原发性细胞衰老的诱导与鉴定
Summary
在这里, 我们讨论了一系列的诱导和确认细胞衰老在培养细胞的协议。我们专注于不同的衰老诱发刺激, 并描述了常见的衰老相关标记的量化。我们提供的技术细节, 以成纤维细胞作为模型, 但协议可以适应各种细胞模型。
Abstract
细胞衰老是一种永久性细胞周期停止激活的反应, 以应对不同的破坏性刺激。细胞衰老的活化是各种病理生理条件的标志, 包括肿瘤抑制、组织重塑和衰老。细胞衰老的诱导体在体内仍然缺乏特征。然而, 一些刺激可以用来促进细胞衰老前体。其中, 最常见的衰老诱导因子是复制衰竭, 电离和非电离辐射, 毒性药物, 氧化应激, 去甲基化和乙酰剂。在这里, 我们将提供有关如何使用这些刺激诱导成纤维细胞衰老的详细说明。该协议可以很容易地适应不同类型的主要细胞和细胞系, 包括癌细胞。我们还描述了不同的方法来验证衰老诱导。特别是, 我们专注于测量溶酶体与衰老相关的β-乳糖酶 (SA β-加仑) 的活性, 使用 5-乙炔-2 ‘-脱氧尿苷 () 的 DNA 合成率, 细胞周期的表达水平抑制剂 p16 和 p21, 以及衰老相关分泌表型 (SASP) 成员的表达和分泌。最后, 给出了实例结果, 并讨论了这些协议的进一步应用。
Introduction
在 1961年, 佛烈克和穆尔黑德报告说, 在连续1段后, 培养的主要成纤维细胞失去了增殖潜能。这一过程是由每细胞分裂后端粒的顺序缩短引起的。当端粒到达临界短的长度时, 它们被 DNA 损伤反应 (DDR) 所识别, 它激活了不可逆转的增殖–也被定义为复制衰老。复制衰老是目前已知的许多刺激之一, 导致一个永久性细胞周期逮捕的状态, 使细胞不敏感的 mitogens 和凋亡信号2,3。衰老程序通常的特点是额外的功能, 包括高溶酶活性, 线粒体功能障碍, 核变化, 染色质重组, 内质网应力, DNA 损伤和衰老相关分泌表型 (SASP)3,4。衰老细胞在体内具有多种功能: 发育、创面愈合和肿瘤抑制2。同样, 众所周知, 它们在衰老和肿瘤进展5中起着重要的作用。衰老的消极和部分矛盾的影响通常归因于 SASP6。
最近, 它表明, 消除衰老细胞的小鼠导致寿命延长和消除许多老化功能7,8,9,10,11,12. 同样, 已经开发了多种药物, 以消除衰老细胞 (senolytics) 或靶向 SASP13,14。抗衰老治疗潜力最近引起了人们的广泛关注。
对细胞衰老相关机制的研究和药理干预的筛选严重依赖于体外模型, 特别是在人原代成纤维细胞中。虽然不同的衰老诱导因子激活了一些共同的特征, 但衰老表型的大变异性被观察到, 并依赖于各种因素, 包括细胞类型, 刺激和时间点3,15, 16,17。研究和定位衰老细胞的异质性是当务之急。因此, 本议定书的目的是提供一系列方法, 以诱发衰老的主要成纤维细胞使用不同的治疗。正如它将解释, 这些方法可以很容易地适应其他细胞类型。
除了复制衰老, 我们还描述了其他五种衰老诱导治疗: 电离辐射, 紫外线 (紫外线) 辐射, 阿霉素, 氧化应激和后生变化 (即促进组蛋白乙酰化或 DNA 去甲基化).电离辐射和紫外线辐射直接导致 DNA 损伤, 并在适当剂量下引发衰老18,19。阿霉素还导致衰老主要是通过锂 dna 损伤的 dna 和干扰拓扑异构酶 II 功能, 从而停止 DNA 修复机制20。衰老的关键基因表达通常由组蛋白乙酰化和 DNA 甲基化控制。因此, 组蛋白乙酰抑制剂 (如丁酸钠和萨哈) 和 DNA 去甲基化 (例如, 5-aza) 剂会导致正常细胞21,22的衰老。
最后, 与衰老细胞相关的四种最常见的标记将被解释为: 衰老相关的乳糖酶 (SA β) 的活性、由免疫组织测定的 DNA 合成率、细胞周期调节器的过度表达和细胞周期蛋白依赖性激酶抑制剂 p16 和 p21, 并表达和分泌的 SASP 成员。
Protocol
1. 一般准备 准备 D10 培养基。补充 DMEM 中 Glutamax 与10% 血清和1% 青霉素/链霉素 (最后浓度: 100 U/毫升)。 准备无菌 PBS。根据制造商的指示, 在水中溶解药片。用高压釜消毒。 准备1x 胰蛋白酶。稀释5毫升的胰蛋白酶-Versene EDTA/10x 1:10 在45毫升的无菌 PBS。注意: 在整个协议中, 我们使用的细胞培养条件, 更接近的生理条件, 主要成纤维细胞。这意味着, 我们孵化的细胞在37摄?…
Representative Results
衰老成纤维细胞中 SA β-gal 染色的富集 β-乳糖酶 (β-加仑) 是一种酶, 表达在所有细胞和具有最佳 pH 值为 4.025,26。然而, 在衰老过程中, 溶酶体体积增大, 衰老细胞积累β-加仑。这种酶的增加使它有可能检测到它的活性, 即使在次优 pH 值 6.025,27?…
Discussion
这里解释的协议被优化了为人的主要成纤维细胞, 特别是 BJ 和 WI-38 细胞。复制衰老, 电离辐射和阿霉素的协议已成功地应用于其他类型的成纤维细胞 (HCA2 和 IMR90) 和其他细胞类型 (即新生儿黑色素细胞和角质形成素或 iPSC 衍生心肌细胞)在我们的实验室里。然而, 可以通过调整一些细节, 如种子细胞数量, 方法和化学物质来帮助细胞附着/分离到塑料支架上, 以及治疗的剂量以避免毒性, 来优化其他细?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢 Demaria 实验室的成员进行了卓有成效的讨论, 并 Thijmen van 弗利特共享关于紫外线诱发衰老的数据和协议。
Materials
| DMEM Media – GlutaMAX | Gibco | 31966-047 | |
| Fetal Bovine Serum | Hyclone | SV30160.03 | |
| Penicillin-Streptomycin (P/S; 10,000 U/ml) | Lonza | DE17-602E | |
| Dimethyl Sulfoxide (DMSO) | Sigma-Aldrich | SC-202581 | |
| Nuclease-Free Water (not DEPC-Treated) | Ambion | AM9937 | |
| T75 flask | Sarstedt | 833911002 | |
| Trypsin/EDTA Solution | Lonza | CC-5012 | |
| PBS tablets | Gibco | 18912-014 | |
| 1.5 ml microcentrifuge tubes | Sigma-Aldrich | T9661-1000EA | |
| Corning 15 mL centrifuge tubes | Sigma-Aldrich | CLS430791 | |
| 6-well plate | Sarstedt | 83.3920 | |
| 24-well plate | Sarstedt | 83.3922 | |
| 13mm round coverslips | Sarstedt | 83.1840.002 | |
| Steriflip | Merck Chemicals | SCGP00525 | |
| Cesium137-source | IBL 637 Cesium-137γ-ray machine | ||
| UV radiation chamber | Opsytec, Dr. Göbel BS-02 | ||
| Doxorubicin dihydrochloride | BioAustralis Fine Chemicals | BIA-D1202-1 | |
| Hydrogen peroxide solution | Sigma-Aldrich | 7722-84-1 | |
| 5-aza-2’-deoxycytidine | Sigma-Aldrich | A3656 | |
| SAHA | Sigma-Aldrich | SML0061 | |
| Sodium Butyrate | Sigma-Aldrich | B5887 | |
| X-gal (5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside) | Fisher Scientific | 7240-90-6 | |
| Citric acid monohydrate | Sigma-Aldrich | 5949-29-1 | |
| Sodium dibasic phosphate | Acros organics | 7782-85-6 | |
| Potassium ferrocyanide | Fisher Scientific | 14459-95-1 | |
| Potassium ferricyanide | Fisher Scientific | 13746-66-2 | |
| Sodium Chloride | Merck Millipore | 7647-14-5 | |
| Magnesium Chloride | Fisher Chemicals | 7791-18-6 | |
| 25% glutaraldehyde | Fisher Scientific | 111-30-8,7732-18-5 | |
| 16% formaldehyde (w/v) | Thermo-Fisher Scientific | 28908 | |
| EdU (5-ethynyl-2’-deoxyuridine) | Lumiprobe | 10540 | |
| Sulfo-Cyanine3 azide (Sulfo-Cy3-Azide) | Lumiprobe | D1330 | |
| Sodium ascorbate | Sigma-Aldrich | A4034 | |
| Copper(II) sulfate pentahydrate (Cu(II)SO4.5H2O) | Sigma-Aldrich | 209198 | |
| Triton X-100 | Acros organics | 215682500 | |
| TRIS base | Roche | 11814273001 | |
| LightCycler 480 Multiwell Plate 384, white | Roche | 4729749001 | |
| Lightcycler 480 sealing foil | Roche | 4729757001 | |
| Sensifast Probe Lo-ROX kit | Bioline | BIO-84020 | |
| UPL Probe Library | Sigma-Aldrich | Various | |
| Human IL-6 DuoSet ELISA | R&D | D6050 | |
| Bio-Rad TC20 | Bio-Rad | ||
| Counting slides | Bio-Rad | 145-0017 | |
| Dry incubator | Thermo-Fisher Scientific | Heratherm | |
| Dimethylformamide | Merck Millipore | 1.10983 | |
| Parafilm 'M' laboratory film | Bemis | #PM992 | |
| Tweezers | |||
| Needles |
Referências
- Hayflick, L., Moorhead, P. S. The serial cultivation of human diploid cell strains. Experimental Cell Research. 25, 585-621 (1961).
- Muñoz-Espín, D., Serrano, M. Cellular senescence: from physiology to pathology. Nature reviews. Molecular cell biology. 15, 482-496 (2014).
- Sharpless, N. E., Sherr, C. J. Forging a signature of in vivo senescence. Nature Reviews Cancer. 15 (7), 397-408 (2015).
- Correia-Melo, C., et al. Mitochondria are required for pro-ageing features of the senescent phenotype. The EMBO Journal. 10, e201592862 (2016).
- Loaiza, N., Demaria, M. Cellular senescence and tumor promotion: Is aging the key?. Biochimica et Biophysica Acta (BBA) – Reviews on Cancer. , (2016).
- Coppe, J. P., et al. Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biology. 6 (12), 2853-2868 (2008).
- Baker, D. J., et al. Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan. Nature. 530 (7589), 184-189 (2016).
- Xu, M., et al. Targeting senescent cells enhances adipogenesis and metabolic function in old age. Elife. 4, e12997 (2015).
- Baker, D. J., et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature. 479 (7372), 232-236 (2011).
- Jeon, O. H., et al. Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. Nature Medicine. 23 (6), 775-781 (2017).
- Demaria, M., et al. Cellular Senescence Promotes Adverse Effects of Chemotherapy and Cancer Relapse. Cancer Discovery. 7 (2), 165-176 (2017).
- Chang, J., et al. Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice. Nature Medicine. 22 (1), 78-83 (2016).
- Soto-Gamez, A., Demaria, M. Therapeutic interventions for aging: the case of cellular senescence. Drug Discov Today. 22 (5), 786-795 (2017).
- Childs, B. G., et al. Senescent cells: an emerging target for diseases of ageing. Nature Reviews Drug Discovery. 16 (10), 718-735 (2017).
- Marthandan, S., et al. Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence. Biological Research. 49, 34 (2016).
- Marthandan, S., et al. Conserved Senescence Associated Genes and Pathways in Primary Human Fibroblasts Detected by RNA-Seq. PLoS One. 11 (5), e0154531 (2016).
- Hernandez-Segura, A., et al. Unmasking Transcriptional Heterogeneity in Senescent Cells. Current Biology. 27 (17), 2652-2660 (2017).
- Le, O. N., et al. Ionizing radiation-induced long-term expression of senescence markers in mice is independent of p53 and immune status. Aging Cell. 9 (3), 398-409 (2010).
- Hall, J. R., et al. C/EBPalpha regulates CRL4(Cdt2)-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint. Cell Cycle. 13 (22), 3602-3610 (2014).
- Nitiss, J. L. Targeting DNA topoisomerase II in cancer chemotherapy. Nature Reviews Cancer. 9 (5), 338-350 (2009).
- Pazolli, E., et al. Chromatin remodeling underlies the senescence- associated secretory phenotype of tumor stromal fibroblasts that supports cancer progression. Pesquisa do Câncer. 72, 2251-2261 (2012).
- Venturelli, S., et al. Differential induction of apoptosis and senescence by the DNA methyltransferase inhibitors 5-azacytidine and 5-aza-2′-deoxycytidine in solid tumor cells. Molecular Cancer Therapeutics. 12, 2226-2236 (2013).
- Tennant, J. R. Evaluation of the Trypan Blue Technique for Determination of Cell Viability. Transplantation. 2, 685-694 (1964).
- Livak, K. J., Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25 (4), 402-408 (2001).
- Lee, B. Y., et al. Senescence-associated β-galactosidase is lysosomal β-galactosidase. Aging Cell. 5, 187-195 (2006).
- Kopp, H. G., Hooper, A. T., Shmelkov, S. V., Rafii, S. Beta-galactosidase staining on bone marrow. The osteoclast pitfall. Histology and Histopathology. 22 (9), 971-976 (2007).
- 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. 92 (20), 9363-9367 (1995).
- Salic, A., Mitchison, T. J. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proceedings of the National Academy of Sciences. 105 (7), 2415-2420 (2008).
- Sherr, C. J., McCormick, F. The RB and p53 pathways in cancer. Cancer Cell. 2 (2), 103-112 (2002).
- Bunz, F., et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science. 282 (5393), 1497-1501 (1998).
- Severino, J., Allen, R. G., Balin, S., Balin, A., Cristofalo, V. J. Is beta-galactosidase staining a marker of senescence in vitro and in vivo. Experimental Cell Research. 257 (1), 162-171 (2000).
- Stolzing, A., Coleman, N., Scutt, A. Glucose-induced replicative senescence in mesenchymal stem cells. Rejuvenation Research. 9 (1), 31-35 (2006).
- Blazer, S., et al. High glucose-induced replicative senescence: point of no return and effect of telomerase. Biochemical and Biophysical Research Communications. 296 (1), 93-101 (2002).
- Wiley, C. D., Campisi, J. From Ancient Pathways to Aging Cells-Connecting Metabolism and Cellular Senescence. Cell Metabolism. 23 (6), 1013-1021 (2016).
- Kumar, R., Gont, A., Perkins, T. J., Hanson, J. E. L., Lorimer, I. A. J. Induction of senescence in primary glioblastoma cells by serum and TGFbeta. Scientific Reports. 7 (1), 2156 (2017).
- Hypoxia Blagosklonny, M. V. MTOR and autophagy: converging on senescence or quiescence. Autophagy. 9 (2), 260-262 (2013).
- Meuter, A., et al. Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen. Journal of Assisted Reproduction and Genetics. 31 (10), 1259-1267 (2014).
- Coppe, J. P., et al. A human-like senescence-associated secretory phenotype is conserved in mouse cells dependent on physiological oxygen. PLoS One. 5 (2), e9188 (2010).
- van Deursen, J. M. The role of senescent cells in ageing. Nature. 509 (7501), 439-446 (2014).
- Kim, Y. M., et al. Implications of time-series gene expression profiles of replicative senescence. Aging Cell. 12, 622-634 (2013).
Citar este artigo
Hernandez-Segura, A., Brandenburg, S., Demaria, M. Induction and Validation of Cellular Senescence in Primary Human Cells. J. Vis. Exp. (136), e57782, doi:10.3791/57782 (2018).