Name: induction and analysis of oxidative stress in sleeping beauty transposon-transfected human retinal pigment epithelial cells
Uploaded: 2020-12-11T13:00:00
Description: Watch this Scientific Journal Video about Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells at JoVE.com

作者要感谢Gregg Sealy和Alain Conti的出色技术援助，以及柏林Max-Delbrück中心的Zsuzsanna Izsvák教授慷慨地提供了pSB100X和pT2-CAGGS-Venus质粒。这项工作得到了瑞士国家科学基金会和欧盟委员会在第七个框架方案的范围内的支持。Z.I由欧洲研究委员会ERC Advanced资助[ERC-2011-ADG 294742]。

收集和使用人眼的程序由州伦理研究委员会批准（编号：2016-01726）。
1. 细胞分离和培养条件
<ol><li>人 ARPE-19 细胞系<ol>
<li>在Dulbecco的改良鹰培养基/营养混合物F-12火腿（DMEM / Ham's F-12）中培养5 x 105 个ARPE-19细胞，这是一种人RPE细胞系，在37°C下在T75烧瓶中加入10%胎牛血清（FBS），80 U / mL青霉素，80μg/ mL链霉素B（完整培养基）在5%CO2 和95%空气的加湿气氛中（其?...

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这里提出的方案提供了一种分析转染细胞产生的PEDF和GM-CSF的抗氧化和保护功能的方法，可以应用于任何假定有益基因转染的细胞。在以通过移植转基因细胞将蛋白质递送到组织的基因治疗策略中，获得有关蛋白质表达水平，表达寿命以及表达蛋白质在疾病模型中的有效性的信息至关重要。在我们的实验室中，这里提出的方案可用于确定PEDF和GM-CSF对氧化应激的有效性，氧化应激已被假设为aAMD<sup cl...

这里描述的模型为评估生物制药剂减少细胞氧化应激的效率提供了一种有用的方法。我们利用该模型研究了PEDF和GM-CSF对H2O2介导的氧化应激对视网膜色素上皮细胞的保护作用，这些细胞暴露于高水平的O2和可见光，以及光感受器外段膜的吞噬作用，产生显着水平的活性氧（ROS）1， 2.它们被认为是缺血性年龄相关性黄斑变性（aAMD）发病机制的主要贡献者3，4，5，6，7，8。此外，RPE合成的神经保护因子减少，特别是色素上皮衍生因子（PEDF），胰岛素样生长因子（IGF）和粒细胞巨噬细胞集落刺激因子（GM-CSF）导致RPE细胞的功能障碍和丢失，其次是光感受器和视网膜神经节细胞（RGC）死亡3，4，5.AMD是一种复杂的疾病，由代谢，功能，遗传和环境因素之间的相互作用引起4。缺乏对aAMD的治疗是工业化国家60岁以上患者失明的主要原因9，10。通过视网膜下移植过表达PEDF和GM-CSF的转基因RPE细胞重建神经保护性和神经源性视网膜环境，有可能通过减轻氧化应激的影响，抑制炎症和支持细胞存活来预防视网膜变性11，12，13，14，15，16.尽管有几种方法将基因递送到细胞，但我们选择了非病毒性过度活跃的睡美人转座子系统将PEDF和GM-CSF基因递送到RPE细胞，因为它的安全性，基因整合到宿主细胞的基因组中，以及它倾向于将递送的基因整合到非转录活性位点中，正如我们之前所展示的那样17， 18，19.
细胞氧化应激可以通过几种氧化剂在体外培养的细胞中诱导，包括过氧化氢（H2O 2），4-氢炔诺（HNE），过氧化氢叔丁酯（tBH），高氧张力和可见光（全光谱或紫外线照射）20，21。高氧张力和光需要特殊的设备和条件，这限制了向其他系统的可转移性。H 2 O2、HNE和 tBH 等试剂可诱导重叠的氧化应激分子和细胞变化。我们选择H2O2来测试PEDF和GM-CSF的抗氧化活性，因为它方便且具有生物学相关性，因为它是由RPE细胞在光感受器外段吞噬作用22期间作为活性氧中间体产生的，并且存在于体内的眼组织中23。由于谷胱甘肽的氧化可能是眼睛中H2O2产生部分原因，因此我们在研究中分析了GSH / 谷胱甘肽的水平，这与H2O2诱导的氧化应激和细胞的再生能力有关21，22。谷胱甘肽水平的分析特别相关，因为它参与眼睛的抗氧化保护机制24。暴露于H2O2经常被用作模型来检查RPE细胞1，25，26，27，28，29，30的氧化应激敏感性和抗氧化活性，此外，它显示出与光诱导的氧化应激损伤的相似性，氧化应激损伤是氧化应激21的"生理"来源。
为了评估神经保护因子的功能和有效性，我们开发了一种体外模型，该模型允许分析以量化由基因修饰以过度表达PEDF和GM-CSF的细胞表达的生长因子的抗氧化作用。在这里，我们表明，与PEDF和GM-CSF基因转染的RPE细胞比未转染的对照细胞更能抵抗H2O2的有害影响，如细胞形态学，密度，活力，谷胱甘肽的细胞内水平和UCP2基因的表达所证明的那样，UCP2基因编码线粒体解偶联蛋白2，已被证明可以减少活性氧（ROS）31。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Greiner</td>
			<td>7657160</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture plate white with clear flat bottom&#160;</td>
			<td>Costar</td>
			<td>3610</td>
			<td>Allows to check the cells before measuring the luminescence (GSH-Glo Assay)</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Corning&#160;</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamid 40%</td>
			<td>Biorad</td>
			<td>161-0144</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>AMIMED</td>
			<td>4-05F00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-GMCSF</td>
			<td>ThermoFisher Scientific</td>
			<td>PA5-24184</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-mouse IgG/IgA/IgM&#160;</td>
			<td>Agilent</td>
			<td>P0260</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-PEDF&#160;</td>
			<td>Santa Cruz Biotechnology Inc</td>
			<td>sc-390172</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-penta-His&#160;</td>
			<td>Qiagen</td>
			<td>34660</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-phospho-Akt</td>
			<td>Cell Signaling Technology</td>
			<td>9271</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-rabbit IgG H&#38;L-HRP</td>
			<td>Abcam</td>
			<td>ab6721</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody donkey anti-rabbit Alexa Fluor 594&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody goat anti-mouse Alexa 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr>
			<td>ARPE-19 cell line</td>
			<td>ATCC</td>
			<td>CRL-2302</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9418-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>chamber culture glass slides</td>
			<td>Corning</td>
			<td>354118</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoTox-Glo Cytotoxicity Assay&#160;</td>
			<td>Promega</td>
			<td>G9291</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/Ham`s F12&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8062</td>
			<td></td>
		</tr>
		<tr>
			<td>Duo Set ELISA kit</td>
			<td>R&#38;D Systems&#160;</td>
			<td>DY215-05</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>78440</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISAquant kit</td>
			<td>BioProducts MD</td>
			<td>PED613-10-Human</td>
			<td></td>
		</tr>
		<tr>
			<td>Eyes (human)</td>
			<td>Lions Gift of Sight Eye Bank (Saint Paul, MN)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS&#160;</td>
			<td>Brunschwig</td>
			<td>P40-37500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount Aqueous Mounting Medium</td>
			<td>Sigma-Aldrich</td>
			<td>F4680-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FLUOstar Omega plate reader&#160;</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism software (version 8.0)</td>
			<td>GraphPad Software, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GSH-Glo Glutathione Assay</td>
			<td>Promega</td>
			<td>V6912</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrogen peroxide (H2O2)</td>
			<td>Merck</td>
			<td>107209</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software (image processing program)</td>
			<td>W.S. Rasband, NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/; 1997&#8211;2014</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazol&#160;</td>
			<td>Axonlab</td>
			<td>A1378.0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMI4000B microscope&#160;</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 Instrument II&#160;</td>
			<td>Roche Molecular Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 SW1.5.1 software</td>
			<td>Roche Molecular Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71376-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Axonlab</td>
			<td>3468.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System 10 &#181;L Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK1096</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Marienfeld-superior</td>
			<td>640010</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA superflow&#160;</td>
			<td>Qiagen</td>
			<td>30410</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose&#160;</td>
			<td>VWR</td>
			<td>732-3197</td>
			<td></td>
		</tr>
		<tr>
			<td>Omega Lum G Gel Imaging System</td>
			<td>Aplegen Life Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P0781-100</td>
			<td></td>
		</tr>
		<tr>
			<td>PerfeCTa SYBR Green FastMix</td>
			<td>Quantabio</td>
			<td>95072-012</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>158127-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers&#160;</td>
			<td>Invitrogen&#160;</td>
			<td></td>
			<td>See Table 1 in Supplementary Materials</td>
		</tr>
		<tr>
			<td>pSB100X (250 ng/&#181;L)</td>
			<td></td>
			<td></td>
			<td>M&#225;t&#233;s et al., 2009. Provide by Prof. Zsuzsanna Izsvak</td>
		</tr>
		<tr>
			<td>pT2-CMV-GMCSF-His plasmid DNA (250 ng/&#181;L)</td>
			<td></td>
			<td></td>
			<td>Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.</td>
		</tr>
		<tr>
			<td>pT2-CMV-PEDF-His plasmid DNA (250 ng/&#181;L)</td>
			<td></td>
			<td></td>
			<td>Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.</td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit</td>
			<td>QIAGEN</td>
			<td>51304</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant hGM-CSF&#160;</td>
			<td>Peprotech</td>
			<td>100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant hPEDF&#160;&#160;</td>
			<td>BioProductsMD</td>
			<td>004-096</td>
			<td></td>
		</tr>
		<tr>
			<td>ReliaPrep RNA Cell Miniprep System</td>
			<td>Promega</td>
			<td>Z6011</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>89901</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free DNase Set</td>
			<td>QIAGEN</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>QIAGEN</td>
			<td>74204</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Applichem</td>
			<td>A2572</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-dry transfer system for WB&#160;</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SuperMix qScript</td>
			<td>Quantabio</td>
			<td>95048-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered saline (TBS)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>15504020</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>AppliChem</td>
			<td>A4975</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween</td>
			<td>AppliChem&#160;</td>
			<td>A1390</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>ThermoFisher Scientific</td>
			<td>29700</td>
			<td></td>
		</tr>
		<tr>
			<td>WesternBright ECL HRP substrate</td>
			<td>Advansta</td>
			<td>K-12045-D50</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman nitrocellulose membrane</td>
			<td>Chemie Brunschwig</td>
			<td>MNSC04530301</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and analysis of oxidative stress in sleeping beauty transposon-transfected human retinal pigment epithelial cells

氧化应激在几种退行性疾病中起着关键作用，包括年龄相关性黄斑变性（AMD），这是一种影响全球约3000万患者的病理。它导致视网膜色素上皮 （RPE） 合成的神经保护因子减少，例如色素上皮衍生因子 （PEDF） 和粒细胞-巨噬细胞集落刺激因子 （GM-CSF），随后 RPE 细胞丢失，最终导致光感受器和视网膜神经节细胞 （RGC） 死亡。我们假设通过转染的RPE细胞过表达PEDF和GM-CSF的视网膜下移植来重建神经保护性和神经源性视网膜环境，有可能通过减轻氧化应激的影响，抑制炎症和支持细胞存活来预防视网膜变性。使用 睡美人 转座子系统（SB100X），人RPE细胞已经转染了 PEDF 和 GM-CSF 基因，并使用qPCR，蛋白质印迹，ELISA和免疫荧光显示出稳定的基因整合，长期基因表达和蛋白质分泌。为了确认转染的RPE细胞分泌的 PEDF 和 GM-CSF 的功能和效力，我们开发了一种体外测定法来量化培养物中H2O2诱导的氧化应激对RPE细胞的减少。通过分析细胞形态、密度、谷胱甘肽的细胞内水平 、UCP2 基因表达和细胞活力来评估细胞保护。与未处理的对照相比，转染的RPE细胞过度表达PEDF和/或GM-CSF以及未转染但用PEDF和/或GM-CSF（市售或从转染细胞中纯化）预处理的细胞显示出显着的抗氧化细胞保护。目前的H2O2模型是一种简单有效的方法来评估可能有效治疗AMD或类似神经退行性疾病的因子的抗氧化作用。

诱导人视网膜色素上皮细胞中的氧化应激 ARPE-19和原代hRPE细胞用不同浓度的H2O2处理24小时，并定量抗氧化剂谷胱甘肽的细胞内水平（图2A，B）。H2O2在50μM和100μM不影响谷胱甘肽的产生，而在350μM时，ARPE-19和原代hRPE细胞中的谷胱甘肽显着降低。细胞毒性分析表明，350μM是H2O2...

Watch this Scientific Journal Video about Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells at JoVE.com

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

我们提出了一种开发和使用氧化应激模型的方案，方法是用H2O2处理视网膜色素上皮细胞，分析细胞形态，活力，密度，谷胱甘肽和UCP-2水平。它是研究转座子转染细胞分泌的蛋白质治疗神经视网膜变性的抗氧化作用的有用模型。

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Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

一种用于兔视网膜下手术分步协议

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

小鼠加速动脉硬化的诱导："电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

磷31磁共振波谱分析：一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...