Name: a model of experimental steatosis in vitro: hepatocyte cell culture in lipid overload-conditioned medium
Uploaded: 2021-05-18T14:00:00
Description: Watch this Scientific Journal Video about A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium at JoVE.com

这项工作由国家委员会和特克诺洛尼亚委员会资助（委员会，CB-221137）。阿德里亚娜·坎波斯是墨西哥国立奥蒂诺马大学生物学博士项目的博士生，并得到科纳西特（CVU：1002502）的支持。

1. 标准和有条件的中等准备
<ol><li>要准备标准RPMI 1640，补充RPMI 1640培养介质与10%（v/v）的胎儿牛血清（FBS，以前热灭活）和1%（v/v）青霉素-链霉素溶液。使用 0.22 μm 过滤器将介质存储在 4 °C.消毒。</li>
	<li>要准备棕榈酸盐库存溶液，请在标准 RPMI 1640 中准备 50 mM 的棕榈酸盐溶液，以前补充 1% 的牛血清白蛋白（无脂）。该库存的 5-10 mL 的体积就足够了。使用 0.22 μm 过滤器对库存溶液进行消毒。?...

<ol>
	<li>Younossi, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-alcoholic+fatty+liver+disease+-+a+global+public+health+perspective.">Non-alcoholic fatty liver disease - a global public health perspective.</a> Journal of Hepatology. 70 (3), 531-544 (2019).</li><li>Younossi, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+perspectives+on+nonalcoholic+fatty+liver+disease+and+nonalcoholic+steatohepatitis.">Global perspectives on nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.</a> Hepatology. 69 (6), 2672-2682 (2019).</li><li>Eslam, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+definition+for+metabolic+dysfunction-associated+fatty+liver+disease:+An+international+expert+consensus+statement.">A new definition for metabolic dysfunction-associated fatty liver disease: An international expert consensus statement.</a> Journal of Hepatology. 73 (1), 202-209 (2020).</li><li>Eslam, M., Sanyal, A. J., George, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAFLD:+A+consensus-driven+proposed+nomenclature+for+metabolic+associated+fatty+liver+disease.">MAFLD: A consensus-driven proposed nomenclature for metabolic associated fatty liver disease.</a> Gastroenterology. 158 (7), 1999-2014 (2020).</li><li>Chalasani, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnosis+and+management+of+nonalcoholic+fatty+liver+disease:+Practice+guidance+from+the+American+association+for+the+study+of+liver+diseases.">The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American association for the study of liver diseases.</a> Hepatology. 67 (1), 328-357 (2018).</li><li>Calzadilla Bertot, L., Adams, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+course+of+non-alcoholic+fatty+liver+disease.">The natural course of non-alcoholic fatty liver disease.</a> International Journal of Molecular Science. 17 (5), 774 (2016).</li><li>Reccia, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-alcoholic+fatty+liver+disease:+A+sign+of+systemic+disease.">Non-alcoholic fatty liver disease: A sign of systemic disease.</a> Metabolism. 72, 94-108 (2017).</li><li>Tomita, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+cholesterol+accumulation+in+hepatic+stellate+cells:+Mechanism+of+liver+fibrosis+aggravation+in+nonalcoholic+steatohepatitis+in+mice.">Free cholesterol accumulation in hepatic stellate cells: Mechanism of liver fibrosis aggravation in nonalcoholic steatohepatitis in mice.</a> Hepatology. 59 (1), 154-169 (2014).</li><li>Byrne, C. D., Targher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nafld:+A+multisystem+disease.">Nafld: A multisystem disease.</a> Journal of Hepatology. 62, 47-64 (2015).</li><li>Ipsen, D. H., Lykkesfeldt, J., Tveden-Nyborg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+hepatic+lipid+accumulation+in+non-alcoholic+fatty+liver+disease.">Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease.</a> Cellular and Molecular Life Sciences. 75 (18), 3313-3327 (2018).</li><li>Diehl, A. M., Day, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cause,+pathogenesis,+and+treatment+of+nonalcoholic+steatohepatitis.">Cause, pathogenesis, and treatment of nonalcoholic steatohepatitis.</a> New England Journal of Medicine. 377 (21), 2063-2072 (2017).</li><li>Zeng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oleic+acid+ameliorates+palmitic+acid+induced+hepatocellular+lipotoxicity+by+inhibition+of+ER+stress+and+pyroptosis.">Oleic acid ameliorates palmitic acid induced hepatocellular lipotoxicity by inhibition of ER stress and pyroptosis.</a> Nutrition and Metabolism. 17, 11 (2020).</li><li>Ore, A., Akinloye, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+antioxidant+biomarkers+in+clinical+and+experimental+models+of+non-alcoholic+fatty+liver+disease.">Oxidative stress and antioxidant biomarkers in clinical and experimental models of non-alcoholic fatty liver disease.</a> Medicina (Kaunas). 55 (2), 26 (2019).</li><li>Paradies, G., Paradies, V., Ruggiero, F. M., Petrosillo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress,+cardiolipin+and+mitochondrial+dysfunction+in+nonalcoholic+fatty+liver+disease.">Oxidative stress, cardiolipin and mitochondrial dysfunction in nonalcoholic fatty liver disease.</a> World Journal of Gastroenterology. 20 (39), 14205-14218 (2014).</li><li>Aravinthan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+senescence+predicts+progression+in+non-alcohol-related+fatty+liver+disease.">Hepatocyte senescence predicts progression in non-alcohol-related fatty liver disease.</a> Journal of Hepatology. 58 (3), 549-556 (2013).</li><li>Gomez, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+hepatocellular+in+vitro+model+to+investigate+steatosis.">A human hepatocellular in vitro model to investigate steatosis.</a> Chemico Biological Interactions. 165 (2), 106-116 (2007).</li><li>Wu, W. K. K., Zhang, L., Chan, M. T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy,+NAFLD+and+NAFLD-related+HCC.">Autophagy, NAFLD and NAFLD-related HCC.</a> Advances in Experimental Medicine and Biology. 1061, 127-138 (2018).</li><li>Levy, G., Cohen, M., Nahmias, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+culture+models+of+hepatic+steatosis.">In vitro cell culture models of hepatic steatosis.</a> Methods in Molecular Biology. 1250, 377-390 (2015).</li><li>Kanuri, G., Bergheim, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+models+of+non-alcoholic+fatty+liver+disease+(NAFLD).">In vitro and in vivo models of non-alcoholic fatty liver disease (NAFLD).</a> International Journal of Molecular Science. 14 (6), 11963-11980 (2013).</li><li>Ricchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effect+of+oleic+and+palmitic+acid+on+lipid+accumulation+and+apoptosis+in+cultured+hepatocytes.">Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes.</a> Journal of Gastroenterology and Hepatology. 24 (5), 830-840 (2009).</li><li>Lee, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+biomarkers+of+de+novo+lipogenesis+fatty+acids+and+incident+heart+failure+in+older+adults:+The+cardiovascular+health+study.">Serial biomarkers of de novo lipogenesis fatty acids and incident heart failure in older adults: The cardiovascular health study.</a> Journal of the American Heart Association. 9 (4), 014119 (2020).</li><li>Alnahdi, A., John, A., Raza, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmentation+of+glucotoxicity,+oxidative+stress,+apoptosis+and+mitochondrial+dysfunction+in+Hepg2+cells+by+palmitic+acid.">Augmentation of glucotoxicity, oxidative stress, apoptosis and mitochondrial dysfunction in Hepg2 cells by palmitic acid.</a> Nutrients. 11 (9), 1979 (2019).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oleic+acid+protects+saturated+fatty+acid+mediated+lipotoxicity+in+hepatocytes+and+rat+of+non-alcoholic+steatohepatitis.">Oleic acid protects saturated fatty acid mediated lipotoxicity in hepatocytes and rat of non-alcoholic steatohepatitis.</a> Life Sciences. 203, 291-304 (2018).</li><li>Xing, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NLRP3+inflammasome+mediate+palmitate-induced+endothelial+dysfunction.">NLRP3 inflammasome mediate palmitate-induced endothelial dysfunction.</a> Life Sciences. 239, 116882 (2019).</li><li>Yan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+Growth+Factor+Binding+Protein+7+accelerates+hepatic+steatosis+and+insulin+resistance+in+non-alcoholic+fatty+liver+disease.">Insulin-like Growth Factor Binding Protein 7 accelerates hepatic steatosis and insulin resistance in non-alcoholic fatty liver disease.</a> Clinical and Experimental Pharmacology and Physiology. 46 (12), 1101-1110 (2019).</li><li>Wang, J., Hu, R., Yin, C., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tanshinone+IIA+reduces+palmitate-induced+apoptosis+via+inhibition+of+endoplasmic+reticulum+stress+in+Hepg2+liver+cells.">Tanshinone IIA reduces palmitate-induced apoptosis via inhibition of endoplasmic reticulum stress in Hepg2 liver cells.</a> Fundamental and Clinical Pharmacology. 34 (2), 249-262 (2020).</li><li>Xiao, Z., Chu, Y., Qin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGFBP5+modulates+lipid+metabolism+and+insulin+sensitivity+through+activating+ampk+pathway+in+non-alcoholic+fatty+liver+disease.">IGFBP5 modulates lipid metabolism and insulin sensitivity through activating ampk pathway in non-alcoholic fatty liver disease.</a> Life Sciences. 256, 117997 (2020).</li><li>Avila, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+effects+of+conjugated+linoleic+acid+(CLA)+on+inflammatory+functions+of+bovine+monocytes.">In vitro effects of conjugated linoleic acid (CLA) on inflammatory functions of bovine monocytes.</a> Journal of Dairy Science. 103 (9), 8554-8563 (2020).</li><li>Malhi, H., Bronk, S. F., Werneburg, N. W., Gores, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+fatty+acids+induce+JNK-dependent+hepatocyte+lipoapoptosis.">Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis.</a> The Journal of Biological Chemistry. 281 (17), 12093-12101 (2006).</li><li>Oh, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+palmitic+acid+on+TNF-alpha-induced+cytotoxicity+in+SK-Hep-1+cells.">Effects of palmitic acid on TNF-alpha-induced cytotoxicity in SK-Hep-1 cells.</a> Toxicology In Vitro: An International Journal Published in Association with BIBRA. 26 (6), 783-790 (2012).</li><li>Stellavato, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assessment+of+nutraceutical+compounds+and+novel+nutraceutical+formulations+in+a+liver-steatosis-based+model.">In vitro assessment of nutraceutical compounds and novel nutraceutical formulations in a liver-steatosis-based model.</a> Lipids in Health and Disease. 17 (1), 24 (2018).</li><li>Pachikian, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implication+of+trans-11,+trans-13+conjugated+linoleic+acid+in+the+development+of+hepatic+steatosis.">Implication of trans-11, trans-13 conjugated linoleic acid in the development of hepatic steatosis.</a> PLoS One. 13 (2), 0192447 (2018).</li><li>Ferreira, T., Rasband, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyze.">Analyze.</a> ImageJ User Guide. , 132-135 (2012).</li><li>Geng, Y., Wu, Z., Buist-Homan, M., Blokzijl, H., Moshage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hesperetin+protects+against+palmitate-induced+cellular+toxicity+via+induction+of+GRP78+in+hepatocytes.">Hesperetin protects against palmitate-induced cellular toxicity via induction of GRP78 in hepatocytes.</a> Toxicology and Applied Pharmacology. , 404 (2020).</li><li>Geng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+metformin+against+palmitate-induced+hepatic+cell+death.">Protective effect of metformin against palmitate-induced hepatic cell death.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. 1866 (3), 165621 (2020).</li><li>Sarnyai, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cis-+and+trans-monounsaturated+fatty+acids+on+palmitate+toxicity+and+on+palmitate-induced+accumulation+of+ceramides+and+diglycerides.">Effect of cis- and trans-monounsaturated fatty acids on palmitate toxicity and on palmitate-induced accumulation of ceramides and diglycerides.</a> International Journal of Molecular Science. 21 (7), 2626 (2020).</li><li>Chen, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrahydrocurcumin+ameliorates+free+fatty+acid-induced+hepatic+steatosis+and+improves+insulin+resistance+in+HepG2+cells.">Tetrahydrocurcumin ameliorates free fatty acid-induced hepatic steatosis and improves insulin resistance in HepG2 cells.</a> Journal of Food and Drug Analysis. 26 (3), 1075-1085 (2018).</li><li>Burhans, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+oleate+regulates+adipose+tissue+lipogenesis+and+fatty+acid+oxidation.">Hepatic oleate regulates adipose tissue lipogenesis and fatty acid oxidation.</a> Journal of Lipid Research. 56 (2), 304-318 (2015).</li><li>Abenavoli, L., Milanovic, M., Milic, N., Luzza, F., Giuffre, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olive+oil+antioxidants+and+non-alcoholic+fatty+liver+disease.">Olive oil antioxidants and non-alcoholic fatty liver disease.</a> Expert Reviews in Gastroenterology and Hepatology. 13 (8), 739-749 (2019).</li><li>Nagarajan, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+and+glucose+metabolism+in+hepatocyte+cell+lines+and+primary+mouse+hepatocytes:+A+comprehensive+resource+for+in+vitro+studies+of+hepatic+metabolism.">Lipid and glucose metabolism in hepatocyte cell lines and primary mouse hepatocytes: A comprehensive resource for in vitro studies of hepatic metabolism.</a> American Journal of Physiology - Endocrinology and Metabolism. 316 (4), 578-589 (2019).</li><li>Hodson, L., Skeaff, C. M., Fielding, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+composition+of+adipose+tissue+and+blood+in+humans+and+its+use+as+a+biomarker+of+dietary+intake.">Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake.</a> Progress in Lipid Research. 47 (5), 348-380 (2008).</li></ol>

该协议旨在提供一个战略，研究体 外硬化。细胞培养是研究暴露在不同条件下的细胞的细胞、分子、生化和毒理学方面的有力工具。通过这种方法，硬化不仅可以被想象为MAFLD复杂疾病的一个阶段，而且可以被想象为肝细胞过度暴露在脂质中以及这种接触可能的结果。因此，它的应用并不局限于MAFLD的生理病理学，而是脂肪肝患者接触到治疗药物、污染物以及其他可能受硬化影响的疾病。?...

脂肪肝与代谢功能障碍有关，在世界范围内非常普遍1，2：据估计，多达25%的人口受到影响3。这种疾病以前被称为非酒精性脂肪肝病（NAFLD），它更新了代谢功能障碍相关的脂肪肝疾病（MAFLD）的命名，以准确反映与肥胖，胰岛素抵抗，糖尿病2型和血脂异常有关的发病机制，以及疾病的可能管理3，4。
无论名称如何，该病包括广泛的组织病理学变化，其特征是肝脏中脂质的异常高积累（肝细胞5中脂肪的>5%），并可能通过通常存在于简单硬化中的脂质积累进展到硬膜炎，这反过来又可能导致纤维化、肝硬化的发展， 肝细胞癌和肝衰竭5，6，7，8。由于其日益流行，MAFLD有望成为肝移植的第一个迹象和肝细胞癌9的主要原因。
虽然它被认为是脂肪肝疾病的良性或温和形式，肝硬化实际上是MAFLD10中的代谢键。不同的代谢途径受肝脏脂质积累的影响，包括但不限于脂质合成、出口和代谢10。胰岛素抵抗、氧化应激、网状应激和细胞功能障碍与肝脂质11、12密切相关。另一方面，脂肪肝细胞是活性氧物种的目标，使代谢物成为过氧化脂、蛋白质碳基和核酸的附庸。在细胞水平上，脂肪肝细胞可能经历线粒体损伤14，细胞衰老15，凋亡16，热细胞增多12，自噬17，以及其他事件。
肝细胞对新陈代谢、排毒和广泛分子的合成负有高度责任。许多这些功能可能因在硬化中观察到的脂质积累而受损。因此，拥有可重复的工具，以便准确评估硬化，是非常重要的。从这个意义上说，体外模型是易于适用和高度可重复的。体外史的斯蒂特病已经用不同的目标16，18，19。HepG2细胞被广泛用作肝细胞系。它具有易于培养和特征良好的优点。也许，HepG2细胞的唯一缺点是它是致癌细胞系，所以在分析结果时必须考虑这一点。在这里，显示了脂肪酸混合物在细胞培养中广泛使用的应用：棕榈酸（PA）和油酸（OA）。PA 和 OA 在文化20中都提供了不同的结果。PA （C 16：0） 是最常见的饱和脂肪酸从饮食16获得。PA被认为是非脂肪生成的生物标志物，是NAFLD21发展的关键一步。PA显示为剧毒22：因此，可能不建议在体外诱发硬化。OA （C 18：1） 是一种单不饱和脂肪酸。与PA相比，OA被建议具有抗炎和抗氧化特性，能够对抗PA12。PA和OA都是甘油三酯中的主要脂肪酸，无论健康状况或疾病16。表1提供了肝细胞培养与PA，OA，及其混合物的例子，以及报告的结果12，23，24，25，26，27。其他脂肪酸也用于肝细胞培养，包括乳酸（C 18：0）28，29，30，亚油酸 （C 18：1）28，30，31及其结合 （CLA）28，32，棕榈酸 （C 16：1）29.然而，他们的使用在文献中最不常被报道，也许是因为他们的肝丰度低于PA和OA16。
同时，这两种脂肪酸类似于体外硬化，提供增殖细胞，与控制条件相比，细胞死亡增加，生存能力降低。值得一提的是，这些脂肪酸的盐是可用的，也可以使用。评估肝细胞培养中的脂质超载时，主要问题之一是毒理模型和最能代表硬化的模型之间的差异。许多模型可以在第一种情况下进行说明。事实上，单单使用PA就可视为其中之一，而高死亡率是最明显的结果12、16、23、24、25、26、27。即使在OA的情况下，使用高剂量也可以被视为毒理学模型。这里显示的协议是根据硬化的发展更高，因为它显示低死亡率相比，观察到的其他模型，并允许它遵循在几天与渐进脂质积累，因为它发生在NAFLD。通过实验条件评估轻度和重度硬化的可能性被认为是另一个优势。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>脂肪酸</td>
			<td colspan="2">条件</td>
			<td colspan="2">结果</td>
			<td>参考</td>
		</tr>
<tr>
<td rowspan="3">宾夕法尼亚州</td>
			<td colspan="2">浓度： 200 μM</td>
			<td colspan="2">脂质积累</td>
			<td rowspan="3">严等人， 201925.</td>
		</tr>
<tr>
<td colspan="2">时间曝光： 24 小时</td>
			<td colspan="2">肝细胞损伤</td>
		</tr>
<tr>
<td colspan="2"></td>
			<td colspan="2">跨氨酶高程</td>
		</tr>
<tr>
<td rowspan="2">宾夕法尼亚州</td>
			<td colspan="2">浓度： 50、100 和 200 μM</td>
			<td colspan="2" rowspan="2">脂质积累</td>
			<td rowspan="2">兴等人， 201924.</td>
		</tr>
<tr>
<td colspan="2">时间曝光： 24 小时</td>
		</tr>
<tr>
<td rowspan="2">宾夕法尼亚州</td>
			<td colspan="2">浓度：250 μM、500 μM、750 μM 和 1，000 μM</td>
			<td colspan="2">脂质积累</td>
			<td rowspan="2">王等人， 202026.</td>
		</tr>
<tr>
<td colspan="2">时间曝光： 24 小时</td>
			<td colspan="2">逐步降低细胞生存能力</td>
		</tr>
<tr>
<td rowspan="3">OA/PA 的混合</td>
			<td colspan="2">浓度： 1mM</td>
			<td colspan="2">脂质积累</td>
			<td rowspan="3">肖等人， 202027.</td>
		</tr>
<tr>
<td colspan="2">时间曝光： 24 小时</td>
			<td colspan="2">不报告脂质</td>
		</tr>
<tr>
<td colspan="2">费率： 2OA：1PA</td>
			<td colspan="2"></td>
		</tr>
<tr>
<td rowspan="4">OA/PA 的混合</td>
			<td colspan="2">第一次刺激 200 μM 和 400 μM 的 PA，然后第二次刺激与 200 μM 的 OA</td>
			<td colspan="2">脂质积累。</td>
			<td rowspan="4">曾等人， 202012.</td>
		</tr>
<tr>
<td colspan="2">浓度：400 μM PA： 200 μM OA</td>
			<td colspan="2">PA 诱发的脂质性的证据因 OA 的刺激而减少。</td>
		</tr>
<tr>
<td colspan="2">价格： 2PA：1OA</td>
			<td colspan="2"></td>
		</tr>
<tr>
<td colspan="2">时间曝光： 24 小时</td>
			<td colspan="2"></td>
		</tr>
<tr>
<td rowspan="3">OA/PA 的混合</td>
			<td colspan="2">浓度： 400 μM PA： 200 μM OA</td>
			<td colspan="2" rowspan="3">脂质积累</td>
			<td rowspan="3">陈等人， 201823.</td>
		</tr>
<tr>
<td colspan="2">价格： 2PA：1OA</td>
		</tr>
<tr>
<td colspan="2">时间曝光： 24 小时</td>
		</tr>
<tr>
<td rowspan="3">OA/PA 的混合</td>
			<td colspan="2">浓度 ：50 和 500 μM</td>
			<td colspan="2">两种类型的硬化的生成：轻度硬化和 严重的硬化。</td>
			<td rowspan="3">坎波斯和古兹曼 2021</td>
		</tr>
<tr>
<td colspan="2">价格： 2PA：1OA</td>
			<td colspan="2">模拟脂质超载的长期阐述</td>
		</tr>
<tr>
<td colspan="2">时间暴露：24小时，2天，3天和4天。</td>
			<td colspan="2"></td>
		</tr>
</tbody></table>表1。肝细胞培养在造菌条件下。 该表介绍了脂肪酸的使用类型、维持的条件以及肝细胞培养的观察结果。PA：棕榈酸。欧亚：油酸。
最后，该模型不仅适用于硬化和脂肪肝的研究，还适用于在硬化背景下的肝代谢、合成和排毒途径。此外， 体外 诱发的硬化可能为确定该疾病的潜在标记和治疗靶点提供证据。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Biosafety cabinet</td>
			<td>ESCO Airstream</td>
			<td>AC2-452+C2:C26</td>
			<td>Class II&#160;Type A2&#160;Biological Safety Cabinet</td>
		</tr>
		<tr>
			<td>Bottle top filter</td>
			<td>Corning, US</td>
			<td>430513</td>
			<td>&#160;Non-pyrogenic, polystyrene, sterile. 1 filter/Bag. 0.22 &#956;m, 500 mL.</td>
		</tr>
		<tr>
			<td>Bovine serum albimun (BSA)</td>
			<td>Gold Biotechnology, US</td>
			<td>A-421-10</td>
			<td>BSA Fatty Acid Free for cell culture</td>
		</tr>
		<tr>
			<td>Culture media RPMI 1640</td>
			<td>ThermoFisher-Gibco, US</td>
			<td>31800-022</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFisher-Gibco, US</td>
			<td>A4766801</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Marienfeld, DE</td>
			<td>640010</td>
			<td>-</td>
		</tr>
		<tr>
			<td>HepG2 cell line</td>
			<td>ATCC, US</td>
			<td>HB-8065</td>
			<td>Hepatocellular carcinoma human cells.</td>
		</tr>
		<tr>
			<td>Humidified incubator</td>
			<td>Thermo Electronic Corporation,US</td>
			<td>Model: 3110</td>
			<td>Temperature (37 &#176;C &#177; 1 &#176;C), humidity (90% &#177; 5%) , CO2 (5% &#177; 1%)</td>
		</tr>
		<tr>
			<td>Inverted microscope Eclipse</td>
			<td>NIKON, JPN</td>
			<td>Model: TE2000-S</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich, US</td>
			<td>I9030-4L</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Oil Red O Kit</td>
			<td>Abcam, US</td>
			<td>ab150678</td>
			<td>Kit for histological visualization of neutral fat.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich, US</td>
			<td>P6148-500G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>ThermoFisher-Gibco, US</td>
			<td>15140-122</td>
			<td>Antibiotics 10,000 U/mL Penicillin, 10,000 &#956;g/mL Streptomycin</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Beckman, US</td>
			<td>Model: 360 PH/Temp/MV Meter</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>ThermoFisher-Gibco, US</td>
			<td>10010-023</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Sarstedt, AUS</td>
			<td>86.1253.001</td>
			<td>&#160;Non-pyrogenic, sterile, 5 mL</td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Sarstedt, AUS</td>
			<td>&#160;86.1254.001</td>
			<td>&#160;Non-pyrogenic, sterile, 10 mL</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich, US</td>
			<td>S5761-1KG</td>
			<td>Preparation of culture media</td>
		</tr>
		<tr>
			<td>Sodium oleate</td>
			<td>Santa Cruz Biotechnology, US</td>
			<td>sc-215879A</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium palmitate</td>
			<td>Santa Cruz Biotechnology, US</td>
			<td>sc-215881</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Syring filter</td>
			<td>Corning, US</td>
			<td>431219</td>
			<td>&#160;Non-pyrogenic, sterile, 28 mm, 0.2 &#956;m.</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma-Aldrich, US</td>
			<td>T6146-25G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Trypsin 0.05% /EDTA 0.53 mM</td>
			<td>Corning, US</td>
			<td>25-052-Cl</td>
			<td>-</td>
		</tr>
		<tr>
			<td>24 well cell culture cluster</td>
			<td>Corning, US</td>
			<td>3524</td>
			<td>Flat bottom with lid. Tissue culture treated. Nonpyrogenic, polystyrene, sterile. 1/Pack.</td>
		</tr>
		<tr>
			<td>96 well cell culture cluster</td>
			<td>Corning, US</td>
			<td>3599</td>
			<td>Flat bottom with lid. Tissue culture treated. Nonpyrogenic, polystyrene, sterile. 1/Pack.</td>
		</tr>
	</tbody>
</table>

a model of experimental steatosis in vitro: hepatocyte cell culture in lipid overload-conditioned medium

代谢功能障碍相关的脂肪肝疾病（MAFLD），以前被称为非酒精性脂肪肝疾病（NAFLD），是全世界最普遍的肝脏疾病，因为它与肥胖症、糖尿病2型和脂质血症有关。肝硬化是肝炎、纤维化和末期肝病中观察到的炎症之前疾病的一个关键特征，它积累在肝炎中。肝细胞中的脂质积累可能会干扰异生物和内源分子的正常代谢，并诱导导致疾病进展的细胞过程。虽然在 体内可以进行硬化实验研究，但 体外 研究硬化的方法是具有不同优势的互补工具。脂质超载介质中的肝细胞培养是研究肝硬化的极好可重复选择，它能够识别与脂质积累相关的细胞过程，如氧化和网状压力、自噬、增殖、细胞死亡等，以及包括药物有效性、毒理学测试等许多其他可能应用的其他测试。在这里，它的目的是描述肝细胞培养在脂质超载调节介质的方法。HepG2细胞在RMPI 1640中型中培养，以棕榈酸钠和油酸钠为条件。重要的是，这两种脂质的比例对于有利于脂质液滴积累至关重要，同时保持细胞增殖和中等死亡率，就像疾病期间肝脏中发生的情况一样。该方法，从脂溶液库存的制备，混合物，除了介质，肝细胞培养表现出来。通过这种方法，可以识别肝细胞中易于观察到的油红色 O 染色剂中的脂质液滴，以及增殖/死亡率曲线。

培养在造菌介质中培养的肝细胞在井表面显示生长：然而，脂肪肝细胞的生长速度低于控制介质培养的细胞。OA和PA的拟议比例和浓度，保证细胞在培养过程中的存活。在24井板中每口井播种1×105 个细胞，如 图1所示，提供最佳汇合。
与对照条件相比，培养细胞在造血组（轻度和重度）中的生存能力较低。事实上，随着文化时间的增加，生存能力...

Watch this Scientific Journal Video about A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium at JoVE.com

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

该协议旨在成为研究硬化症和分子，生化，细胞变化产生的肝细胞过度暴露 在体外脂质的工具。

体外实验性硬化模型：脂质超载介质中的肝细胞培养

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver ...

本协议适用于肝硬化的研究，以及肝病，代谢，合成和毒化途径在硬化的背景下的研究。这种方法的主要优点是其可重复性和获得成果所需的时间短。再加上它提供了两个级别的硬化，轻度和严重。体外诱发的硬化可能为确定诊断疾病的潜在标记以及治疗目标提供证据。此方法旨在应用于硬化结果。然而，它可以调整到广泛的不同细胞受脂质过度暴露的影响，如在肥胖和脂质血症期间。准备标准 RPMI 1640。补充RPMI 1640培养基与10%的热激活FBS，和1%的青霉素链霉素溶液。使用 0.22 微米过滤器对它进行消毒，并将补充剂存储在 4 摄氏度。要准备棕榈酸盐库存溶液，在标准RPMI 1640中准备50毫摩尔的棕榈酸盐溶液，并辅以1%的无脂 BSA。使用 0.22 微米过滤器对库存溶液进行约 5 到 10 毫升的消毒，并将储存在 4 摄氏度的高温下，防止光线照射长达一个月。要准备油酸盐库存溶液，应在补充的 RPMI 1640 中准备 50 毫摩尔油酸盐溶液。使用 0.22 微米过滤器对库存溶液进行消毒。将其存放在零下20摄氏度，保护光线长达一个月。要从先前准备的库存中制备造血介质，应准备 100 毫米的 50 微摩尔混合物，其中一部分为棕榈酸盐，另一种为油酸盐。对于温和水平，在标准RPMI 1640中倒入500微摩尔混合物，其中一部分为棕榈酸盐，两部分油酸盐为严重水平。使用 0.22 微米过滤器进行消毒。在摄氏四度下储存长达一周。或者，使用免费脂质白蛋白，用各自的脂肪酸制备库存溶液，将棕榈酸或油酸盐溶解在两毫升绝对乙醇中，然后混合在标准RPMI 1640的最终体积中。通过存储在标准RPMI 1640培养介质中直接溶解油酸盐。允许乙醇蒸发，在70摄氏度的水浴中孵化，并彻底混合。使用 0.22 微米过滤器对两种库存溶液进行消毒，并将棕榈酸库存溶液存储在 4 摄氏度。并在零下20摄氏度的油酸盐库存溶液。在 24 井板中每口油井可容纳 010 万个 Hep G2 细胞。添加一毫升标准RPMI 1640。在37摄氏度和5%的二氧化碳下孵育24小时，允许细胞附着。培养前后，丢弃标准RPMI 1640介质。并添加造质介质。丢弃超高超，每24小时添加一次新鲜的造菌介质。为了执行生存能力和形态评估，通过添加 500 微升 0.05% 试穿 EDTA，将超纳米和分离细胞从井中分离。在37摄氏度和5%的二氧化碳下孵育5分钟。在微管中收集重新悬存的细胞。离心机的离心机为300倍G.然后丢弃超高音，加入200微升的标准RPMI 1640，并重新悬浮细胞。在新鲜的微管中加入 15 微升 0.4% 的试穿蓝色溶液。添加并混合前一个单元格悬浮器的 15 微升。在血细胞计中计算染色细胞和非染色细胞，并计算生存能力和死亡率。将细胞培养盖滑入24井板中的每口井中，并按文本手稿中描述种子 Hep G2 细胞。适当孵化后，用一毫升PBS清洗细胞，并丢弃超高分子。在PBS中将它们与一毫升4%的乙醛混合，在室温下孵育一小时。丢弃多余的副甲醛，用一毫升蒸馏水冲洗细胞。然后加入一毫升70%异丙酚，孵育5分钟。丢弃多余的异丙酚，加入一毫升油红O溶液，孵育30分钟。丢弃多余的油红 O 溶液，然后用一毫升蒸馏水冲洗。以400倍的放大倍数观察显微镜下的细胞。随机从井的整个区域选择并捕获 10 个光学场的照片。重复每口井的成像。要评估红色染色区域的百分比，请打开 ImageJ 软件并导入所需的文件，然后单击调整和使用颜色阈值"工具，定义红色检测的色调参数。然后调整图像的饱和度和亮度。使用分析粒子"工具"将染色区域与光学场的完整区域进行比较，并计算每口油井的平均百分比。在24口井板中每口油井可容纳10万肝细胞，可提供最佳的汇合度。随着文化时间的增加，生存能力逐渐减弱，在严重硬化的四天里达到60%的最低点。因此，在造菌条件下培养的肝细胞的死亡率较高。随着接触脂质的时间，它逐渐增加。由于增殖，细胞数量逐渐增加。然而，在三天和四天轻度硬化中，增殖率较低。相比之下，严重的硬化与24小时的低增殖有关。用油红 O 染色细胞表明，在造血条件下培养的细胞中，脂质液滴至少增加了两倍。细胞内脂肪根据在造菌介质中接触培养物的时间而增加。在轻度硬化中，脂质含量从第二天开始增加，而在严重硬化中，从24小时开始，脂质含量显著高。细胞培养需要以前的经验。在使用此协议之前，在标准条件下培养 Hep G2 细胞可能会有所帮助。脂肪酸库存的准备也是一个关键点。棕榈酸在室温下是固体，在管理之前需要加热。这种方法应允许对不同的途径进行分子分析，包括但不限于增殖、凋亡、自噬、氧化应激以及药理学和毒理学分析。

Research

JoVE Journal

Medicine

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

科研

医学

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

使用体外活体细胞成像技术研究脂纳米粒子化疗

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

A Cell Culture Model of Resistance Arteries

阻力动脉的细胞培养模型

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

小鼠尿的解剖和外植体培养对囊附着的作用分析

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...