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

Os autores gostariam de agradecer a Gregg Sealy e Alain Conti pela excelente assistência técnica e prof. Zsuzsanna Izsvák do Centro Max-Delbrück em Berlim por fornecer gentilmente os plasmídeos pSB100X e pT2-CAGGS-Venus. Este trabalho foi apoiado pela Fundação Nacional de Ciências suíça e pela Comissão Europeia no contexto do Sétimo Programa-Quadro. Z.I foi financiado pelo European Research Council, ERC Advanced [ERC-2011-ADG 294742].

Os procedimentos de coleta e uso de olhos humanos foram aprovados pela Comissão de Ética Cantonnal de Pesquisa (nº 2016-01726).
1. Condições de isolamento celular e cultura
<ol><li>Linha celular HUMANA ARPE-19<ol>
<li>Cultura 5 x 105 células ARPE-19, uma linha celular RPE humana, na mistura de médio/nutrientes da Águia Modificada de Dulbecco F-12 Ham (DMEM/Ham's F-12) suplementada com 10% de soro bovino fetal (FBS), penicilina U/mL de 80 U/mL, Estreptomicina de 80 μg/mL, ...

<ol>
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O protocolo aqui apresentado oferece uma abordagem para analisar a função antioxidante e protetora do PEDF e GM-CSF produzido por células transfeinadas, que podem ser aplicadas a células transfeinadas com qualquer gene benéfico putativo. Em estratégias terapêuticas genéticas que têm o objetivo de fornecer proteínas ao tecido por meio do transplante de células geneticamente modificadas, é fundamental obter informações quanto ao nível de expressão proteica, à longevidade da expressão e à eficácia da pro...

O modelo descrito aqui, oferece uma abordagem útil para avaliar a eficiência dos agentes biofarmacêuticos para reduzir o estresse oxidativo nas células. Utilizamos o modelo para investigar os efeitos protetores do PEDF e do GM-CSF no estresse oxidativo mediado H2O2nas células epiteliais do pigmento retinal, que estão expostas a altos níveis de O2, luz visível, e a fagocitose das membranas do segmento externo fotoreceptor, gerando níveis significativos de espécies reativas de oxigênio (ROS)1, 2. São considerados um dos principais contribuintes para a patogênese da degeneração macular avascular relacionada à idade (aAMD)3,4,5,6,7,8. Além disso, há uma diminuição nos fatores neuroprotetores sintetizados por RPE, especificamente o fator derivado do epitélio pigmento (PEDF), fatores de crescimento semelhantes à insulina (IGFs) e fator estimulante da colônia-colônia granucyte (GM-CSF) levando à disfunção e perda de células RPE, seguidos pela morte de células fotorreceptoras e gânglios da retina (RGC)3,4,5 . A AMD é uma doença complexa que resulta da interação entre fatores metabólicos, funcionais, genéticos e ambientais4. A falta de tratamentos para a AAMD é a principal causa de cegueira em pacientes com mais de 60 anos de idade em países industrializados9,10. A reconstituição do ambiente neuroprotetor e neurogênico da retina pelo transplante subretinal de células RPE geneticamente modificadas que superexpressam PEDF e GM-CSF tem o potencial de prevenir a degeneração da retina, mitigando os efeitos do estresse oxidativo, inibindo inflamação e apoiando a sobrevivência celular11,12,13,14,15,16 . Embora existam várias metodologias para entregar genes às células, escolhemos o sistema transposon da Bela Adormecida hiperativo não viral para entregar os genes PEDF e GM-CSF às células RPE por causa de seu perfil de segurança, a integração dos genes no genoma das células hospedeiras e sua propensão a integrar os genes entregues em locais não transcricionalmente, como mostramos anteriormente17, 18,19.
O estresse oxidativo celular pode ser induzido em células cultivadas in vitro por vários agentes oxidativos, incluindo peróxido de hidrogênio (H2O2),4-hidroynonenal (HNE), tertbutylhydroperoxide (tBH), altas tensões de oxigênio e luz visível (espectro completo ou irradiação UV)20,21. Altas tensões de oxigênio e luz requerem equipamentos e condições especiais, o que limita a transferência para outros sistemas. Agentes como H2O2,HNE e tBH induzem alterações moleculares e celulares por estresse oxidativo. Escolhemos H2O2 para testar a atividade antioxidante do PEDF e GM-CSF porque é conveniente e biologicamente relevante, pois é produzido por células RPE como um intermediário de oxigênio reativo durante a fagocitose do segmento externo fotoreceptor22 e é encontrado em tecidos oculares in vivo23. Como a oxidação da glutationa pode ser parcialmente responsável pela produção de H2O2 no olho, analisamos os níveis de GSH/glutationa em nossos estudos, que estão ligados ao estresse oxidativo induzido por H2O2e à capacidade regenerativa das células21,22. A análise dos níveis de glutationa é especialmente relevante, pois participa dos mecanismos de proteção antioxidantes no olho24. A exposição a H2O2 é utilizada com frequência como modelo para examinar a suscetibilidade ao estresse oxidativo e a atividade antioxidante das células RPE1,25,26,27,28,29,30, e, além disso, apresenta semelhanças com danos oxidativos induzidos pela luz, fonte "fisiológica" de estresse oxidativo21.
Para avaliar a funcionalidade e a eficácia dos fatores neuroprotetores, desenvolvemos um modelo in vitro que permite que a análise quantifique o efeito antioxidante dos fatores de crescimento expressos por células geneticamente modificadas para superexpressar o PEDF e o GM-CSF. Aqui, mostramos que as células RPE transfeminadas com os genes para PEDF e GM-CSF são mais resistentes aos efeitos nocivos de H2O2 do que são células de controle não transfectadas, como evidenciado pela morfologia celular, densidade, viabilidade, nível intracelular de glutationa e expressão do gene UCP2, que codifica a proteína mitocondrial de desacoplamento 2 que tem sido demonstrada para reduzir espécies reativas de oxigênio (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

O estresse oxidativo desempenha um papel crítico em várias doenças degenerativas, incluindo a degeneração macular relacionada à idade (AMD), uma patologia que afeta ~30 milhões de pacientes em todo o mundo. Leva a uma diminuição no epitélio do pigmento da retina (RPE)-fatores neuroprotetores sintetizados, por exemplo, fator derivado do epitélio pigmento (PEDF) e fator estimulante da colônia granutócito-macrófago (GM-CSF), seguido pela perda de células RPE, e eventualmente fotorreceptor e morte de células gânglios de retina (RGC). Temos a hipótese de que a reconstituição do ambiente neuroprotetor e neurogênico da retina pelo transplante subretinal de células RPE transfecidas que superexpressam PEDF e GM-CSF tem o potencial de prevenir a degeneração da retina, mitigando os efeitos do estresse oxidativo, inibindo inflamação e apoiando a sobrevivência celular. Usando o sistema transposon da Bela Adormecida (SB100X)as células RPE humanas foram transfectadas com os genes PEDF e GM-CSF e mostraram integração genética estável, expressão genética de longo prazo e secreção de proteínas usando qPCR, mancha ocidental, ELISA e imunofluorescência. Para confirmar a funcionalidade e a potência do PEDF e gm-CSF secretados pelas células RPE transfeinadas, desenvolvemos um ensaio in vitro para quantificar a redução do estresse oxidativo induzido por H2O2em células RPE na cultura. A proteção celular foi avaliada pela análise da morfologia celular, densidade, nível intracelular de glutationa, expressão genética UCP2 e viabilidade celular. Ambas as células RPE transfectadas que expressam o FPE e/ou GM-CSF e as células não transfetizadas, mas pré-tratadas com PEDF e/ou GM-CSF (comercialmente disponíveis ou purificadas de células transfetizadas) apresentaram proteção celular antioxidante significativa em comparação com controles não tratados. O atual modelo H2O2é uma abordagem simples e eficaz para avaliar o efeito antioxidante de fatores que podem ser eficazes para tratar a AMD ou doenças neurodegenerativas similares.

Indução do estresse oxidativo nas células epiteliais do pigmento da retina humana As células ARPE-19 e hRPE primária foram tratadas com concentrações variadas de H2O2 por 24 h e o nível intracelular da glutationa antioxidante foi quantificado(Figura 2A,B). H2O2 a 50 μM e 100 μM não afetaram a produção de glutationa, enquanto em 350 μM houve uma diminuição signific...

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

Apresentamos um protocolo para o desenvolvimento e uso de um modelo de estresse oxidativo, tratando células epiteliais de pigmento da retina com H2O2, analisando morfologia celular, viabilidade, densidade, glutationa e nível UCP-2. É um modelo útil para investigar o efeito antioxidante de proteínas secretadas por células transfeminadas transposon para tratar a degeneração neuroretinal.

Indução e Análise do Estresse Oxidativo em Células Epiteliais de Pigmento Epitelial Humano Transposon-Transfected Da Bela Adormecida

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Research

JoVE Journal

Medicine

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

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Identificação de<em&gt; A Bela Adormecida</em&gt; Inserções de transposões em tumores sólidos utilizando Linker mediada por PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Medicina

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Análise de Transcriptoma Humanos espécimes cirúrgicos retina utilizando Jornal

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

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Isolamento, criopreservação e cultivo de células epiteliais humanas Amnion para aplicações clínicas

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

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Realizando Subretinal Injeções em roedores para entregar epitélio pigmentar da retina células em suspensão

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

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Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

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

A Passo a Passo Protocolo de Cirurgia sub-retiniana em Coelhos

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

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

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

Indução de Aterosclerose Acelerada em Camundongos: O Modelo de "Lesão por Fio"

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

Fósforo-31 Magnetic Resonance Spectroscopy: uma ferramenta para medir<em&gt; In Vivo</em&gt; Capacidade mitocondrial fosforilação oxidativo no músculo esquelético humano

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