基因型衰老过程中正常人成纤维细胞活性氧和衰老相关分泌表型的定量测定
Summary
胞内活性氧在细胞衰老诱导中起着重要作用。在这里, 我们描述了一个敏感的测定细胞衰老过程中的 ROS 水平。我们还提供了评估衰老相关分泌表型的协议, 据报告, 这有助于各种年龄相关的功能障碍。
Abstract
细胞衰老被认为是一种不可逆的生长状态, 在衰竭的增生能力或接触到各种压力。最近的研究扩大了细胞衰老在各种生理过程中的作用, 包括发育, 伤口愈合, 免疫监测和年龄相关的组织功能障碍。虽然细胞周期骤停是细胞衰老的一个重要标志, 但增加的细胞内活性氧 (ROS) 的生产也被证明对细胞衰老的诱导起着重要作用。此外, 最近的研究表明衰老细胞通过衰老相关的分泌表型 (SASP) 在邻近细胞和组织中表现出强有力的分泌活动。对细胞衰老的治疗策略的兴趣急剧增加强调需要对衰老机制, 包括胞内活性氧和 SASP 进行精确的了解。在这里, 我们描述了用 ros 敏感荧光染料和流式细胞仪定量评估 h-Ras 诱导的细胞衰老过程中细胞内 ROS 含量的协议。此外, 我们还介绍了敏感技术, 以分析诱导的 mRNA 表达和分泌的 SASP 因素。这些协议可应用于各种细胞衰老模型。
Introduction
50年前, 佛烈克和穆尔黑德发现, 正常细胞在细胞分裂1后, 在其增殖潜能耗尽后进入不可逆转的生长。这种现象现在被称为复制衰老, 并被认为与生物体衰老有强烈的关联2。虽然端粒的渐进侵蚀被认为是复制衰老的主要原因, 但据报道, 各种细胞应力, 如 DNA 损伤、致癌活化和氧化应激等, 都被报告诱发另一种细胞衰老。称为 “过早衰老” 或 “应力诱发衰老”。有趣的是, 过早衰老对肿瘤的激活起到了强有力的抑制作用, 如 H-Ras 和 BRAF。对小鼠模型和人体组织的研究已经产生了强有力的证据, 表明细胞衰老的生物标志物主要存在于前病变中, 在那里致癌 Ras 和 BRAF 被激活, 但在恶性癌症中被减少, 从这些病变3,4,5。除了它在衰老和肿瘤抑制方面的作用, 细胞衰老已经在以前的研究中显示, 在各种生理过程中发挥作用, 包括伤口愈合, 组织修复, 免疫监测, 胚胎发育6。
尽管生长骤停已被广泛研究为7细胞衰老的标志, 但大量的证据表明胞内活性氧 (ROS) 也有助于细胞衰老8。在不同类型的细胞衰老过程中, ROS 水平的升高, 包括复制衰老和癌基因引起的衰老, 最初是在数十年前的9,10。一个更直接, 外源治疗与致死剂量的 H2O2诱导衰老11,12。抑制 ROS 清除酶, 如 SOD1, 也导致过早衰老13。相比之下, 低环境氧条件和增加 ROS 清除延缓衰老的开始10,14,15。这些结果无疑表明, ROS 是细胞衰老诱导的重要调解人或决定因素。然而, ros 是如何促进细胞衰老的诱导以及在细胞衰老过程中 ros 水平的升高需要进一步的研究。
最近的研究表明, 衰老细胞通过 SASP16、17, 在邻近细胞和组织中具有强大的分泌活性。在衰老的组织中, 衰老细胞通过 SASP 的多种途径促进与年龄相关的组织功能障碍, 除了对增生细胞的自主耗尽之外。衰老细胞分泌的各种促炎因子, 如 IL-6、IL-8、TGFβ和基质金属蛋白酶 (金属蛋白酶), 通过组织稳态的损伤、组织结构的破坏, 引起与年龄相关的组织功能障碍,邻近细胞衰老, 无菌炎症18,19。然而, SASPs 可以产生有益的影响, 取决于生物环境。此外, SASPs 的 heterogenetic 性质取决于衰老细胞类型和细胞阶段, 强调需要进一步研究19。
在这里, 我们描述了快速和灵敏的细胞技术, 以评估细胞内活性氧的水平。此外, 还介绍了利用定量实时聚合酶链反应 (qPCR) 和 ELISA 法分析 SASP 因子的方法。
Protocol
1. 诱发癌基因引起的衰老 制备 RasV12 逆转录病毒 在室温下添加2毫升0.001% 聚 l-赖氨酸/磷酸盐缓冲盐水 (PBS), 将 100 mm 培养皿涂成5分钟。 使用连接到真空的玻璃吸管去除聚 l-赖氨酸溶液, 并通过添加2毫升 1x PBS 来洗涤培养皿。 板 3 x 106 ecotropic BOSC-23 包装细胞在涂层培养皿与 Dulbecco 的改性鹰培养基 (DMEM) 含有10% 胎牛血清 (血清) 和1% 青霉素/链霉素,…
Representative Results
图 1显示了 H-Ras 引起的衰老的例子。WI-38 正常人成纤维细胞感染 H-RasV12 逆转录病毒诱发戏剧性形态学改变 (图 1B)。此外, 如图 1C所示, 在 H-RasV12 表达时, SA β-gal 染色活性显著增加。在 H-RasV12 逆转录病毒感染后, 超过70% 的细胞显示 SA β-gal 染色活性 6 d, 表明 H-RasV12 的表达成功地诱导 WI-38 细胞的细胞衰老, 正…
Discussion
在这里, 我们提出了监测 WI-38 正常人成纤维细胞在 H-Ras 诱导衰老过程中胞内活性氧水平的方法。用细胞渗透性试剂 DCF-DA 和流式细胞仪定量测定活细胞内的活性氧水平。细胞吸收后, 脱乙酰度细胞内酯, 然后由 ROS 氧化, 形成高度荧光 2 ‘, 7 ‘-dichlorofluorescein (dcf)。用 FL1 检测器 (绿色荧光) 检测流式细胞仪可以探测到 DCF 荧光。利用 DCF-DA 染色法, 成功检测了 WI-38 正常人成纤维细胞在 H-Ras 诱导的细胞衰?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了韩国国家研究基金会 (2015R1D1A1A01060839) 和韩国政府 (NRF) 资助的韩国国家研究基金会 (MSIT) 的资助 (不 2016R1A2B2008887, 不。2016R1A5A2007009) (Jeanho 云)。
Materials
| REAGENTS | |||
| poly-L-lysine | Sigma-Aldrich | P2636 | |
| BOSC 23 | ATCC | CRL-11269 | |
| FBS | GIBCO | 16000-044 | |
| penicillin/streptomycin | wellgene | LS202-02 | |
| PBS | Hyclone | SH30013.02 | |
| DMEM | GIBCO | 12800-082 | |
| OPTI-MEM | GIBCO | 31985-070 | |
| pBabe puro-H-RasV12 | Addgene | 1768 | |
| pGAG/pol | Addgene | 14887 | |
| pVSVG | Addgene | 1733 | |
| Turbofect | Thermo Fisher Scientific | R0531 | |
| polybrene | Sigma-Aldrich | H9268 | 8 mg/ml |
| puromycin | Sigma-Aldrich | P8833 | 2 mg/ml |
| formaldehyde | Sigma-Aldrich | F8775 | |
| 5-bromo-4-chloro-3-indolyl β D-galactopyranoside (X-gal) | Sigma-Aldrich | B4252 | |
| potassium ferrocyanide | Sigma-Aldrich | B4252 | |
| potassium ferricyanide | Sigma-Aldrich | P9387 | |
| trypsin-EDTA | wellgene | LS015-01 | |
| DCF-DA | Sigma-Aldrich | D6883 | 10 mM |
| Trizol | Thermo Fisher Scientific | 15596026 | |
| MMLV Reverse transcriptase | Promega | M1701 | |
| SYBR Green PCR master 2X mix | Takara | PR820A | |
| Random Primer | Promega | C118A | |
| Tween-20 | Sigma-Aldrich | P9416 | |
| Ultra-pure distilled water | Invitrogen | 10977015 | |
| Human IL-6 ELISA assay | PeproTech | #900-TM16 | |
| Human IL-8 ELISA assay | PeproTech | #900_TM18 | |
| EQUIPMENTS | |||
| 0.45 μm syringe filter | sartorius | 16555 | |
| Parafilm | BEMIS | PM-996 | |
| Microscope | NIKON | TS100 | |
| Flow cytometer | BD Bioscience | LSR Fortessa | |
| Amicon Ultra-4ml | Merk Millipore | UFC800324 | |
| NanoDrop spectrophotometer | BioDrop | 80-3006-61 | |
| Real-time PCR System | Applied Biosystems | ABI Prism 7500 | |
| ELISA Reader | Molecular Devices | EMax microplate reader |
Riferimenti
- Hayflick, L., Moorhead, P. S. The serial cultivation of human diploid cell strains. Experimental Cell Research. 25, 585-621 (1961).
- Campisi, J. Aging, cellular senescence, and cancer. Annual Review of Physiology. 75, 685-705 (2013).
- Braig, M., et al. Oncogene-induced senescence as an initial barrier in lymphoma development. Nature. 436 (7051), 660-665 (2005).
- Michaloglou, C., et al. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature. 436 (7051), 720-724 (2005).
- Collado, M., et al. Tumour biology: senescence in premalignant tumours. Nature. 436 (7051), 642 (2005).
- Malaquin, N., Martinez, A., Rodier, F. Keeping the senescence secretome under control: Molecular reins on the senescence-associated secretory phenotype. Experimental Gerontology. 82, 39-49 (2016).
- Kuilman, T., Michaloglou, C., Mooi, W. J., Peeper, D. S. The essence of senescence. Genes & Development. 24 (22), 2463-2479 (2010).
- Lu, T., Finkel, T. Free radicals and senescence. Experimental Cell Research. 314 (9), 1918-1922 (2008).
- Furumoto, K., Inoue, E., Nagao, N., Hiyama, E., Miwa, N. Age-dependent telomere shortening is slowed down by enrichment of intracellular vitamin C via suppression of oxidative stress. Life Sciences. 63 (11), 935-948 (1998).
- Lee, A. C., et al. Ras proteins induce senescence by altering the intracellular levels of reactive oxygen species. The Journal of Biological Chemistry. 274 (12), 7936-7940 (1999).
- Chen, Q., Ames, B. N. Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proceedings of the National Academy of Sciences of the United States of America. 91 (10), 4130-4134 (1994).
- Dumont, P., et al. Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast. Free Radical Biology & Medicine. 28 (3), 361-373 (2000).
- Blander, G., de Oliveira, R. M., Conboy, C. M., Haigis, M., Guarente, L. Superoxide dismutase 1 knock-down induces senescence in human fibroblasts. The Journal of Biological Chemistry. 278 (40), 38966-38969 (2003).
- Packer, L., Fuehr, K. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature. 267 (5610), 423-425 (1977).
- Serra, V., von Zglinicki, T., Lorenz, M., Saretzki, G. Extracellular superoxide dismutase is a major antioxidant in human fibroblasts and slows telomere shortening. The Journal of Biological Chemistry. 278 (9), 6824-6830 (2003).
- Rodier, F., Campisi, J. Four faces of cellular senescence. The Journal of Cell Biology. 192 (4), 547-556 (2011).
- Munoz-Espin, D., Serrano, M. Cellular senescence: from physiology to pathology. Nature Reviews. Molecular Cell Biology. 15 (7), 482-496 (2014).
- Tchkonia, T., Zhu, Y., van Deursen, J., Campisi, J., Kirkland, J. L. Cellular senescence and the senescent secretory phenotype: therapeutic opportunities. The Journal of Clinical Investigation. 123 (3), 966-972 (2013).
- van Deursen, J. M. The role of senescent cells in ageing. Nature. 509 (7501), 439-446 (2014).
- 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).
- Kim, Y. Y., et al. Cooperation between p21 and Akt is required for p53-dependent cellular senescence. Aging Cell. 16 (5), 1094-1103 (2017).
- Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D., Lowe, S. W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell. 88 (5), 593-602 (1997).
- Wu, D., Yotnda, P. Production and detection of reactive oxygen species (ROS) in cancers. Journal of Visualized Experiments. (57), (2011).
- Wojtala, A., et al. Methods to monitor ROS production by fluorescence microscopy and fluorometry. Methods in Enzymology. 542, 243-262 (2014).
- Duncan, F. E., et al. Age-associated dysregulation of protein metabolism in the mammalian oocyte. Aging Cell. 16 (6), 1381-1393 (2017).
- Yang, L., Song, T., Chen, L., Soliman, H., Chen, J. Nucleolar repression facilitates initiation and maintenance of senescence. Cell Cycle. 14 (22), 3613-3623 (2015).
- 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).
- Kosar, M., et al. Senescence-associated heterochromatin foci are dispensable for cellular senescence, occur in a cell type- and insult-dependent manner and follow expression of p16(ink4a). Cell Cycle. 10 (3), 457-468 (2011).
- Sharpless, N. E., Sherr, C. J. Forging a signature of in vivo senescence. Nature Reviews. Cancer. 15 (7), 397-408 (2015).
- Baker, D. J., et al. Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency. Nature Cell Biology. 10 (7), 825-836 (2008).
- Baker, D. J., et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature. 479 (7372), 232-236 (2011).
- 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).
- Farr, J. N., et al. Targeting cellular senescence prevents age-related bone loss in mice. Nature Medicine. 23 (9), 1072-1079 (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).
- Yosef, R., et al. Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL. Nature Communications. 7, 11190 (2016).
- 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).
Citazione di questo articolo
Kim, Y. Y., Um, J., Yun, J. A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence. J. Vis. Exp. (138), e57890, doi:10.3791/57890 (2018).