在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量
Summary
Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.
Abstract
生物钟是所有光敏感的生物功能，允许通过预测日常环境变化的适应外部世界。在我们的生物钟与生理的许多方面之间的紧密联系的理解相当大的进展已在该领域已经取得了在过去十年。不过，这解开在underlies人体昼夜节律振荡器功能的分子基础保持最高的技术挑战。这里，我们提供了长期（2-5天）的生物发光的记录和流出介质收集培养的人原代细胞的实验方法的详细描述。为了这个目的，我们已转导原代细胞用慢病毒萤光素酶报道即下一个核心时钟基因启动子，其允许激素分泌和昼夜生物发光的并行评估的控制。此外，我们描述的条件扰乱公关生物钟imary人体细胞转染的siRNA靶向时钟 。我们对人类胰岛分泌胰岛素的节律性的结果，人类骨骼肌细胞分泌myokine，在这里提出来说明这一方法的应用。这些设置可用于研究人末梢时钟的分子组成和分析的生理或病理生理条件下的原代细胞及其功能的影响。
Introduction
昼夜定时系统（来自拉丁美洲“近似行乐”）已经出现在所有的光敏感的生物，如自适应机制，地球的旋转。在哺乳动物中，它在一个分层的方式组织，包括中央时钟，这是坐落在腹侧丘脑的视交叉上核和外设（或从站）是在不同的器官手术振荡器。此外，这些细胞中自主自我维持的振荡器是在主体1的几乎每一个细胞中有功能。光的信号代表的SCN神经元支配同步提示（ 授时 ），而来自SCN发出神经，体液的信号复位外设时钟。除了休息，活动节律，这反过来喂养空腹开车的周期，对于外设时钟2进一步同步。据我们目前的了解，核心时钟的分子组成是基于转录和translaTIONAL反馈回路，这是生物体之间是保守的。这包括转录激活因子BMAL1和CLOCK，它们共同激活负内核时钟PER和CRY基因的转录。 PER和CRY蛋白质含量高将通过抑制BMAL1 / CLOCK复杂的抑制自己的转录。辅助回路包括核受体REV-ERBS和RORS，这也规范BMAL1和CLOCK的转录。此外，翻译后的事件，包括磷酸化，SUMO化，乙酰化，O-GlcNAc糖基，降解和核心时钟蛋白质的入核建立了24小时的振荡周期3代表一个附加的重要的调节层。
越来越多的证据来自于啮齿动物模型研究茎和突出了昼夜系统中的代谢和内分泌功能4-5的协调的关键作用。许多的LARG电子秤转录组分析表明，馈送-空腹循环发挥周振荡器6-8的同步的中心作用。在这些研究中的协议，在啮齿动物和人类代谢组学和脂质组分析已经揭示，大量的代谢产物在组织，血浆和唾液振荡在一个昼夜方式9-11。重要的是，大多数表现出的激素在血液中5,12-13昼夜节律。此外，生产外周组织相应的激素生物钟可能局部调节激素的分泌。细胞自主昼夜振荡器在啮齿动物和人类的胰岛细胞14-16被描述。这些振荡器在调节胰岛转录发挥重要的作用和功能15,17-18。此外，由人骨骼肌管myokine分泌最近已证实表现出昼夜节律模式，它是由细胞自主oscillato调节RS在这些细胞中19可操作。
为在体内的人类研究的生理节律的几种方法已被广泛使用。例如，血浆褪黑激素或皮质醇水平以及胸部皮肤表面温度（在参考文献3,20中综述）进行了研究，以评估内源性生物钟。虽然这些方法可以研究在体内系统性的昼夜波动，他们远离提供在不同器官和组织自由运行的自主昼夜节律的可靠评估。然而，来自全身调节这种夹层会理解对这些细胞的功能的细胞内分子的时钟的具体效果的不可或缺的工具。因此，大量的努力已经进行制定体外同步永生或原代培养细胞研究人类钟表可靠的方法。重要的是，它已被证明在原代培养的皮肤成纤维细胞测量时钟特性密切地反映整个机体21的单个时钟性能。荧光和生物发光昼夜记者的发展极大地推进这种做法22-27。此外，个从不同的外围器官衍生的研究主小区时钟允许人类组织特异性时钟3,5,16,19-20,28的分子性质的研究。因此，在体外同步初级外植体或细胞生物钟的评估，通过使用生物发光的记者，表示一个非常有用的方法来研究人末梢时钟的分子组成和它们对器官功能的影响。
在本文中，我们将介绍详细的协议， 用于体外同步以及自主胞脉的影响，对人体主要的胰岛细胞和骨骼肌细胞评估节律基因表达中断对这些细胞的分泌功能。
Protocol
Representative Results
Discussion
此处所述的实验设置是由慢病毒递送的昼夜生物发光记者到培养的人原代细胞中，接着后续体外同步和几天生物发光的连续记录，并通过在同一细胞激素分泌的平行分析。他们代表了探索的分子机制和人原代细胞的生物钟功能方面的有效方法。
供体材料的质量是可行的主组织培养的制剂的一个重要问题。人胰岛的质量应在开始实验前每次进行评估。不推荐这些实验与?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们非常感谢我们来自日内瓦大学的同事：雅克·菲利普对这项工作的建设性意见，Ueli SCHIBLER用于与灌流系统的发展和科学的激励宝贵的帮助，安德烈利亚尼对于已经构思设计，制造和调试灌注系统，莱萨科技有限公司公司在灌流系统和滴灌biolumicorder软件开发的协助下，乔治·塞韦用于与灌流实验援助，乌苏拉Loizides – 曼戈尔德批判地阅读手稿，和安妮 – 玛丽·马赫卢夫的慢病毒制剂;到艾蒂安Lefai，萧蔷Chanon和休伯特·维达尔（INSERM，里昂）制备人主要成肌细胞;和多梅尼科黄宗泽和蒂埃里·伯尼（人胰岛移植中心，日内瓦大学医院）提供人胰岛。这项工作是由瑞士国家科学基金会批准号资助31003A_146475 / 1，硅nergia瑞士国家科学基金会批准号CRSII3-154405，基金会罗曼德倒拉河畔RECHERCHE糖尿病反，博Hjelt基金会，基金会Ernst等露西斯密德亨尼，和兴业Académique日内瓦（CD）。
Materials
| Trypsin-EDTA | Invitrogen | 25300-054 | For muscle biopsy digestion |
| DPBS no calcium no magnesium | Invitrogen | 14190-094 | |
| HAM F-10 | Invitrogen | 41550-021 | For myoblasts culture |
| FBS | Invitrogen | 10270 | Supplement to culture medium |
| Penicillin-Streptomycin | Sigma | P0781-100 | Supplement to culture medium |
| Gentamycin | Axon | A1492.0001 | Supplement to culture medium |
| Fungizone | Invitrogen | 15290-026 | Amphotericin B, supplement to culture medium |
| DMEM 1g/L glucose + Na pyruvate + glutamax | Invitrogen | 21885-025 | For myotubes culture |
| DMEM 1g/L glucose -Na Pyruvate – glutamax | Invitrogen | 11880-028 | Recording medium for LumiCycle |
| Glutamax | Invitrogen | 35050-028 | L-alanyl-L-glutamine dipeptide, supplement to recording medium |
| Accutase | Innovative Cell Technologies | AT-104 | Cell detachment solution, for islet cell dissociation |
| CMRL | Gibco | 21530-027 | Culture medium for islet cells |
| Sodium Pyruvate | Gibco | 11360-039 | Supplement to culture medium |
| 15 ml High-Clarity Polipropylene Conical Tube | Falcon | 352096 | |
| F75 flask | BD Falcon | 353136 | |
| 3.5 cm Petri dish | BD Falcon | 353001 | |
| Foskolin | Sigma | F6886 | Adenylyl cyclase activator, used for synchronization |
| Luciferin | Prolume LTD | 260150 | Supplement to recording medium |
| OptiMEM | Invitrogen | 51985-026 | Serum-free Minimal Essential Medium (MEM) used for human islet cells transfection |
| Lipofectamine RNAiMAX reagent | Invitrogen | 13778-150 | Transfection reagent |
| HiPerFect reagent | Qiagen | 301705 | Transfection reagent |
| ON-TARGET plus siCLOCK smartpool | Dharmacon | L-008212-00 | |
| ON-TARGET plus non targeting siRNA #1 (siControl) | Dharmacon | D-001810-01 | |
| DNeasy Blood & Tissue Kit | Qiagen | 69504 | For myotubes DNA extraction |
| RNeasy Plus Mini kit | Qiagen | 74104 | For myotubes RNA extraction |
| QIAshredder | Qiagen | 79654 | For myotubes RNA extraction |
| 2 ml collecting tubes | Axygen | 311-10-051 | To collect the medium with the perifusion |
| Tissue culture Plate, 6 Well | BD Falcon | 353046 | To collect the medium with the perifusion |
| RNeasy Plus Micro kit | Qiagen | 74034 | For islet RNA extraction |
| Human IL-6 Instant ELISA kit | eBioscience | 88-7066-22 | |
| Human Insulin Kit Mercodia | Mercodia | 10-1113-01 | |
| Hydrochloric acid, min,37%,p.a. | Acros organics | 124630010 | Used for preparation of lysis buffer (375ml Ethanol+7.5%HCl+117.5%H2O) |
| Ethanol (>99.8%) | Fluka Analytical | 02860-1L | Used for preparation of lysis buffer (375ml Ethanol+7.5%HCl+117.5%H2O) |
| Human Islets for Research | Prodo Laboratories | ||
| Name | Company | Catalog Number | Comments |
| Equipment: | |||
| Centrifuge | Heraeus | Megafuge 1.0R | |
| Water bath | VWR | 1112A | at 37 °C |
| Tissu culture hood | Faster | SafeFastElite | |
| Tissu culture incubator | Heraeus | HeraCell 150 | 5% CO2 at 37 °C, no water due to the LumiCycle installation |
| Tissu culture incubator | Heraeus | HeraCell 150 | 5% CO2 at 37 °C, no water due to the LumiCycle installation |
| Tissu culture incubator | Thermo Scientific | Hera Cell 150i | 5% CO2 at 37 °C |
| Shaker | Heidolph Instruments | Unimax 1010 | For agitation of the siRNA mix |
| LumiCycle | Actimetrics | ||
| LumiCycle software | Actimetrics | ||
| CosinorJ software | EPFL | Freely available at: http://bigwww.epfl.ch/algorithms/cosinorj/ | |
| Rheodyne titan MX | ERC GmbH | Control software that controls the timing of the automated switch | |
References
- Albrecht, U. Timing to perfection: the biology of central and peripheral circadian clocks. Neuron. 74 (2), 246-260 (2012).
- Dibner, C., Schibler, U., Albrecht, U. The mammalian circadian timing system: organization and coordination of central and peripheral clocks. Annu Rev Physiol. 72, 517-549 (2010).
- Dibner, C., Schibler, U. Circadian timing of metabolism in animal models and humans. J Intern Med. , (2015).
- Marcheva, B., et al. Circadian clocks and metabolism. Handb Exp Pharmacol. (217), 127-155 (2013).
- Philippe, J., Dibner, C. Thyroid circadian timing: roles in physiology and thyroid malignancies. J Biol Rhythms. 30 (2), 76-83 (2015).
- Andrews, J. L., et al. CLOCK and BMAL1 regulate MyoD and are necessary for maintenance of skeletal muscle phenotype and function. Proc Natl Acad Sci U S A. 107 (44), 19090-19095 (2010).
- McCarthy, J. J., et al. Identification of the circadian transcriptome in adult mouse skeletal muscle. Physiol Genomics. 31 (1), 86-95 (2007).
- Shostak, A., Husse, J., Oster, H. Circadian regulation of adipose function. Adipocyte. 2 (4), 201-206 (2013).
- Dallmann, R., Viola, A. U., Tarokh, L., Cajochen, C., Brown, S. A. The human circadian metabolome. Proc Natl Acad Sci U S A. 109 (7), 2625-2629 (2012).
- Adamovich, Y., et al. Circadian clocks and feeding time regulate the oscillations and levels of hepatic triglycerides. Cell Metab. 19 (2), 319-330 (2014).
- Chua, E. C., et al. Extensive diversity in circadian regulation of plasma lipids and evidence for different circadian metabolic phenotypes in humans. Proc Natl Acad Sci U S A. 110 (35), 14468-14473 (2013).
- Kalsbeek, A., Fliers, E. Daily regulation of hormone profiles. Handb Exp Pharmacol. (217), 185-226 (2013).
- Hastings, M., O’Neill, J. S., Maywood, E. S. Circadian clocks: regulators of endocrine and metabolic rhythms. J Endocrinol. 195 (2), 187-198 (2007).
- Muhlbauer, E., Wolgast, S., Finckh, U., Peschke, D., Peschke, E. Indication of circadian oscillations in the rat pancreas. FEBS Lett. 564 (1-2), 91-96 (2004).
- Marcheva, B., et al. Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes. Nature. 466 (7306), 627-631 (2010).
- Pulimeno, P., et al. Autonomous and self-sustained circadian oscillators displayed in human islet cells. Diabetologia. 56 (3), 497-507 (2013).
- Perelis, M., et al. Pancreatic beta cell enhancers regulate rhythmic transcription of genes controlling insulin secretion. Science. 350 (6261), (2015).
- Saini, C., et al. A functional circadian clock is required for proper insulin secretion by human pancreatic islet cells. Diabetes Obes Metab. , (2015).
- Perrin, L., et al. Human skeletal myotubes display a cell-autonomous circadian clock implicated in basal myokine secretion. Mol Metab. 4 (11), 834-845 (2015).
- Saini, C., Brown, S. A., Dibner, C. Human peripheral clocks: applications for studying circadian phenotypes in physiology and pathophysiology. Front Neurol. 6, 95 (2015).
- Brown, S. A., et al. Molecular insights into human daily behavior. Proc Natl Acad Sci U S A. 105 (5), 1602-1607 (2008).
- Asher, G., et al. SIRT1 regulates circadian clock gene expression through PER2 deacetylation. Cell. 134 (2), 317-328 (2008).
- Dibner, C. On the robustness of mammalian circadian oscillators. Cell Cycle. 8 (5), 681-682 (2009).
- Dibner, C., et al. Circadian gene expression is resilient to large fluctuations in overall transcription rates. EMBO J. 28 (2), 123-134 (2009).
- Nagoshi, E., et al. Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells. Cell. 119 (5), 693-705 (2004).
- Sage, D., Unser, M., Salmon, P., Dibner, C. A software solution for recording circadian oscillator features in time-lapse live cell microscopy. Cell Div. 5, (2010).
- Kowalska, E., Moriggi, E., Bauer, C., Dibner, C., Brown, S. A. The circadian clock starts ticking at a developmentally early stage. J Biol Rhythms. 25 (6), 442-449 (2010).
- Mannic, T., et al. Circadian clock characteristics are altered in human thyroid malignant nodules. J Clin Endocrinol Metab. 98 (11), 4446-4456 (2013).
- Parnaud, G., et al. Proliferation of sorted human and rat beta cells. Diabetologia. 51 (1), 91-100 (2008).
- Agley, C. C., Rowlerson, A. M., Velloso, C. P., Lazarus, N. L., Harridge, S. D. Isolation and quantitative immunocytochemical characterization of primary myogenic cells and fibroblasts from human skeletal muscle. J Vis Exp. (95), e52049 (2015).
- Liu, A. C., et al. Redundant function of REV-ERBalpha and beta and non-essential role for Bmal1 cycling in transcriptional regulation of intracellular circadian rhythms. PLoS Genet. 4 (2), e1000023 (2008).
- Hughes, M. E., Hogenesch, J. B., Kornacker, K. JTK_CYCLE: an efficient nonparametric algorithm for detecting rhythmic components in genome-scale data sets. J Biol Rhythms. 25 (5), 372-380 (2010).
- Dyar, K. A., et al. Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock. Mol Metab. 3 (1), 29-41 (2014).
- Innominato, P. F., et al. The circadian timing system in clinical oncology. Ann Med. 46 (4), 191-207 (2014).
- Chitikova, Z., et al. Identification of new biomarkers for human papillary thyroid carcinoma employing NanoString analysis. Oncotarget. 6 (13), 10978-10993 (2015).
- Pagani, L., et al. The physiological period length of the human circadian clock in vivo is directly proportional to period in human fibroblasts. PLoS One. 5 (10), e13376 (2010).
