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

De auteurs willen Gregg Sealy en Alain Conti bedanken voor hun uitstekende technische assistentie en Prof. Zsuzsanna Izsvák van het Max-Delbrück Center in Berlijn voor het leveren van de pSB100X en pT2-CAGGS-Venus plasmiden. Dit werk werd ondersteund door de Zwitserse National Sciences Foundation en de Europese Commissie in het kader van het zevende kaderprogramma. Z.I werd gefinancierd door european research council, ERC Advanced [ERC-2011-ADG 294742].

Procedures voor het verzamelen en gebruiken van menselijke ogen werden goedgekeurd door de Kantonnale Ethische Commissie voor Onderzoek (nr. 2016-01726).
1. Celisolatie en kweekomstandigheden
<ol><li>Menselijke ARPE-19 cellijn<ol>
<li>Kweek 5 x 105 ARPE-19 cellen, een menselijke RPE-cellijn, in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (DMEM/Ham's F-12) aangevuld met 10% foetaal runderserum (FBS), 80 U/ml penicilline, 80 μg/ml streptomycine en 2,5 μg/ml amfote...

<ol>
	<li>Zareba, M., Raciti, M. W., Henry, M. M., Sarna, T., Burke, 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=Oxidative+stress+in+ARPE-19+cultures:+Do+melanosomes+confer+cytoprotection.">Oxidative stress in ARPE-19 cultures: Do melanosomes confer cytoprotection.</a> Free Radical Biology and Medicine. 40 (1), 87-100 (2006).</li><li>Gong, X., Draper, C. S., Allison, G. S., Marisiddaiah, R., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+macular+carotenoid+lutein+in+human+retinal+pigment+epithelial+cells.">Effects of the macular carotenoid lutein in human retinal pigment epithelial cells.</a> Antioxidants. 6 (4), (2017).</li><li>Sacconi, R., Corbelli, E., Querques, L., Bandello, F., Querques, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+current+and+future+management+of+geographic+atrophy.">A Review of current and future management of geographic atrophy.</a> Ophthalmology and Therapy. 6, 69-77 (2017).</li><li>Al-Zamil, W. M., Yassin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+age-related+macular+degeneration:+a+review.">Recent developments in age-related macular degeneration: a review.</a> Clinical Interventions in Aging. 12, 1313-1330 (2017).</li><li>Kumar-Singh, R. <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+complement+membrane+attack+complex+in+dry+and+wet+AMD+-+From+hypothesis+to+clinical+trials.">The role of complement membrane attack complex in dry and wet AMD - From hypothesis to clinical trials.</a> Experimental Eye Research. 184, 266-277 (2019).</li><li>Ung, L., Pattamatta, U., Carnt, N., Wilkinson-Berka, J. L., Liew, G., White, A. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+reactive+oxygen+species.">Oxidative stress and reactive oxygen species.</a> Clinical Science. 131, 2865-2883 (2017).</li><li>Beatty, S., Koh, H. H., Phil, M., Henson, D., Boulton, 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+oxidative+stress+in+the+pathogenesis+of+age-related+macular+degeneration.">The role of oxidative stress in the pathogenesis of age-related macular degeneration.</a> Survey of Ophthalmology. 45 (2), 115-134 (2000).</li><li>Alhasani, R. H., 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=Gypenosides+protect+retinal+pigment+epithelium+cells+from+oxidative+stress.">Gypenosides protect retinal pigment epithelium cells from oxidative stress.</a> Food and Chemical Toxicology. 112, 76-85 (2018).</li><li>. National Institute of Health Available from: <a target="_blank" href="https://nei.nih.gov/learn-about-eye-health/resources-for-health-educators/eye-health-data-and-statistics/age-related-macular-degeneration-amd-data-and-statistics">https://nei.nih.gov/learn-about-eye-health/resources-for-health-educators/eye-health-data-and-statistics/age-related-macular-degeneration-amd-data-and-statistics</a> (2020)</li><li>Mitchell, P., Liew, G., Gopinath, B., Wong, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration.">Age-related macular degeneration.</a> The Lancet. 392, 1147-1159 (2018).</li><li>He, Y., Leung, K. W., Ren, Y., Jinzhi, P., Jian, G., Tombran-Tink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEDF+improves+mitochondrial+function+in+RPE+cells+during+oxidative+stress.">PEDF improves mitochondrial function in RPE cells during oxidative stress.</a> Investigative Ophthalmology &amp; Visual Science. 55, 6742-6755 (2014).</li><li>Cao, S., Walker, G. B., Wang, X., Cui, J. Z., Matsubara, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+cytokine+profiles+of+human+retinal+pigment+epithelium:+Oxidant+injury+and+replicative+senescence.">Altered cytokine profiles of human retinal pigment epithelium: Oxidant injury and replicative senescence.</a> Molecular Vision. 19, 718-728 (2013).</li><li>Farnoodian, M., Sorenson, C. M., Sheibani, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEDF+expression+affects+the+oxidative+and+inflammatory+state+of+choroidal+endothelial+cells.">PEDF expression affects the oxidative and inflammatory state of choroidal endothelial cells.</a> Amercian Journal of Physiology and Cell Physiology. 314 (4), 456-472 (2018).</li><li>Polato, F., Becerra, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+Degenerative+Diseases:+Mechanisms+and+Experimental+Therapies.">Retinal Degenerative Diseases: Mechanisms and Experimental Therapies.</a> Retinal Degenerative Diseases. , 699-706 (2016).</li><li>Schallenberg, M., Charalambous, P., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM-CSF+regulates+the+ERK1/2+pathways+and+protects+injured+retinal+ganglion+cells+from+induced+death.">GM-CSF regulates the ERK1/2 pathways and protects injured retinal ganglion cells from induced death.</a> Experimental Eye Research. 89, 665-677 (2009).</li><li>Schallenberg, M., Charalambous, P., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM-CSF+protects+rat+photoreceptors+from+death+by+activating+the+SRC-dependent+signalling+and+elevating+anti-apoptotic+factors+and+neurotrophins.">GM-CSF protects rat photoreceptors from death by activating the SRC-dependent signalling and elevating anti-apoptotic factors and neurotrophins.</a> Graefes Archives for Clinical and Experimental Ophthalmology. 250, 699-712 (2012).</li><li>Thumann, G., 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=Engineering+of+PEDF-expressing+primary+pigment+epithelial+cells+by+the+SB+transposon+system+delivered+by+pFAR4+plasmids.">Engineering of PEDF-expressing primary pigment epithelial cells by the SB transposon system delivered by pFAR4 plasmids.</a> Molecular Therapy - Nucleic Acids. 6, 302-314 (2017).</li><li>Garcia-Garcia, L., 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=Long-term+PEDF+release+in+rat+iris+and+retinal+epithelial+cells+after+Sleeping+Beauty+transposon-mediated+gene+delivery.">Long-term PEDF release in rat iris and retinal epithelial cells after Sleeping Beauty transposon-mediated gene delivery.</a> Molecular Therapy - Nucleic Acids. 9, 1-11 (2017).</li><li>Johnen, S., 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=Antiangiogenic+and+neurogenic+activities+of+Sleeping+Beauty-mediated+PEDF-transfected+RPE+cells+in+vitro+and+in+vivo.">Antiangiogenic and neurogenic activities of Sleeping Beauty-mediated PEDF-transfected RPE cells in vitro and in vivo.</a> BioMed Research International. 2015, (2015).</li><li>Weigel, A. L., Handa, J. T., Hjelmeland, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+of+H2O2-,+HNE-,+or+tBH-treated+ARPE-19+cells.">Microarray analysis of H2O2-, HNE-, or tBH-treated ARPE-19 cells.</a> Free Radical Biology &amp; Medicine. 33 (10), 1419-1432 (2002).</li><li>Allen, R. G., Tresini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+gene+regulation.">Oxidative stress and gene regulation.</a> Free Radical Biology and Medicine. 28 (3), 463-499 (2000).</li><li>Tate, D. J., Miceli, M. V., Newsome, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+and+H2O2+induce+catalase+and+metallothionein+gene+expression+in+human+retinal+pigment+epithelial+cells.">Phagocytosis and H2O2 induce catalase and metallothionein gene expression in human retinal pigment epithelial cells.</a> Investigative Ophthalmology and Visual Science. 36 (7), 1271-1279 (1995).</li><li>Halliwell, B., Clement, M. V., Long, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+in+the+human+body.">Hydrogen peroxide in the human body.</a> FEBS Letters. 486 (1), 10-13 (2000).</li><li>Giblin, F. J., McCready, J. P., Kodama, T., Reddy, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+correlation+between+the+levels+of+ascorbic+acid+and+H2O2+in+aqueous+humor.">A direct correlation between the levels of ascorbic acid and H2O2 in aqueous humor.</a> Experimental Eye Research. 38, 87-93 (1984).</li><li>Geiger, R. C., Waters, C. M., Kamp, D. W., Glucksberg, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KGF+prevents+oxygen-mediated+damage+in+ARPE-19+cells.">KGF prevents oxygen-mediated damage in ARPE-19 cells.</a> Investigative Ophthalmology and Visual Science. 46, 3435-3442 (2005).</li><li>Campochiaro, P. 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=Lentiviral+vector+gene+transfer+of+endostatin/angiostatin+for+macular+degeneration+(GEM)+study.">Lentiviral vector gene transfer of endostatin/angiostatin for macular degeneration (GEM) study.</a> Human Gene Therapy. 28, 99-111 (2017).</li><li>Chen, X. -. D., Su, M. -. Y., Chen, T. -. T., Hong, H. -. Y., Han, A. -. D., Li, W. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+affects+retinal+pigment+epithelial+cell+survival+through+epidermal+growth+factor+receptor/AKT+signaling+pathway.">Oxidative stress affects retinal pigment epithelial cell survival through epidermal growth factor receptor/AKT signaling pathway.</a> International Journal of Ophthalmology. 10 (4), 507-514 (2017).</li><li>Tu, G., 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=Allicin+attenuates+H2O2+-+induced+cytotoxicity+in+retinal+pigmented+epithelial+cells+by+regulating+the+levels+of+reactive+oxygen+species.">Allicin attenuates H2O2 - induced cytotoxicity in retinal pigmented epithelial cells by regulating the levels of reactive oxygen species.</a> Molecular Medicine Reports. 13, 2320-2326 (2016).</li><li>Hao, Y., Liu, J., Wang, Z., Yu, L., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piceatannol+protects+human+retinal+pigment+epithelial+cells+against+hydrogen+peroxide+induced+oxidative+stress+and+apoptosis+through+modulating.">Piceatannol protects human retinal pigment epithelial cells against hydrogen peroxide induced oxidative stress and apoptosis through modulating.</a> Nutrients. 11, 1-13 (2019).</li><li>Ballinger, S. W., Van Houten, B., Conklin, C. A., Jin, G. F., Godley, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+causes+significant+mitochondrial+DNA+damage+in+human+RPE+cells.">Hydrogen peroxide causes significant mitochondrial DNA damage in human RPE cells.</a> Experimental Eye Research. 68 (6), 765-772 (1999).</li><li>Ma, S., 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=Transgenic+overexpression+of+uncoupling+protein+2+attenuates+salt-induced+vascular+dysfunction+by+inhibition+of+oxidative+stress.">Transgenic overexpression of uncoupling protein 2 attenuates salt-induced vascular dysfunction by inhibition of oxidative stress.</a> American Journal of Hypertension. 27 (3), 345-354 (2014).</li><li>Johnen, S., 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=Sleeping+Beauty+transposon-mediated+transfection+of+retinal+and+iris+pigment+epithelial+cells.">Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells.</a> Investigative Ophthalmology and Visual Science. 53 (8), 4787-4796 (2012).</li><li>M&#225;t&#233;s, L., 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=Molecular+evolution+of+a+novel+hyperactive+Sleeping+Beauty+transposase+enables+robust+stable+gene+transfer+in+vertebrates.">Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates.</a> Nature Genetics. 41 (6), 753-761 (2009).</li><li>. Marienfeld Technical information Neubauer-improved Available from: <a target="_blank" href="https://www.marienfeld-superior.com/information-about-our-counting-chambers.html">https://www.marienfeld-superior.com/information-about-our-counting-chambers.html</a> (2020)</li><li>. Electron Microscopy Sciences. Neubauer Haemocytometry Available from: <a target="_blank" href="https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx">https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx</a> (2020)</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^(-&#916;&#916;CT)+method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2^(-&#916;&#916;CT) method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Bascuas, T., 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=Non-virally+transfected+primary+human+pigment+epithelium+cells+overexpressing+the+oxidative+stress+reduction+factors+PEDF+and+GM-CSF+to+treat+retinal+neurodegeneration+neurodegenerationl.">Non-virally transfected primary human pigment epithelium cells overexpressing the oxidative stress reduction factors PEDF and GM-CSF to treat retinal neurodegeneration neurodegenerationl.</a> Human Gene Therapy. 30 (11), (2019).</li><li>Zhuge, C. C., 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=Fullerenol+protects+retinal+pigment+epithelial+cells+from+oxidative+stress-induced+premature+senescence+via+activating+SIRT1.">Fullerenol protects retinal pigment epithelial cells from oxidative stress-induced premature senescence via activating SIRT1.</a> Investigative Ophthalmology &amp; Visual Science. 55 (7), 4628-4638 (2014).</li><li>Kaczara, P., Sarna, T., Burke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+H2O2+Availability+to+ARPE-19+cultures+in+models+of+oxidative+stress.">Dynamics of H2O2 Availability to ARPE-19 cultures in models of oxidative stress.</a> Free Radical Biology and Medicine. 48 (8), 1068-1070 (2010).</li><li>Gorrini, C., Harris, I. S., Mak, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+oxidative+stress+as+an+anticancer+strategy.">Modulation of oxidative stress as an anticancer strategy.</a> Nature Reviews Drug Discovery. 12, 931-947 (2013).</li><li>Wang, X., 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=PEDF+protects+human+retinal+pigment+epithelial+cells+against+oxidative+stress+via+upregulation+of+UCP2+expression.">PEDF protects human retinal pigment epithelial cells against oxidative stress via upregulation of UCP2 expression.</a> Molecular Medicine Reports. 19 (1), 59-74 (2019).</li><li>Donadelli, M., Dando, I., Fiorini, C., Palmieri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UCP2,+a+mitochondrial+protein+regulated+at+multiple+levels.">UCP2, a mitochondrial protein regulated at multiple levels.</a> Cellular and Molecular Life Sciences. 71, 1171-1190 (2014).</li></ol>

Het hier gepresenteerde protocol biedt een benadering om de anti-oxidatieve en beschermende functie van PEDF en GM-CSF geproduceerd door getransfecteerde cellen te analyseren, die kunnen worden toegepast op cellen die zijn getransfecteerd met elk vermeend gunstig gen. In gentherapeutische strategieën die tot doel hebben eiwitten aan weefsel te leveren door genetisch gemodificeerde cellen te transplanteren, is het van cruciaal belang om informatie te verkrijgen over het niveau van eiwitexpressie, de levensduur van expres...

Het hier beschreven model biedt een nuttige benadering om de efficiëntie van biofarmaceutische middelen te evalueren voor het verminderen van oxidatieve stress in cellen. We hebben het model gebruikt om de beschermende effecten van PEDF en GM-CSF op de H2O2-gemedieerde oxidatieve stress op retinale pigmentepitheelcellen te onderzoeken, die worden blootgesteld aan hoge niveaus van O2en zichtbaar licht, en de fagocytose van fotoreceptor buitenste segmentmembranen, die aanzienlijke niveaus van reactieve zuurstofsoorten (ROS) genereren1, 2. Ze worden beschouwd als een belangrijke bijdrage aan de pathogenese van avasculaire leeftijdsgebonden maculaire degeneratie (aAMD)3,4,5,6,7,8. Bovendien is er een afname van RPE-gesynthetiseerde neuroprotectieve factoren, met name de pigmentepitheel-afgeleide factor (PEDF), insuline-achtige groeifactoren (IGF's) en granulocyt macrofaag-kolonie-stimulerende factor (GM-CSF) die leidt tot de disfunctie en het verlies van RPE-cellen, gevolgd door fotoreceptor en retinale ganglioncel (RGC) dood3,4,5 . AMD is een complexe ziekte die het gevolg is van de interactie tussen metabole, functionele, genetische en omgevingsfactoren4. Het gebrek aan behandelingen voor aAMD is de belangrijkste oorzaak van blindheid bij patiënten ouder dan 60 jaar in geïndustrialiseerde landen9,10. De reconstitutie van de neuroprotectieve en neurogene retinale omgeving door de subretinale transplantatie van genetisch gemodificeerde RPE-cellen die PEDF en GM-CSF overexpressie geven, heeft het potentieel om retinale degeneratie te voorkomen door de effecten van oxidatieve stress te verminderen, ontstekingen te remmen en celoverleving te ondersteunen11,12,13,14,15,16 . Hoewel er verschillende methodologieën zijn om genen aan cellen te leveren, hebben we gekozen voor het niet-virale hyperactieve Doornroosje transposonsysteem om de PEDF- en GM-CSF-genen aan RPE-cellen te leveren vanwege het veiligheidsprofiel, de integratie van de genen in het genoom van de gastheercellen en de neiging om de afgeleverde genen te integreren in niet-transcriptioneel actieve sites zoals we eerder hebben aangetoond17, 18,19.
Cellulaire oxidatieve stress kan worden geïnduceerd in cellen die in vitro zijn gekweekt door verschillende oxidatieve middelen, waaronder waterstofperoxide (H2O2),4-hydroynonenaal (HNE), tertbutylhydroperoxide (tBH), hoge zuurstofspanningen en zichtbaar licht (volledig spectrum of UV-straling)20,21. Hoge zuurstofspanningen en licht vereisen speciale apparatuur en omstandigheden, die de overdraagbaarheid naar andere systemen beperken. Middelen zoals H 2 O2,HNE en tBH induceren overlappende oxidatieve stress moleculaire en cellulaire veranderingen. We kozen H2O2 om de antioxiderende activiteit van PEDF en GM-CSF te testen omdat het handig en biologisch relevant is omdat het wordt geproduceerd door RPE-cellen als een reactief zuurstoftussenproduct tijdens fotoreceptor buitenste segment fagocytose22 en het wordt gevonden in oculaire weefsels in vivo23. Aangezien de oxidatie van glutathion gedeeltelijk verantwoordelijk kan zijn voor de productie van H2O2 in het oog, hebben we de niveaus van GSH / glutathion in onze studies geanalyseerd, die verband houden met H2O2-geïnduceerde oxidatieve stress en het regeneratieve vermogen van cellen21,22. De analyse van glutathionspiegels is vooral relevant omdat het deelneemt aan de anti-oxidatieve beschermingsmechanismen in het oog24. Blootstelling aan H2O2 wordt vaak gebruikt als een model om de oxidatieve stressgevoeligheid en antioxiderende activiteit van RPE-cellen1,25,26,27,28,29,30te onderzoeken en bovendien vertoont het overeenkomsten met door licht geïnduceerde oxidatieve stressschade, een "fysiologische" bron van oxidatieve stress21.
Om de functionaliteit en de effectiviteit van neuroprotectieve factoren te evalueren, hebben we een in vitro model ontwikkeld dat de analyse mogelijk maakt om het anti-oxidatieve effect van groeifactoren uitgedrukt door cellen die genetisch gemodificeerd zijn om PEDF en GM-CSF te overexpressie te kwantificeren. Hier laten we zien dat RPE-cellen getransfecteerd met de genen voor PEDF en GM-CSF beter bestand zijn tegen de schadelijke effecten van H2O2 dan niet-getransfecteerde controlecellen, zoals blijkt uit celmorfologie, dichtheid, levensvatbaarheid, intracellulair niveau van glutathion en expressie van het UCP2-gen, dat codeert voor het mitochondriale ontkoppelingseiwit 2 waarvan is aangetoond dat het reactieve zuurstofsoorten (ROS) vermindert31.

<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

Oxidatieve stress speelt een cruciale rol bij verschillende degeneratieve ziekten, waaronder leeftijdsgebonden maculaire degeneratie (AMD), een pathologie die wereldwijd ~ 30 miljoen patiënten treft. Het leidt tot een afname van retinale pigmentepitheel (RPE) - gesynthetiseerde neuroprotectieve factoren, bijvoorbeeld pigmentepitheel-afgeleide factor (PEDF) en granulocyt-macrofaag kolonie-stimulerende factor (GM-CSF), gevolgd door het verlies van RPE-cellen, en uiteindelijk fotoreceptor en retinale ganglioncel (RGC) dood. We veronderstellen dat de reconstitutie van de neuroprotectieve en neurogene retinale omgeving door de subretinale transplantatie van getransfecteerde RPE-cellen die PEDF en GM-CSF overexpresseren, het potentieel heeft om retinale degeneratie te voorkomen door de effecten van oxidatieve stress te verminderen, ontsteking te remmen en celoverleving te ondersteunen. Met behulp van het Doornroosje transposonsysteem(SB100X)zijn menselijke RPE-cellen getransfecteerd met de PEDF- en GM-CSF-genen en hebben ze stabiele genintegratie, genexpressie op lange termijn en eiwitsecretie aangetoond met behulp van qPCR, western blot, ELISA en immunofluorescentie. Om de functionaliteit en de potentie van de PEDF en GM-CSF uitgescheiden door de getransfecteerde RPE-cellen te bevestigen, hebben we een in vitro assay ontwikkeld om de reductie van H2O2-geïnduceerde oxidatieve stress op RPE-cellen in cultuur te kwantificeren. Celbescherming werd geëvalueerd door celmorfologie, dichtheid, intracellulair niveau van glutathion, UCP2-genexpressie en levensvatbaarheid van cellen te analyseren. Zowel getransfecteerde RPE-cellen die PEDF en/of GM-CSF overexpressie geven als cellen die niet-getransfecteerd zijn maar voorbehandeld met PEDF en/of GM-CSF (in de handel verkrijgbaar of gezuiverd uit getransfecteerde cellen) vertoonden een significante antioxidant celbescherming in vergelijking met niet-behandelde controles. Het huidige H2O2-model is een eenvoudige en effectieve benadering om het antioxiderende effect te evalueren van factoren die effectief kunnen zijn om AMD of vergelijkbare neurodegeneratieve ziekten te behandelen.

Inductie van oxidatieve stress in menselijke retinale pigmentepitheelcellen ARPE-19 en primaire hRPE-cellen werden behandeld met verschillende concentraties van H2O2 gedurende 24 uur en het intracellulaire niveau van de antioxidant glutathion werd gekwantificeerd (Figuur 2A,B). H2O2 bij 50 μM en 100 μM had geen invloed op de glutathionproductie, terwijl er bij 350 μM een signi...

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

We presenteren een protocol voor de ontwikkeling en het gebruik van een oxidatieve stress-model door retinale pigmentepitheelcellen te behandelen met H2O2, waarbij celmorfologie, levensvatbaarheid, dichtheid, glutathion en UCP-2-niveau worden geanalyseerd. Het is een nuttig model om het antioxiderende effect te onderzoeken van eiwitten die worden uitgescheiden door getransfecteerde cellen om neuroretinale degeneratie te behandelen.

Inductie en analyse van oxidatieve stress in doornroosje Transposon-getransfecteerde menselijke retinale pigmentepitheelcellen

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

Identificatie van<em&gt; Sleeping Beauty</em&gt; Transposon inserties in vaste tumoren met Linker-gemedieerde PCR

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

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Transcriptomic Analyse van de Mens Retina Chirurgische Specimens gebruiken Journal

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

Isolatie, cryopreservatie en cultuur van de mens Amnion epitheelcellen voor klinische toepassingen

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

Presterende Subretinal Injecties in Knaagdieren om retinale pigment epitheel cellen leveren in Suspension

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

Studeren alvleesklier- Stamcel Kenmerken voor het ontwikkelen van nieuwe behandelingsstrategieën

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

Een Step by Step Protocol voor Subretinal Surgery in Rabbits

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

Uitputting van de Muis Cellen van geënte, humane tumoren verbetert Downstream Analyse van Target Cellen

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

Inductie van versnelde atherosclerose bij muizen: het "Wire-Injury" Model

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

Fosfor-31 magnetische resonantie spectroscopie: een instrument voor het meten<em&gt; In Vivo</em&gt; Mitochondrial Oxydatieve Phosphorylation Capacity in Human Skeletal Muscle

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

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Op elektroporatie gebaseerde genetische modificatie van primaire menselijke pigmentepitheelcellen met behulp van het Sleeping Beauty Transposon-systeem