sanyal, A. J. Translating scientific discovery: the need for preclinical models of nonalcoholic steatohepatitis. Hepatology International. 12 (1), 6-16 (2018).
- Ramboer, E., Vanhaecke, T., Rogiers, V., Vinken, M. Immortalized human hepatic cell lines for in vitro testing and research purposes. Methods in Molecular Biology. 1250, 53-76 (2015).
- Gómez-Lechón, M. J., Donato, M. T., Castell, J. V., Jover, R. Human hepatocytes in primary culture: the choice to investigate drug metabolism in man. Current Drug Metabolism. 5 (5), 443-462 (2004).
- Marion, M. J., Hantz, O., Durantel, D. The HepaRG cell line: biological properties and relevance as a tool for cell biology, drug metabolism, and virology studies. Methods in Molecular Biology. 640, 261-272 (2010).
- Gripon, P., et al. Infection of a human hepatoma cell line by hepatitis B virus. Proceedings of the National Academy of Sciences. 99 (24), 15655-15660 (2002).
- Anthérieu, S., Rogue, A., Fromenty, B., Guillouzo, A., Robin, M. A. Induction of vesicular steatosis by amiodarone and tetracycline is associated with up-regulation of lipogenic genes in HepaRG cells. Hepatology. 53 (6), 1895-1905 (2011).
- Rouge, A., et al. PPAR agonists reduce steatosis in oleic acid-overloaded HepaRG cells. Toxicology and Applied Pharmacology. 276 (1), 73-81 (2014).
- Belloni, L., et al. Targeting a phospho-STAT3-miRNAs pathway improves vesicular hepatic steatosis in an in vitro and in vivo model. Scientific Reports. 8, 13638 (2018).
- Nunn, A. D. G., et al. The histone deacetylase inhibiting drug Entinostat induces lipid accumulation in differentiated HepaRG cells. Scientific Reports. 6, 28025 (2016).
- Donato, M. T., et al. Cytometric analysis for drug-induced steatosis in HepG2 cells. Chemico-Biological Interactions. 181 (3), 417-423 (2009).
- Hellerer, T., Axäng, C., Brackmann, C., Hillertz, P., Pilon, M., Enejder, A. Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy. Proceedings of the National Academy of Sciences. 104 (37), 14658-14663 (2007).
- Le, T. T., Ziemba, A., Urasaki, Y., Brotman, S., Pizzorno, G. Label-free evaluation of hepatic microvesicular steatosis with multimodal coherent anti-Stokes Raman scattering microscopy. PLoS One. 7 (11), 51092 (2012).
- Guillouzo, A., Corlu, A., Aninat, C., Glaise, D., Morel, F., Guguen-Guillouzo, C. The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chemico-Biological Interactions. 168 (1), 66-73 (2007).
- Nelson, L. J., et al. Human hepatic HepaRG cells maintain an organotypic phenotype with high intrinsic CYP450 Activity/metabolism and significantly outperform standard HepG2/C3A cells for pharmaceutical and therapeutic applications. Basic & Clinical Pharmacology & Toxicology. 120 (1), 30-37 (2017).
- Gerets, H. H. J., Tilmant, K., Gerin, B., Chanteux, H., Depelchin, B. O., Dhalluin, S., Atienzar, F. A. Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biology and Toxicology. 28 (2), 69-87 (2012).
- Pusl, T., et al. Free fatty acids sensitize hepatocytes to bile acid-induced apoptosis. Biochemical and Biophysical Research Communications. 371 (3), 441-445 (2008).
- Gentile, C. L., Pagliassotti, M. J. The role of fatty acids in the development and progression of nonalcoholic fatty liver disease. The Journal of Nutritional Biochemistry. 19 (9), 567-576 (2008).
- Mei, S., et al. Differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes. Journal of Pharmacological Experimental Therapy. 339, 487-498 (2011).
- Malhi, H., Bronk, S. F., Werneburg, N. W., Gores, G. J. Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis. Journal of Biological Chemistry. 281, 12093-12101 (2006).
- Moravcová, A., Červinková, Z., Kučera, O., Mezera, V., Rychtrmoc, D., Lotková, H. The effect of oleic and palmitic acid on induction of steatosis and cytotoxicity on rat hepatocytes in primary culture. Physiological Research. 64, 627-636 (2015).
- Ohsaki, Y., Shinohara, Y., Suzuki, M., Fujimoto, T. A pitfall in using BODIPY dyes to label lipid droplets for fluorescence microscopy. Histochemistry and Cell Biology. 133 (4), 477-480 (2010).
- Rinia, H. A., Burger, K. N., Bonn, M., Müller, M. Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy. Biophysical Journal. 95 (10), 4908-4914 (2008).
- Waschatko, G., Billecke, N., Schwendy, S., Jaurich, H., Bonn, M., Vilgis, T. A., Parekh, S. H. Label-free in situ imaging of oil body dynamics and chemistry in germination. Journal of The Royal Society Interface. 13 (123), (2016).
- Jüngst, C., Winterhalder, M. J., Zumbusch, A. Fast and long term lipid droplet tracking with CARS microscopy. Journal of Biophotonics. 4 (6), 435-441 (2011).
- Welte, M. A., Gould, A. P. Lipid droplet functions beyond energy storage. Biochimica et Biophysica Acta – Molecular and Cell Biology of Lipids. 1862 (10), 1260-1272 (2017).
- Cohen, S. Lipid droplets as organelles. International Review of Cell and Molecular Biology. 337, 83-110 (2018).
- Farese, R. V., Walther, T. C. Lipid droplets finally get a little R-E-S-P-E-C-T. Cell. 139 (5), 855-860 (2009).
