Name: a quantitative measurement of reactive oxygen species and senescence-associated secretory phenotype in normal human fibroblasts during oncogene-induced senescence
Uploaded: 2018-08-12T13:00:00
Description: Watch this Scientific Journal Video about A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senesc

この作品は (若い淵金) に韓国の国立研究財団 (2015R1D1A1A01060839) からの助成金によって、韓国政府 (MSIT) によって資金を供給された国立研究財団の韓国 (NRF) の助成金によって支えられた (いいえ 2016R1A2B2008887 号2016R1A5A2007009) (に Jeanho ゆん)。

1. 癌遺伝子が誘導する老化を誘導します。
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H の準備-RasV12 レトロ ウイルス
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<li>0.001% ポリ-L-リジン/リン酸 2 mL を追加することにより 100 mm ディッシュのコートは、室温で 5 分の生理食塩水 (PBS) をバッファーしました。</li>
		<li>真空に接続されているガラス ピペットを使用してポリ L リジン ソリューションを削除し、2 mL の 1x PBS を追加することによって培養皿を洗っ?...

<ol>
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Molecular Cell Biology. 15 (7), 482-496 (2014).</li><li>Tchkonia, T., Zhu, Y., van Deursen, J., Campisi, J., Kirkland, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence+and+the+senescent+secretory+phenotype:+therapeutic+opportunities.">Cellular senescence and the senescent secretory phenotype: therapeutic opportunities.</a> The Journal of Clinical Investigation. 123 (3), 966-972 (2013).</li><li>van Deursen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+senescent+cells+in+ageing.">The role of senescent cells in ageing.</a> Nature. 509 (7501), 439-446 (2014).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Kim, Y. 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=Cooperation+between+p21+and+Akt+is+required+for+p53-dependent+cellular+senescence.">Cooperation between p21 and Akt is required for p53-dependent cellular senescence.</a> Aging Cell. 16 (5), 1094-1103 (2017).</li><li>Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D., Lowe, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenic+ras+provokes+premature+cell+senescence+associated+with+accumulation+of+p53+and+p16INK4a.">Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.</a> Cell. 88 (5), 593-602 (1997).</li><li>Wu, D., Yotnda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+detection+of+reactive+oxygen+species+(ROS)+in+cancers.">Production and detection of reactive oxygen species (ROS) in cancers.</a> Journal of Visualized Experiments. (57), (2011).</li><li>Wojtala, 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=Methods+to+monitor+ROS+production+by+fluorescence+microscopy+and+fluorometry.">Methods to monitor ROS production by fluorescence microscopy and fluorometry.</a> Methods in Enzymology. 542, 243-262 (2014).</li><li>Duncan, F. 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P., 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=Senescence-associated+secretory+phenotypes+reveal+cell-nonautonomous+functions+of+oncogenic+RAS+and+the+p53+tumor+suppressor.">Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.</a> PLoS Biology. 6 (12), 2853-2868 (2008).</li><li>Kosar, 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=Senescence-associated+heterochromatin+foci+are+dispensable+for+cellular+senescence,+occur+in+a+cell+type-+and+insult-dependent+manner+and+follow+expression+of+p16(ink4a).">Senescence-associated heterochromatin foci are dispensable for cellular senescence, occur in a cell type- and insult-dependent manner and follow expression of p16(ink4a).</a> Cell Cycle. 10 (3), 457-468 (2011).</li><li>Sharpless, N. 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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=Clearance+of+p16Ink4a-positive+senescent+cells+delays+ageing-associated+disorders.">Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders.</a> Nature. 479 (7372), 232-236 (2011).</li><li>Baar, M. P., 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=Targeted+Apoptosis+of+Senescent+Cells+Restores+Tissue+Homeostasis+in+Response+to+Chemotoxicity+and+Aging.">Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging.</a> Cell. 169 (1), 132-147 (2017).</li><li>Farr, J. 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=Targeting+cellular+senescence+prevents+age-related+bone+loss+in+mice.">Targeting cellular senescence prevents age-related bone loss in mice.</a> Nature Medicine. 23 (9), 1072-1079 (2017).</li><li>Chang, 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=Clearance+of+senescent+cells+by+ABT263+rejuvenates+aged+hematopoietic+stem+cells+in+mice.">Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice.</a> Nature Medicine. 22 (1), 78-83 (2016).</li><li>Yosef, 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=Directed+elimination+of+senescent+cells+by+inhibition+of+BCL-W+and+BCL-XL.">Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL.</a> Nature Communications. 7, 11190 (2016).</li><li>Jeon, O. 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ここでは、我々 は H Ras 誘導する老化は WI 38 正常ひと線維芽細胞の中に細胞内 ROS レベルを監視する方法を提案しました。生細胞における細胞内 ROS レベル DCF DA の細胞膜透過性の試薬を用いて測定することができ、フローサイトメトリーします。細胞内取り込みに DCF DA 細胞内エステラーゼによってされたきちん、その後、高い蛍光 2', 7'-ジクロルフルオレッセン (DCF) を形成する活性酸素に?...

50 年以上も前、ヘイフリック、ムーアヘッドは、正常な細胞が細胞分裂1数後の増殖能の枯渇に不可逆的な増殖停止を入力することを明らかにしました。この現象は、複製老化として今知られているし、個体老化2と強く相関すると考えられています。DNA の損傷、発癌活性化、酸化ストレスなど、さまざまな細胞のストレスを別の種類の細胞の老化を誘導するために報告されているテロメアの進歩的な侵食は複製老化の主な原因と考えられるが、「老化」や「ストレス老化」と呼ばれます。興味深いことに、時期尚早の老化は、H-ras など BRAF 遺伝子の活性化に強力な腫瘍抑制の役割を果たしています。マウス モデルと人間の組織の研究は細胞老化バイオ マーカーは前癌病変、発癌の Ras および BRAF がアクティブになりますがから開発された悪性の癌では減少した主に存在した強力な証拠を作り出した。これら病変3,4,の5。その高齢化と腫瘍抑制における役割を超えて細胞の老化は、創傷治癒、組織修復、免疫監視および萌芽期の開発6などを含む様々 な生理的プロセスで役割を果たす以前の研究で示されています。
増殖停止は、細胞の老化が7の特徴として広く研究されているが証拠の重要なボディは、その細胞内の活性酸素種 (ROS) は、細胞の老化の8にも貢献して示唆しています。ROS レベルの標高複製老化遺伝子が誘導する老化 (OIS) など、細胞の老化の様々 なタイプの中にもともと数十年前は報告された9,10。もっと直接、H2O2の致死線量と外因性治療は老化11,12誘導します。SOD1 などの活性酸素消去酵素の阻害は早期老化13も発生します。対照的に、周囲の酸素条件を低いし、ROS 清掃遅延老化10,14,15の発症の増加します。これらの結果は間違いなく ROS が重要なメディエーターまたは細胞の老化誘導の決定要因であることを示します。ただし、ROS は細胞の老化や細胞の老化中 ROS レベルを昇格する方法の誘導に貢献方法必要さらに調査。
最近の研究では、老化細胞が隣接細胞や SASP16,17を通じて組織の強力なパラクリン活動あるが明らかにしました。高齢者組織老化細胞組織の加齢に伴う機能障害を介して増殖細胞の自律的枯渇に加えて多くの経路 SASP を通じて推進します。様々 な炎症性要因、組織アーキテクチャの破壊組織恒常性が損なわれ、組織の加齢に伴う機能障害を引き起こす IL-8、TGFβ、およびマトリックスメタロプロテアーゼ (Mmp)、老化の細胞によって分泌されるが IL-6 など隣接するセル、および無菌性炎症18,19の老化。しかし、SASPs は、生物学的文脈によって有益な効果を持つことができます。さらに、SASPs の担体の性質は、老化細胞型と19のさらなる研究の必要性を強調し、細胞の段階に依存します。
ここでは、OIS の中に細胞内 ROS レベルを評価するため迅速・高感度のフローサイトメトリーを用いた手法について述べる。さらに、量的なリアルタイムのポリメラーゼの連鎖反応 (qPCR) や ELISA を用いた SASP 因子の分析方法を紹介しています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>REAGENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P2636</td>
			<td></td>
		</tr>
		<tr>
			<td>BOSC 23</td>
			<td>ATCC</td>
			<td>CRL-11269</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>wellgene</td>
			<td>LS202-02</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hyclone</td>
			<td>SH30013.02</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>12800-082</td>
			<td></td>
		</tr>
		<tr>
			<td>OPTI-MEM&#160;</td>
			<td>GIBCO</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>pBabe puro-H-RasV12&#160;</td>
			<td>Addgene</td>
			<td>1768</td>
			<td></td>
		</tr>
		<tr>
			<td>pGAG/pol</td>
			<td>Addgene</td>
			<td>14887</td>
			<td></td>
		</tr>
		<tr>
			<td>pVSVG</td>
			<td>Addgene</td>
			<td>1733</td>
			<td></td>
		</tr>
		<tr>
			<td>Turbofect</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0531</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td>8 mg/ml</td>
		</tr>
		<tr>
			<td>puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td>2 mg/ml&#160;</td>
		</tr>
		<tr>
			<td>formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>5-bromo-4-chloro-3-indolyl &#946; D-galactopyranoside (X-gal)</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium ferrocyanide</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium ferricyanide</td>
			<td>Sigma-Aldrich</td>
			<td>P9387</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>wellgene</td>
			<td>LS015-01</td>
			<td></td>
		</tr>
		<tr>
			<td>DCF-DA</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D6883</td>
			<td>10 mM&#160;</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>MMLV Reverse transcriptase</td>
			<td>Promega</td>
			<td>M1701</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green PCR master 2X mix</td>
			<td>Takara</td>
			<td>PR820A</td>
			<td></td>
		</tr>
		<tr>
			<td>Random Primer</td>
			<td>Promega</td>
			<td>C118A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-pure distilled water</td>
			<td>Invitrogen</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-6 ELISA assay</td>
			<td>PeproTech</td>
			<td>#900-TM16</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-8 ELISA assay</td>
			<td>PeproTech</td>
			<td>#900_TM18</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#956;m syringe filter</td>
			<td>sartorius</td>
			<td>16555</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>BEMIS&#160;</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>NIKON</td>
			<td>TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Bioscience</td>
			<td>LSR Fortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-4ml</td>
			<td>Merk Millipore</td>
			<td>UFC800324</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop spectrophotometer</td>
			<td>BioDrop</td>
			<td>80-3006-61</td>
			<td></td>
		</tr>
		<tr>
			<td>Real-time PCR System</td>
			<td>Applied Biosystems</td>
			<td>ABI Prism 7500</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA Reader</td>
			<td>Molecular Devices</td>
			<td>EMax microplate reader</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative measurement of reactive oxygen species and senescence-associated secretory phenotype in normal human fibroblasts during oncogene-induced senescence

細胞の老化は、増殖能力や様々 なストレスへの暴露の枯渇に不可逆的な増殖停止の状態を考えられています。最近の研究では、開発、創傷治癒、免疫監視、加齢に伴う組織の機能不全など、様々 な生理的過程に細胞の老化の役割を拡張しています。細胞周期の停止は、細胞の老化の重要な特徴は、増加の細胞内活性酸素種 (ROS) 生産は、細胞老化の誘導に重要な役割を果たすことも実証されています。さらに、最近の研究では、老化細胞が隣接細胞と老化関連の分泌形質 (SASP) を通じて組織に強力な傍分泌活動を示すこと明らかにしました。細胞の老化に対する治療戦略に関する関心の急激な増加は、細胞内 ROS および、SASP など老化メカニズムの正確な理解の必要性を強調しています。ここでは、活性酸素感受性蛍光色素とフローサイトメトリーを使用して H Ras 誘導性細胞老化にともなう細胞内 ROS レベルを定量的に評価するためのプロトコルについて述べる。さらに、mRNA の発現の誘導と SASP 因子の分泌の解析のための高感度技術を紹介します。これらのプロトコルは、さまざまな細胞の老化モデルに適用できます。

H Ras 誘導する老化の例は、図 1に示します。WI 38 H RasV12 レトロ ウイルスの培養細胞への感染には、劇的な形態学的変化 (図 1 b) が誘導されます。さらに、図 1のように、SA β gal 染色活動著しく H RasV12 式に増加しました。細胞の 70% 以上を示した SA β gal H RasV12 レトロ ウイルス感染後 6 d の活動を染色、...

Watch this Scientific Journal Video about A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senesc

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

細胞内 ROS は、細胞老化の誘導に重要な役割を果たすに示されています。ここでは、細胞の老化にともなう ROS レベルを定量化するための敏感な試金について述べる。伝えられる様々 な加齢に伴う機能不全に貢献する老化関連分泌形質を評価するためのプロトコルをいたします。

癌遺伝子が誘導する老化の中に活性酸素種および分泌老化関連の表現型正常ひと線維芽細胞の定量測定

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

Research

JoVE Journal

Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

ヒト骨格筋からの一次筋原細胞および線維芽細胞の単離および定量的免疫組織化学的特性評価

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

非形質転換ヒト線維芽細胞から増殖性四倍体細胞の樹立

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

やっぱりアオモンイトトンボ精子における活性酸素種の細胞内局在の評価

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

細胞分化中のマイトジェン活性化プロテインキナーゼ経路の光媒介可逆的調節<em&gt;アフリカツメガエル</em&gt;胚発生

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

A Method for Characterizing Embryogenesis in Arabidopsis

A Method for Characterizing Embryogenesis in Arabidopsis

シロイヌナズナの胚発生の特性評価法

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

傷害による老化と生体内での骨格筋におけるリプログラミングの評価

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

セルショウジョウバエ切り込みをトランスとの結合を評価する集計の試金-配位子とCisによるその阻害-配位子

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

人間の腸 Organoids 上皮関門の透過性のリアルタイム計測

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Induction and Validation of Cellular Senescence in Primary Human Cells

ひと初代細胞における細胞老化の誘導性

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

RNA に基づく誘導多能性幹細胞に主線維芽細胞のプログラムし直す

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...