SA-β-加拉基沙酶筛查检测,用于识别治疗药物
Summary
细胞衰老是慢性年龄相关疾病发展的关键因素。针对衰老细胞的治疗药物的鉴定为延长健康衰老提供了希望。在这里,我们提出了一个新的测定,以筛选基于测量与衰老相关的β-加拉托西沙酶在单细胞中的异质疗法的识别。
Abstract
细胞衰老是已知对健康寿命产生负面影响的衰老特征之一。能够专门杀死细胞培养中的衰老细胞的药物,称为溶酶,可以减轻体内的衰老细胞负担,延长健康范围。迄今为止,已经确定了多种溶血剂,包括HSP90抑制剂、Bcl-2系列抑制剂、管龙胺、FOXO4抑制肽和达萨替尼/克雷塞汀的组合。在增加的溶血球pH值下检测SA-β-Gal是检测衰老细胞的最佳特征标记之一。使用荧光基质C12FDG结合确定总细胞数,结合DNA间质化Hoechst染料对衰老相关β-乳糖酶(SA-β-Gal)活性的活细胞测量,为通过杀死衰老细胞(溶血)或通过抑制SA-β-Gal和其他衰老细胞表型(异形性)来降低整体SA-β-Gal活性的脱氧治疗药物的屏幕。使用高含量荧光图像采集和分析平台,可快速、高通量地筛选药物库,从而对SA-β-Gal、细胞形态和细胞数量产生影响。
Introduction
伦纳德·海夫利克和保罗·摩尔黑德首次描述了细胞衰老,他们表明正常细胞在培养中增殖的能力有限。尽管存在营养物质、生长因子和缺乏接触抑制,衰老细胞仍不能增殖,但仍然具有代谢活性2。这种现象被称为复制性衰老,主要归因于端粒缩短,至少在人类细胞3。进一步的研究表明,细胞也可以诱导进行衰老,以回应其他刺激,如致癌应激(致癌基因诱导衰老,OIS),DNA损伤,细胞毒性药物,或辐照(应力诱发衰老,SIS)4,5,6.为了应对DNA损伤,包括端粒侵蚀,细胞要么衰老,开始不受控制的细胞生长,或者在损伤无法修复时发生凋亡。在这种情况下,细胞衰老似乎是有益的,因为它以肿瘤抑制方式2。相反,由于细胞损伤的积累(包括DNA损伤),衰老会随着衰老而增加。由于衰老细胞可以分泌细胞因子、金属蛋白酶和生长因子,称为衰老相关分泌表型(SASP),这种年龄依赖性增加的细胞衰老和SASP有助于减少组织平衡和随后老化。此外,这种年龄依赖增加的衰老负担已知诱发代谢性疾病,压力敏感性,前列腺综合征,和受损的愈合7,8,并在一定程度上,负责与年龄相关的众多疾病,如动脉粥样硬化,骨关节炎,肌肉退化,溃疡形成,和阿尔茨海默氏病9,10,11,12,13。消除衰老细胞可以帮助预防或延缓组织功能障碍,延长健康范围14。这已经在转基因小鼠模型14,15,16以及通过使用溶血药物和药物组合,发现通过药物筛选努力和生物信息分析在衰老细胞17、18、19、20、21、22中专门诱导的通路。确定更优的脱压治疗药物,能够更有效地减轻衰老细胞负担,是开发健康衰老治疗方法的重要下一步。
在培养和体内,衰老细胞都表现出特征的型板和分子特征。这些衰老标记可能是衰老诱导的原因或结果,或者是这些细胞中分子变化的副产品。然而,在衰老细胞中没有特别的标记。目前,衰老相关β-乳糖酶(SA-β-Gal)检测是测量体外和体内衰老的最佳特征和既定单细胞方法之一。SA-β-Gal是一种溶酶体水解酶,在pH4时具有最佳酶活性。测量其活性在pH6是可能的,因为衰老细胞显示增加的细胞体活性23,24。对于活细胞,通过脂质体碱化获得增加的液化pH与真空H+-ATPase抑制剂巴菲洛霉素A1或内生菌酸化抑制剂氯奎因25,26。5-多地诺霉素二β-D-角质拉诺赛德(C12FDG)用作活细胞中的基质,因为它由于其12种亲碳性小鼠25而保留了细胞中的切块产物。重要的是,SA-β-Gal活性本身与在衰老细胞中识别的任何通路没有直接关系,也没有必要诱导衰老。通过这种测定,即使在异质细胞群和老化组织中,如老年人的皮肤活检,也可以识别衰老细胞。它也被用来显示细胞衰老和老化23之间的相关性,因为它是一个可靠的标志,在若干生物体和条件27,28,29衰老细胞检测, 30.在这里,描述了基于荧光基质C12FDG的高通量SA-β-Gal筛选测定,使用具有强氧应激诱导细胞衰老的原小鼠胚胎成纤维细胞(MEF)进行,其优缺点讨论。虽然这种测定可以在不同的细胞类型中进行,但使用Ercc1缺陷的DNA修复受损的MEF允许在氧化应激条件下更快速地诱导衰老。在小鼠中,脱氧核糖核酸修复内糖酶ERCC1-XPF的表达减少导致DNA修复受损,内源性DNA损伤加速积累,ROS升高,线粒体功能障碍,衰老细胞负担增加,干细胞功能丧失,过早衰老,类似于自然老化31,32。同样,Ercc1缺陷MEF在文化中发生衰老的速度更快17。衰老MEF测定的一个重要特征是,每口井都有衰老和非衰老细胞的混合物,可以清楚地显示衰老细胞特异性效应。然而,尽管我们认为在原发细胞中使用氧化应激诱导衰老更具有生理性,但是这种测定也可用于细胞系,其中衰老是用DNA破坏剂(如etoposide或辐照)诱导的。
Protocol
Representative Results
Discussion
SA-β-Gal 是一种定义良好的细胞衰老生物标志物,最初由 Dimri等人发现。(1995) 表明,与增殖细胞相比,在pH 623测定时,衰老的人类成纤维细胞增加了SA-β-Gal的活性。同时,为不同细胞类型和组织建立了SA-β-Gal的体外和体内测定25、39、40。该协议中描述的基于荧光的单细胞方法测量活细胞中的SA-β-Gal,是影响细胞<…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH赠款AG043376(项目2和核心A,PDR;PDR;以及PDR;项目2和核心A;项目2;项目A;项目2;项目A;项目2;项目A;项目2;项目A;项目2;项目A;项目2;项目A;项目2;项目项目1和核心B、LJN和AG056278(项目3和核心A、PDR;项目2,LJN)和格伦基金会(LJN)的赠款。
Materials
| DMEM | Corning | 10-013-CV | medium |
| Ham's F10 | Gibco | 12390-035 | medium |
| fetal bovine serum | Tissue Culture Biologics | 101 | serum |
| 1x non-essential amino acids | Corning | 25-025-Cl | amino-acids |
| bafilomycin A1 | Sigma | B1793 | lysosomal inhibitor |
| C12FDG | Setareh Biotech | 7188 | b-Gal substrate |
| Hoechst 33342 | Life Technologies | H1399 | DNA intercalation agent |
| 17DMAG | Selleck Chemical LLC | 50843 | HSP90 inhibitor |
| InCell6000 Cell Imaging System | GE Healthcare | High Content Imaging System |
Riferimenti
- Hayflick, L., Moorhead, P. S. The serial cultivation of human diploid cell strains. Experimental Cell Research. 25, 585-621 (1961).
- Campisi, J., di Fagagna, F. D. Cellular senescence: when bad things happen to good cells. Nature Reviews Molecular Cell Biology. 8 (9), 729-740 (2007).
- Harley, C. B., Futcher, A. B., Greider, C. W. Telomeres shorten during ageing of human fibroblasts. Nature. 345 (6274), 458-460 (1990).
- Zhu, J., Woods, D., McMahon, M., Bishop, J. M. Senescence of human fibroblasts induced by oncogenic Raf. Genes and Development. 12 (19), 2997-3007 (1998).
- Dimri, G. P., Itahana, K., Acosta, M., Campisi, J. Regulation of a senescence checkpoint response by the E2F1 transcription factor and p14(ARF) tumor suppressor. Molecular and Cellular Biology. 20 (1), 273-285 (2000).
- Michaloglou, C., et al. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature. 436 (7051), 720-724 (2005).
- Niedernhofer, L. J. Tissue-specific accelerated aging in nucleotide excision repair deficiency. Mechanisms of Ageing and Development. 129 (78), 408-415 (2008).
- Gitenay, D., et al. Glucose metabolism and hexosamine pathway regulate oncogene-induced senescence. Cell Death & Disease. 5, e1089 (2014).
- Erusalimsky, J. D., Kurz, D. J. Cellular senescence in vivo: its relevance in ageing and cardiovascular disease. Experimental Gerontology. 40 (8-9), 634-642 (2005).
- Kassem, M., Marie, P. J. Senescence-associated intrinsic mechanisms of osteoblast dysfunctions. Aging Cell. 10 (2), 191-197 (2011).
- Campisi, J., Andersen, J. K., Kapahi, P., Melov, S. Cellular senescence: A link between cancer and age-related degenerative disease. Seminars in Cancer Biology. 21 (6), 354-359 (2011).
- Golde, T. E., Miller, V. M. Proteinopathy-induced neuronal senescence: a hypothesis for brain failure in Alzheimer’s and other neurodegenerative diseases. Alzheimers Research & Therapy. 1 (2), 5 (2009).
- Telgenhoff, D., Shroot, B. Cellular senescence mechanisms in chronic wound healing. Cell Death & Differentiation. 12 (7), 695-698 (2005).
- Baker, D. J., et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature. 479 (7372), 232-236 (2011).
- Baker, D. J., et al. Naturally occurring p16-positive cells shorten healthy lifespan. Nature. , (2016).
- Childs, B. G., et al. Senescent intimal foam cells are deleterious at all stages of atherosclerosis. Science. 354 (6311), 472-477 (2016).
- Fuhrmann-Stroissnigg, H., et al. Identification of HSP90 inhibitors as a novel class of senolytics. Nature Communications. 8 (1), 422 (2017).
- Zhu, Y., et al. New agents that target senescent cells: the flavone, fisetin, and the BCL-XL inhibitors, A1331852 and A1155463. Aging. 9 (3), 955-963 (2017).
- Zhu, Y., et al. Identification of a Novel Senolytic Agent, Navitoclax, Targeting the Bcl-2 Family of Anti-Apoptotic Factors. Aging Cell. , (2015).
- Zhu, Y., et al. The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs. Aging Cell. , (2015).
- Baar, M. P., et al. Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging. Cell. 169 (1), 132-147 (2017).
- Jeon, O. H., et al. Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. Nature. , (2017).
- Dimri, G. P., et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proceedings of the National Academy of Sciences USA. 92 (20), 9363-9367 (1995).
- Itahana, K., Campisi, J., Dimri, G. P. Methods to detect biomarkers of cellular senescence: the senescence-associated beta-galactosidase assay. Methods in Molecular Biology. 371, 21-31 (2007).
- Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J., Toussaint, O. Protocols to detect senescence-associated beta-galactosidase (SA-[beta]gal) activity, a biomarker of senescent cells in culture and in vivo. Nature Protocols. 4 (12), 1798-1806 (2009).
- Cahu, J., Sola, B. A sensitive method to quantify senescent cancer cells. Journal of Visualized Experiments. 78 (78), (2013).
- Collado, M., et al. Tumour biology: Senescence in premalignant tumours. Nature. 436 (7051), 642 (2005).
- Krishnamurthy, J., et al. Ink4a/Arf expression is a biomarker of aging. The Journal of Clinical Investigation. 114 (9), 1299-1307 (2004).
- Mishima, K., et al. Senescence-associated beta-galactosidase histochemistry for the primate eye. Investigative Ophthalmology, Visual Science. 40 (7), 1590-1593 (1999).
- Melk, A., et al. Expression of p16INK4a and other cell cycle regulator and senescence associated genes in aging human kidney. Kidney International. 65 (2), 510-520 (2004).
- Niedernhofer, L. J., et al. A new progeroid syndrome reveals that genotoxic stress suppresses the somatotroph axis. Nature. 444 (7122), 1038-1043 (2006).
- Wang, J., Clauson, C. L., Robbins, P. D., Niedernhofer, L. J., Wang, Y. The oxidative DNA lesions 8,5′-cyclopurines accumulate with aging in a tissue-specific manner. Aging Cell. 11 (4), 714-716 (2012).
- Jozefczuk, J., Drews, K., Adjaye, J. Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells. Journal of Visualized Experiments. (64), (2012).
- Jagannathan, L., Cuddapah, S., Costa, M. Oxidative stress under ambient and physiological oxygen tension in tissue culture. Current Pharmacology Reports. 2 (2), 64-72 (2016).
- Meuter, A., et al. Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen. J Assist Reprod Genet. 31 (10), 1259-1267 (2014).
- Robinson, A. R., et al. Spontaneous DNA damage to the nuclear genome promotes senescence, redox imbalance and aging. Redox Biology. 17, 259-273 (2018).
- Zhang, J. H., Chung, T. D., Oldenburg, K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. Journal Of Biomolecular Screening. 4 (2), 67-73 (1999).
- Zhao, H., Darzynkiewicz, Z. Biomarkers of cell senescence assessed by imaging cytometry. Methods in Molecular Biology. 965, 83-92 (2013).
- Yang, N. -. C., Hu, M. -. L. A fluorimetric method using fluorescein di-β-d-galactopyranoside for quantifying the senescence-associated β-galactosidase activity in human foreskin fibroblast Hs68 cells. Analytical Biochemistry. 325 (2), 337-343 (2004).
- Zhao, J., et al. Quantitative Analysis of Cellular Senescence in Culture and In Vivo. Current Protocols in Cytometry. 79, (2017).
- Capparelli, C., et al. CDK inhibitors (p16/p19/p21) induce senescence and autophagy in cancer-associated fibroblasts, "fueling" tumor growth via paracrine interactions, without an increase in neo-angiogenesis. Cell Cycle. 11 (19), 3599-3610 (2012).
- Coppe, J. P., Desprez, P. Y., Krtolica, A., Campisi, J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annual Review of Pathology. 5, 99-118 (2010).
- Mah, L. J., El-Osta, A., Karagiannis, T. C. GammaH2AX as a molecular marker of aging and disease. Epigenetics. 5 (2), 129-136 (2010).
- Hewitt, G., et al. Telomeres are favoured targets of a persistent DNA damage response in ageing and stress-induced senescence. Nature Communications. 3, 708 (2012).
- Narita, M., et al. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell. 113 (6), 703-716 (2003).
- Georgakopoulou, E. A., et al. Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues. Aging-Us. 5 (1), 37-50 (2013).
- Severino, J., Allen, R. G., Balin, S., Balin, A., Cristofalo, V. J. Is β-Galactosidase Staining a Marker of Senescence in Vitro and in Vivo?. Experimental Cell Research. 257 (1), 162-171 (2000).
